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Multimodal Chromatography – Handbook

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Chromatography
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Life Sciences Multimodal
immobilized pH gradients Handbook Chromatography
Principles and Methods 18-1157-58 Handbook
80-6429-60 29-0548-08
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Principles and Methods
GST Gene Multimodal
Fusion System Chromatography
Handbook Handbook

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Principles and Methods High-throughput
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Principles and Methods 28-9403-58 Nucleic Acid Sample
Preparation for
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Downstream Analyses
Principles and Methods

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Life Sciences Hydrophobic Interaction GE Healthcare
Life Sciences Protein Sample
Chromatography Systems and Reversed Phase Preparation
Instrument Management Chromatography Handbook
Handbook Principles and Methods 28-9887-41
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and Reversed Phase
Chromatography
11-0012-69 Protein Sample
ÄKTA™ Laboratory-scale Preparation
Chromatography Systems Principles and Methods Handbook
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Life Sciences Antibody Purification GE Healthcare
Life Sciences Imaging GE Healthcare
Life Sciences Purifying Challenging
Handbook 473 532 635 650 685 785 Principles and Methods Proteins
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Imaging Challenging Proteins
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Biacore™ Assay Handbook Handbook Chromatography and Purification Handbook
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Principles and Methods
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Multimodal
Chromatography
Handbook
Foreword
Biopharmaceuticals represent the success of the modern pharma industry and are forecasted
to take over as the most important category of drugs in the near future. After 30 years of
research/development focused on therapeutic proteins, the industry’s maturation is visible
through increasing competition and a general drive to improve the efficiency of process
development and manufacturing operations (technology upgrades and fine tuning of solutions
established over the years). The cost of a legacy process launched in the 1990s can be reduced
by 80% to 90%, with downstream processing improvements playing a significant role in these
attempts, next only to cell culture productivity increases. Flexible facility design and 10- to
100-fold more efficient processing tools are available to those working in today’s development
arena for biologics. These design and tool improvements enable the manufacturer to leapfrog
the cost situation of their legacy competition.
Purification of biologics, whether with industrial or research scope, is powered by selectivity
provided through the features of the ligands on a chromatography support. From that
perspective, multimodal chromatography media represent one of the most powerful additions
to the process development scientist’s toolkit in the recent two decades.
Thus, in addition to addressing the increasing technical challenges arising from high-biomass
upstream processes and the growing economic pressure on manufacturing operations, the
biopharma industry is discovering that the truly new selectivities with broad applicability that
multimodal materials offer are rare innovations, and is adopting them throughout the industry.
Operating conditions for multimodal chromatography media are not as easily predicted as
for classic ion exchangers, a fact that initially delayed their acceptance. However, with the
availability of high-throughput process development (HTPD) approaches for screening for
optimum conditions, these conditions can be found in a very short amount of time despite the
fact that a larger number of conditions needs to be investigated.
During the development of a multimodal purification step, an operating window can usually
be identified that, more often than not, enables reduction of the number of purifications steps
by employing the new selectivities the multimodal ligands offer. Combining hydrophobic
and/or other types of interactions with an ion exchange modality encourages the search
for operating conditions that would eliminate the need for conditioning of process streams,
thus simplifying the corresponding installation of process hardware. As with standard ion
exchangers, multimodal chromatography media can be operated in both flowthrough and
bind/elute modes. For the former, smaller columns can be used to handle large quantities of
target product, as the steps serve as impurity scavengers. In conclusion, use of multimodal
chromatography media enables effective and economical purification processes.
We hope you will find this handbook to be a helpful guide to the vast number of opportunities
that multimodal selectivities offer to your work in purification development. Working with modern
tools will help you unlock great cost savings and even open up growing revenue opportunities
for your facility. Use them wisely and with scientific rigor. You will find them worth your time!

Günter Jagschies
Strategic Customer Relations Leader
Contents
Introduction...................................................................................................................................... 5
Common acronyms and abbreviations..............................................................................................................6
Chapter 1
Chromatography principles and process development........................................................... 7
Protein purification methods...................................................................................................................................7
Protein purification strategies.........................................................................................................................9
Limiting the number of steps in a purification procedure ............................................................ 10
Bind/elute vs flowthrough mode................................................................................................................ 10
Performance parameters...............................................................................................................................11
Workflow—screening, optimization, and scale-up....................................................................................13
Large-scale purification.......................................................................................................................................... 14
Practical considerations in scale-up......................................................................................................... 14
Chapter 2
Overview of multimodal chromatography ............................................................................... 17
Multimodal ligand approach.................................................................................................................................18
Multimodal chromatography in a purification workflow........................................................................19
Determining optimal experimental conditions............................................................................................20
Bind/elute vs flowthrough mode in multimodal chromatography ...................................................20
Salt types and additives..........................................................................................................................................22
Chapter 3
Multimodal chromatography media.......................................................................................... 23
Capto adhere ...............................................................................................................................................................25
pH operating window.......................................................................................................................................25
Removal of aggregates...................................................................................................................................26
Viral clearance......................................................................................................................................................26
Removal of other impurities and contaminants................................................................................. 27
Salt type and additives.................................................................................................................................... 27
Regeneration........................................................................................................................................................28
Capto adhere ImpRes...............................................................................................................................................28
Fast mass transfer.............................................................................................................................................28
High resolution and small pool volumes.................................................................................................29
Regeneration........................................................................................................................................................30
Capto MMC.....................................................................................................................................................................30
High salt tolerance.............................................................................................................................................30
Unique selectivity............................................................................................................................................... 31
Salt type and additives....................................................................................................................................33
Regeneration........................................................................................................................................................34
Capto MMC ImpRes....................................................................................................................................................34
Fast mass transfer.............................................................................................................................................34
Salt tolerance........................................................................................................................................................34
High resolution and high capacity............................................................................................................. 35
Regeneration........................................................................................................................................................ 35
Capto Core 700............................................................................................................................................................ 35
Designed for flowthrough chromatography......................................................................................... 35
Improved productivity......................................................................................................................................36
Regeneration........................................................................................................................................................ 37
Multimodal libraries................................................................................................................................................... 37
Multimodal libraries from Custom Designed Media (CDM)............................................................ 37
Custom Designed Products (CDP)...............................................................................................................38
Formats of multimodal chromatography products.................................................................................. 39

29-0548-08 AA 3
Chapter 4
Applications..................................................................................................................................... 41
Capto adhere applications..................................................................................................................................... 41
1. Optimization of loading conditions on Capto adhere using DoE............................................. 41
Method design and optimization........................................................................................................... 41
Results.................................................................................................................................................................45
Conclusions—optimal loading conditions and general trends...............................................48
2. Development of operational excellence in MAb process development and
manufacturing using Capto adhere.......................................................................................................49
2a. HTS and optimization of a multimodal polishing step in a MAb purification
process using Capto adhere.....................................................................................................................49
Materials and methods...............................................................................................................................50
Results and discussion................................................................................................................................53
Conclusions.......................................................................................................................................................60
2b. Scale-up of a downstream MAb purification process using
HiScreen and AxiChrom columns with Capto adhere................................................................. 61
2c. A flexible antibody purification process based on ReadyToProcess products
with Capto adhere.........................................................................................................................................63
3. Viral clearance using Capto adhere.......................................................................................................64
Capto adhere ImpRes applications...................................................................................................................65
1. Polishing of MAbs using Capto adhere ImpRes in bind/elute mode........................................65
Materials and methods...............................................................................................................................65
Results and discussion................................................................................................................................67
Conclusions.......................................................................................................................................................73
2. Viral clearance using Capto adhere ImpRes...................................................................................... 74
Capto MMC applications......................................................................................................................................... 74
1. HTS and process development for capture of recombinant pro-insulin from
E. coli using Capto MMC.............................................................................................................................. 74
Materials and methods............................................................................................................................... 74
Results and discussion................................................................................................................................ 76
Conclusions.......................................................................................................................................................82
2. Evaluation of three media for capture of recombinant human serum albumin
from Pichia pastoris and scale-up using Capto MMC...................................................................83
Capto MMC ImpRes applications........................................................................................................................84
1. Polishing of MAbs using Capto MMC ImpRes in bind/elute mode............................................84
Materials and methods...............................................................................................................................84
Results and discussion................................................................................................................................85
Conclusions....................................................................................................................................................... 92
2. Effective polishing of domain antibodies (DAbs) using Capto MMC ImpRes.......................92
Capto Core 700 application...................................................................................................................................94
Purification of influenza A/H1N1 using Capto Core 700.............................................................94
References....................................................................................................................................... 97
Appendix 1
Characteristics of multimodal chromatography media.......................................................100
Appendix 2
Maintenance of media and storage conditions......................................................................103
Appendix 3
Use of multimodal media for MAb purification.......................................................................106
Product index.................................................................................................................................108
Related literature.........................................................................................................................109
Ordering information...................................................................................................................110

4 29-0548-08 AA
Introduction
This handbook explores the advantages of using multimodal chromatography in the field of
large-scale (bioprocess) protein purification. In multimodal chromatography, the medium (resin)
provides more than one type of interaction between ligand and sample components. Although
use of traditional media (such as affinity, ion exchange, hydrophobic interaction, etc.) is fine in
most instances, under certain purification challenges the required level of purity is not reached.
Challenges arise, for example, when there is a need for salt-tolerant ion exchange media
for capture of recombinant proteins or when the goal is to effectively remove aggregates,
host cell protein (HCP), viruses, or protein A in purification of monoclonal antibodies (MAbs).
Because multimodal media can be uniquely designed, multimodal chromatography offers an
alternative to traditional media, providing new selectivities for specific targets. Multimodal
chromatography may also improve performance.
The Multimodal Chromatography handbook is targeted toward scientists working in the fields
of process development and large-scale (bioprocess) protein purification who desire to learn
more about the benefits of adding multimodal chromatography to their arsenal of purification
strategies. The handbook begins in Chapter 1 with a brief overview of protein purification
terminology, methods, and strategies, to put the principles of multimodal chromatography
in context with other purification tools. It continues with an overview of multimodal
chromatography itself (Chapter 2), followed by a description of multimodal chromatography
media from GE Healthcare (Chapter 3). Chapter 4 introduces both theoretical and practical
aspects of incorporating multimodal chromatography into a purification strategy, by providing
application examples using multimodal media from GE Healthcare. Appendices provide
additional support information.

29-0548-08 AA 5
Common acronyms and abbreviations

A280 UV absorbance at specified wavelength (in this example, 280 nanometers)

AC affinity chromatography
AIEX anion exchange chromatography
CDM Custom Designed Media
CDP Custom Designed Products
CHO Chinese hamster ovary
CIEX cation exchange chromatography
CIP cleaning-in-place
CIPP capture, intermediate purification, polishing
CCS cell culture supernatant
CV column volume(s)
D/A dimers and aggregates
DAb/DAbs domain antibody/antibodies
DoE design of experiment(s)
DBC dynamic binding capacity
ECP E. coli protein
GF gel filtration (also referred to as SEC, size exclusion chromatography)
HA hemagglutinin
HIC hydrophobic interaction chromatography
HCP host cell protein(s)
HTPD high-throughput process development
HTS high-throughput screening
IEX ion exchange chromatography
IMAC immobilized metal ion affinity chromatography
IPA isopropyl alcohol
kD kilodaltons
LOQ limit of quantitation
MAb/MAbs monoclonal antibody/antibodies
mAU milli absorbance units
MDCK Madin-Darby canine kidney
MF microfiltration
MM multimodal
MMC multimodal chromatography
mS millisiemens
MuLV Murine Leukemia Virus
MVM Minute Virus of Mice
ND not determined
pI isoelectric point
QbD quality by design
RPC reversed phase chromatography
SBC static binding capacity
SEC size exclusion chromatography (referred to in this handbook as gel filtration, GF)
TCID tissue culture infectious dose
UF/DF ultrafiltration/diafiltration

6 29-0548-08 AA
Chapter 1
Chromatography principles and
process development
In order to appreciate where multimodal chromatography fits into the overall scheme of
protein purification, it is helpful to review current protein purification terminology, methods,
and strategies.
In protein purification, the stationary phase is termed the chromatography medium
(sometimes also called chromatography resin). The medium is composed of a porous matrix to
which a ligand can be coupled. This coupling is often referred to as functionalizing the matrix.
The matrix, generally in the form of spherical beads, and the coupled ligand that contains a
specific molecular group giving a tailored function to the chromatography medium, are utilized
for binding of either the target protein or contaminants. With gel filtration (GF), no ligand is
present, and separation of molecules is achieved based on the accessibility of the bead pores
for the different sized molecules.
The term multimodal, sometimes also referred to as mixed-mode, is broadly used in the
context of an object having more than one mode of action. These different modes of action
can operate independently of one another or in concert. In the field of protein purification,
multimodal chromatography refers to media that provide more than one type of interaction
between ligand and sample components. Several such media from GE Healthcare are
discussed in Chapter 3. In addition, a special type of multimodal medium (Capto™ Core 700)
is described for which both multimodal ligand interaction and principles of size exclusion are
used for the separation of molecules.

Protein purification methods

Biomolecules are purified using methods that separate according to differences in specific
properties. The main properties upon which protein purification is based are listed in Table 1.1.
For more information, refer to the numerous handbooks from GE Healthcare (see “Related
literature” at the end of this handbook).

29-0548-08 AA 7
Table 1.1. Protein properties exploited in chromatographic purification

Protein property Method Description

Various (e.g., charge, Multimodal chromatography Separation through at least one ligand
hydrophobicity, and type that has more than one interaction
hydrogen bonding) site. Different modes of interaction can
be expected depending on experimental
conditions, e.g., electrostatic, hydrophobic,
π-π interaction, hydrogen bonding, and
thiophilic interaction. These interactions can
cooperate or work independently.
Specific ligand Affinity chromatography (AC) Separation through specific interaction
recognition between target molecule and an immobilized
affinity ligand.
Immobilized metal ion affinity Separation through affinity of proteins with
chromatography (IMAC) His, Cys, or Trp amino acid residues and
immobilized metal ions.
For further information refer to the
handbooks Affinity Chromatography:
Principles and Methods (18-1022-29) and
Antibody Purification (18-1037-46) from
GE Healthcare (see also “Related literature”
at the end of this handbook).
Charge Ion exchange chromatography (IEX), Separation based on electrostatic
encompassing anion and cation interactions between solutes and
exchange chromatography (AIEX chromatography medium.
and CIEX, respectively) For further information refer to the handbook
Ion Exchange Chromatography and
Chromatofocusing: Principles and Methods
(11-0004-21 from GE Healthcare (see also
“Related literature” at the end of this handbook).
Size Gel filtration ([GF], also Separation of solutes according to size.
referred to as size exclusion For further information refer to the handbook
chromatography [SEC]) Gel Filtration: Principles and Methods
(18-1022-18 from GE Healthcare (see also
“Related literature” at the end of this handbook).
Hydrophobicity Hydrophobic interaction Separation based on hydrophobic interactions.
chromatography (HIC) HIC is run in aqueous solutions while RPC is
and run in combination with organic solvents.
Reversed phase chromatography For further information refer to the handbook
(RPC) Hydrophobic Interaction and Reversed Phase
Chromatography: Principles and Methods
(11-0012-69) from GE Healthcare (see also
“Related literature” at the end of this handbook).
Isoelectric point (pI) Chromatofocusing Separation based on pI.
For further information refer to the handbook
Ion Exchange Chromatography and
Chromatofocusing: Principles and Methods
(11-0004-21) from GE Healthcare (see also
“Related literature” at the end of this handbook).

8 29-0548-08 AA
Protein purification strategies
Regardless of the technique chosen to purify a target protein (or proteins), the scientist
generally faces the need to obtain the protein with sufficient purity and quantity in an efficient
and economical manner. The purification strategy Capture, Intermediate Purification, and
Polishing (CIPP) (Fig 1.1) has been developed to both describe a protein purification process
by assigning a specific task to each unit operation and to simplify planning and execution of
protein purification at any scale. The strategy gives guidelines for how to combine purification
methods optimally to reach the set goals.
Purity

Polishing
Achieve final
Intermediate high-level purity
purification
Remove bulk
Capture impurities

Isolate, concentrate,
Preparation, and stabilize
extraction,
clarification
Step
Fig 1.1. Illustration of the different stages in a purification process.

Sample preparation is the starting point of any purification strategy. The purpose of sample
preparation is generally to obtain a clarified extract of the source material, although techniques
are available to address situations in which the starting material is unclarified. The extract
should be prepared under or adjusted to conditions that are compatible with the first
chromatography step.
In the capture stage, the objectives are to isolate, concentrate, and stabilize the target product.
The product should be concentrated and transferred to an environment that will conserve
potency/activity. At best, significant removal of critical contaminants can also be achieved.
During the intermediate purification stage, the key objective is to remove most of the bulk
impurities, such as other proteins and nucleic acids, endotoxins, and viruses. If the capture step
is highly efficient, the intermediate purification stage is often omitted in favor of one or more
polishing steps.
The objective of the polishing stage is to achieve final purity. In this stage, remaining impurities,
often at trace levels, and possibly also undesirable product variants and proteins closely related
to the target protein, are removed, and the target protein may be transferred to conditions
suitable for use or storage.
General advice:
• Apply a systematic approach to development of a purification strategy.
• Assign a specific objective to each step within the purification process.

29-0548-08 AA 9
Limiting the number of steps in a purification procedure
The purification strategy described above does not mean that all protocols must have a set
number of chromatography purification steps. The number of steps to be included will depend
on the purity requirements, intended use of the protein, and the complexity of the starting
material. Keep in mind that increasing the number of purification steps will often decrease the
overall protein yield (Fig 1.2) and that more steps mean a longer purification time, which can
be detrimental to protein stability and activity. For most laboratory-scale and bioprocess-scale
work, a two- or three-step purification protocol will be sufficient, although difficult purifications
may require additional steps.
100

80
95%/step
Yield (%)

60

90%/step
40
85%/step
20 80%/step
75%/step
0
1 2 3 4 5 6 7 8 9 10
Number of steps
Fig 1.2. Total yield versus number of purification steps.

Because multimodal media are characterized by selectivities that are different from those of
“traditional” ligands, their use opens up new opportunities for solving challenging purification
problems. Multimodal media can often reduce the number of steps needed to reach the
required level of purity.

Bind/elute vs flowthrough mode

The mode of operation also plays a part in the successful multimodal chromatography
purification scheme. Using IEX as an example method, the bind/elute mode of separation
works by binding sample components to the chromatography medium based on electrostatic
charges. If the medium has negatively charged functional groups (as in CIEX), sample
components with positively charged ions will bind to it; if the medium has positively charged
functional groups (as in AIEX), then protein sample components with negatively charged ions
will bind to it. Once sample components are bound, the medium is washed, and nonbound
material is washed through, after which the bound material is eluted under conditions of
increasing ionic strength or change in pH. With increasing salt concentration, salt ions in
the buffer compete for binding with the charges on the medium, and the bound material
is displaced and eluted. Alternatively, when pH is changed, bound proteins are titrated and
eventually become noncharged or have the same charge as the ligand, leading to repulsion
and elution of the bound protein.
In flowthrough mode, on the other hand, the pH of the sample and buffer is selected to modifiy
the charge of the protein of interest or the chromatography medium such that the protein
will not bind but will rather flow through the column, leaving most impurities bound. Thus,
the advantages of flowthrough mode are that higher loads can be used and there are fewer
washing and elution steps—one needs mainly to be concerned with maximizing recovery and
binding of impurities. The purity of the protein of interest found in the flowthrough fractions
can be increased by optimizing conditions such as pH, buffer, and salt. A wash step is used for
increasing the yield of the target protein by allowing weakly bound proteins to be collected.
An elution step is sometimes used to remove/elute some bound contaminants before the
cleaning-in-place (CIP) step is applied.

10 29-0548-08 AA
Performance parameters
For optimal productivity in process-scale chromatography, four important performance
parameters should be considered when planning each purification step: resolution, capacity,
speed, and yield. The importance of each parameter will vary depending on whether a
purification step is used for capture, intermediate purification, or polishing. Purification
methods should be selected and optimized to meet the objectives for each purification step.
Purity of the final product can never be compromised, but the way this purity is achieved must
keep productivity and process economy in mind. In today’s competitive environment in the
pharmaceutical and biotech industries, all process as well as individual unit operations need
to be optimized for these factors. Productivity, often defined as gram or kilogram of product
produced per hour or day per liter of chromatography medium used, needs to be high to
facilitate a good process economy (e.g., cost per gram of product produced).
Resolution depends on the selectivity and efficiency of the packed bed, sample, and
conditions (e.g., flow rate) used. In general, high resolution is more important at the final stage
of purification because at this point the impurities and target protein are likely to have very
similar properties.
Capacity refers to how much sample can be loaded onto the column. The amount of sample
that can be loaded may be limited by volume (as in GF) or by total amount of target protein
and impurities that can be bound to the column without loss or reduction of purity. (Purity
may decrease with high sample loads.) The amount of sample and usually also the volume of
sample decreases toward the final stage of the purification.
Speed is most important at the beginning of purification where contaminants, such as
proteases, must be removed as quickly as possible.
Yield becomes increasingly important as the purification proceeds because of the increased
value of the purified product. Yield may be decreased by destructive processes in the sample
and by unfavorable conditions during the purification.
Each protein purification method has inherent characteristics that determine how it should
be optimized for the key performance parameters described above: resolution, capacity,
speed, and yield. Table 1.2 is a guide to the suitability of each purification method for the
stages in CIPP. Refer to the GE Healthcare handbooks (see “Related literature” at the end of this
handbook) for in-depth discussion of these and other chromatography techniques.
AC is the method of choice and the most common capture step when a specific ligand is
available against the target protein, for example, protein A media for antibody purification.
AC can often combine resolution, capacity, speed, and yield in a single purification step,
although it is more common that it is followed by at least one polishing step.
IMAC is an excellent capture step for histidine-tagged proteins and is used with or without a
subsequent polishing step. The technique is generally not used in bioprocess applications.
GF is seldom used as a capture step because of limitation in sample volume, but it is sometimes
used as a polishing step despite the sample volume limitation.
IEX is a common method for any purification stage. IEX columns are suitable for the capture
stage because they have high binding capacity, allow high flow rates, and are resistant to
harsh cleaning conditions that may be needed after purification of crude samples. CIEX is
more common in capture than AIEX, and generally also has higher capacity than AIEX. IEX is
frequently used as a polishing step.

29-0548-08 AA 11
 Table 1.2. Suitability of purification methods for CIPP

Typical characteristics Purification phase Conditions

Selectivity Capacity Capture Intermediate Polishing Sample start conditions Sample end conditions

12 29-0548-08 AA
pH depends on protein and type
pH and ionic strength depends
Multimodal +++ ++ +++ +++ +++ of ligand; salt tolerance of binding can in
on protein and ligand type
some cases be expected (see Table 1.1)

AC +++++ +++ +++ ++ + Various binding conditions Specific elution conditions

Low concentration of imidazole High concentration of imidazole,

IMAC +++ ++ +++ ++ +
and high concentration of NaCl pH>7, 500 mM NaCl

Most conditions acceptable;

GF +++ + + +++ Buffer exchange possible, diluted sample
limited sample volume

Low ionic strength; pH depends

IEX +++ +++ +++ +++ +++ High ionic strength and/or pH changed
on protein and IEX type

High ionic strength; addition

HIC +++ ++ ++ +++ +++ Low ionic strength
of salt required

Ion-pair reagents and organic Organic solvents (risk for loss

RPC ++++(+) ++ (+) + +++
modifiers may be required of biological activity)

Polybuffer
Chromatofocusing +++ ++ + + ++ Low ionic strength
Low ionic strength
HIC can be an excellent capture step, especially after ammonium sulfate precipitation. The salt
concentration and the total sample volume will be significantly reduced after elution from the
HIC column. HIC can be used at any stage.
RPC is very rarely used as a capture step because the method will usually bind too many of
the sample components in an extract. On the other hand, RPC is often used for purification of
therapeutic peptides and other small biomolecules. If the target protein is sufficiently stable,
RPC can be efficient for polishing.
Chromatofocusing is a high-resolution method with moderate binding capacity and is therefore
rarely used as a capture step. The method can be used for the polishing stage. It has yet to find
its place in bioprocess applications.
Multimodal chromatography can be used advantageously in CIPP at various stages. It offers
new options, for example, high conductivity binding and new selectivity, when faced with
challenging conditions or separations in a purification workflow. See Chapter 2 for a detailed
description of multimodal chromatography.
General advice:
• Combine methods that apply different separation mechanisms.
• Minimize sample handling between purification steps by combining methods to avoid the
need for sample conditioning before the next step. The product should ideally be eluted from
the first column in a buffer suitable for the starting conditions required for the next method.

Workflow—screening, optimization, and scale-up

Modern chromatographic processes are increasingly driven by economic factors. The need to
shorten time to market means that the process development must be fast and inexpensive without
compromising quality. With this in mind, a good development workflow is important and will
ensure a robust process where critical parameters have been identified and are under control.
Figure 1.3 shows an example of a typical workflow. It starts with screening of conditions in
high-throughput formats such as 96-well filter plates or mini-columns, followed by optimization
in small columns and finally scale-up to final column size. For further information on high-
throughput 96-well filter plates refer to the handbook High throughput process development
with PreDictor™ plates (28-9403-58) from GE Healthcare (see also “Related literature” at the end
of this handbook). For more information on mini-columns, see “Formats” in Chapter 3.

HTPD screening Column optimization Large scale

Fig 1.3. A typical workflow. Initial screening is performed in a high-throughput format, followed by optimization
using a small column and verification in large scale. A discussion of Design of Experiments (DoE) and Monte
Carlo simulations is provided in the text below.

29-0548-08 AA 13
High-throughput process development (HTPD) shortens development time at the same
time as it increases the amount of knowledge about the purification process and the
influence of different process parameters (such as pH, conductivity, load, etc.). In multimodal
chromatography, the availability of high-throughput process tools is especially valuable
because of the complexity of interactions. Experimental conditions should be carefully
screened in order to fully exploit the potential of the media and understand critical parameters.
When appropriate conditions have been found with the HTPD formats, further screening and
optimization is performed in small columns. DoE (see Box, G. E. P. and Draper, N. R. [1987] and
Metropolis, N. and Ulam, S. [1949] in “References”) is a statistical tool used during optimization
to plan experiments to maximize the information extracted. It allows for detailed quantitation
of the cause and effect relationship between process inputs and process outputs. It is very
well complemented by Monte Carlo simulation, which allows for quantitation of how variation
in the process inputs (including random process variation) is translated into variation in the
process outputs. DoE together with Monte Carlo simulations greatly increases the likelihood
that a good purification process will be established. The use of high-throughput screening (HTS)
technology is becoming the generally accepted approach for improving process understanding
and for finding and defining the experimental space for a DoE study. More on this subject can
be found in Chapter 4.
This workflow is in line with Quality by Design (QbD) guidelines, which offer a systematic
approach to process development that emphasizes product and process understanding,
process control, and quality risk management (see Guidance for Industry; Q8[R2]
Pharmaceutical Development {2009} in “References”).

Large-scale purification
As previously discussed, key concerns in large-scale purification differ to some extent
from those typical at laboratory scale. The emphasis in large-scale purification is on the
development of robust and cost-effective protocols with a minimum number of unit operations
in order to improve overall process economy, all without ever compromising the quality
(purity) of the desired product. When going from laboratory scale to production scale, buffer
consumption will be an issue, and choice of chromatography medium must be thoroughly
considered based on economics, security of supply, and adherence to regulatory requirements.
If the purification process is going to be scaled up, the scale-up perspective must be considered
already at the research stage during the development of a new biopharmaceutical so as to
avoid problems at later stages, for example, scalability of separation methods, packability of
chromatography media, and process economy.
Below we provide a brief introduction to large-scale purification. For further reading, refer
to the Handbook of Process Chromatography: Development, Manufacturing, Validation and
Economics by Lars Hagel, Günter Jagschies, and Gail Sofer, 2nd ed., 2008, Academic Press.

Practical considerations in scale-up

It is important to define the parameters during process development to obtain an efficient
process with high productivity and to know how changes influence the process and the final
product. Established conditions are used as the basis for scale-up. Scale factors between 10
and 100 per step are recommended. There are a number of chromatography parameters that
have to be maintained to ensure conformity in performance between laboratory scale and final
production scale:

14 29-0548-08 AA
• residence time
• maintenance of gradient slope (gradient volume/media volume)
• sample concentration and composition
• ratio of sample volume to media volume

Scale-up is obtained by increasing:

• column dimensions
• volumetric flow rate
• sample volume proportionally to column volume
• gradient volume proportionally to column volume
Increasing the bed volume by increasing the column diameter, and increasing volumetric flow,
sample load volume, and gradient volume accordingly, will ensure the same performance
and cycle time as in laboratory scale during method development. The bed volume can also
be increased by increasing the bed height and keeping the residence time constant, but
chromatographic behavior may in some rare cases differ, with possible impact on purity.
In IEX, HIC, and AC, adequate productivity is normally obtained with columns having a bed
height of 10 to 20 cm. In some cases, if the ideal column diameter is not available, it might
be an advantage to increase the bed height using a column with a diameter smaller than the
diameter of the ideal column, but an increase in the pressure drop must be anticipated.
Numerous applications that involve scale-up are presented in Chapter 4.

29-0548-08 AA 15
16 29-0548-08 AA
Chapter 2
Overview of multimodal chromatography
In multimodal chromatography, ligands interact with the target molecule through two or more
modes of action. These different modes of action can operate independently or in concert.
Electrostatic interactions are commonly involved, but hydrogen bonding and hydrophobic
interactions can be significant. The strength of these individual interactions depends on the
target molecule and on the overall process conditions.
Multimodal chromatography media are characterized by selectivities that are different from
those of “traditional” ligands (i.e., those seen in AC, IEX, HIC, etc.), thereby opening up new
opportunities for solving challenging purification problems. At the same time, the higher
complexity of multimodal media normally requires process optimization studies in order to take
full advantage of the potential of this technology. Having efficient HTPD tools and methodology
facilitates this optimization work.
Multimodal chromatography media can be prepared in different ways (Fig 2.1):
1) The stochastic approach, in which two or more different interactions are introduced
independently on the matrix. Use of this approach provides a very efficient way to study
the influence of a second interaction because in this construction the ratio of the different
groups can be gradually modified. With this approach, the homogeneity of the medium and
the three-dimensional interactions cannot be guaranteed.
2) The multimodal ligand approach, in which groups promoting different interactions are
connected via a scaffold, resulting in a well-defined three-dimensional structure, and in
which the stoichiometric ratio (e.g., 1:1) between the groups is fixed.
3) Use of a responsive material that will, according to the conditions, exhibit different primary
interactions. In the example in Figure 2.1, the medium changes from a hydrophobic to a
hydrophilic one through temperature change, where the hydrophobic group is less exposed
(buried in the structure). Other types of physically and/or chemically induced changes could
possibly also be employed.

Stochastic Multimodal ligand Responsive material

O
O O- Induced change:
temp, pH
O O
O O
O- Scaffold

Fig 2.1. Schematics showing examples of creating a multimodal medium or column based on the three types
of preparation methods described above.

29-0548-08 AA 17
In a category by itself, Capto Core 700 (Fig 2.2) from GE Healthcare combines GF and multimodal
anion exchange characteristics, as described in Chapter 3.

(A) Shell
Core

(B)
H
Core Spacer N+
O
H

Fig 2.2. (A) Schematic representation of the principle for Capto Core 700 showing a bead with the inactive
porous shell and the ligand-activated core. Small proteins and contaminants (colored green, yellow, and
purple) penetrate the core while target viruses (red) and larger proteins are excluded from the medium and
are collected in the flowthrough. (B) The immobilized ligand in the core.

Multimodal ligand approach

To create multimodal chromatography media using the multimodal ligand approach, ligands
that have multiple modes of interaction are immobilized on the chromatography matrix
(Fig 2.3). These interactions are introduced via a chemical scaffold that links the new interaction
with the primary one, resulting in a well-defined three-dimensional new multimodal ligand.
The interactions introduced can be quite diverse, for example, electrostatic, hydrophobic,
π-π, hydrogen bonding, and thiophilic interactions. Thus, an IEX medium might be modified to
include another functionality providing the option of hydrophobic interactions, with the result
being that in one step a purification based on both electrostatic and hydrophobic properties
could occur.
The well-defined primary functionality ensures a degree of control over the chromatographic
behavior of the multimodal medium, which at the same time allows for the modification of
chromatographic performance in ways that can be advantageously used in specific cases.
Typical positive effects associated with the multimodal approach include differences in binding
capacities under specific conditions, changes in elution conditions, and enhanced selectivities.
The current multimodal product offering from GE Healthcare consists of Capto MMC,
Capto MMC ImpRes, Capto adhere, Capto adhere ImpRes (all developed using the multimodal
ligand approach), and Capto Core 700, with layered functionality. The stochastic approach
has also been used for some of the media included in the multimodal libraries available from
GE Healthcare (available via Custom Designed Media, CDM; see Chapter 3). Figure 2.3 compares
traditional and multimodal media, using examples of GE Healthcare products.

18 29-0548-08 AA
(A) (B)
Traditional media Multimodal media
Scaffold

CM Sepharose™ Fast Flow Capto™ MMC

O O
O OH OH
O- O O S NH

Q Sepharose Fast Flow

OH OH O O-
O O N+
Capto adhere

OH OH
Phenyl Sepharose HP O O N+
OH
O

Octyl Sepharose Fast Flow Capto Core 700

H
O
Core Spacer N+
O
H

Fig 2.3. Comparison of traditional and multimodal media. (A) Schematic of traditional media and examples
(CM Sepharose Fast Flow, Q Sepharose Fast Flow, Phenyl Sepharose HP, and Octyl Sepharose Fast Flow).
(B) Multimodal media. Capto MMC, Capto adhere, and Capto Core are shown as examples. Note that the
Capto Core 700 ligand is found only in the core of the bead.

Chromatographic performance will be strongly dependent on the importance of the primary

and secondary modes of interaction, respectively. For example, the traditional ion exchange
behavior of a strong anion exchanger is affected by the gradual increase of hydrophobic
groups or H-bond groups.
See Chapter 3 for a description of the functionalities of multimodal media and custom libraries
developed by GE Healthcare.

Multimodal chromatography in a purification workflow

Multimodal chromatography offers new solutions in purification workflows by widening the
window of operation in circumstances where traditional media are not as effective as desired.
Such circumstances may be encountered, for example, when the loading conductivity of the
sample is too high for traditional ion exchange media, when there is a need to reduce the
number of purification steps, or when the selectivity of traditional media is insufficient to
provide the required purity of the target protein.
When working with process development of a purification process and exploring the introduction
of multimodal chromatography into a purification workflow, early decision points include:
• determining which multimodal chromatography medium to select (discussed in Chapter 3)
• choosing the format that will best suit the user’s needs (bulk media, prepacked columns, or
plates) (discussed in Chapter 3)

29-0548-08 AA 19
• deciding (for bulk media and prepacked columns) between flowthrough and bind/elute
mode (discussed in Chapters 1 and 2)
• determining conditions that will optimize the purification (discussed in Chapters 2 and 4)
• planning for the eventual scale-up of the optimized purification protocols (discussed in Chapter 4)
Given the higher complexity of multimodal media compared with traditional media, somewhat
more emphasis on process optimization is required in order to take full advantage of the
potential of this technology. Having efficient HTPD tools and methodology and making use of
DoE and Monte Carlo simulations facilitate this optimization work (see Chapter 1).

Determining optimal experimental conditions

It is recommended to explore a wide range of chromatography conditions early, to increase
process understanding and increase the likelihood of developing a robust purification process.
This is the case for both traditional and multimodal chromatography.
Consider the differences between multimodal and traditional IEX and HIC media (Fig 2.4),
and several questions may come to mind: What will happen on a medium that contains both
interactions? Will the protein of interest bind in a high-conductivity environment? Under which
conditions will it be possible to elute the target protein?
With multimodal media, it is a priori more difficult to predict the answers to these questions.
The answers will be determined by the multimodal functionality of the media, by the operating
conditions, and by the target molecule itself. Thus optimization is critical for success.

Traditional media
OH
O O O
SO3-

Low conductivity IEX Low conductivity Low Int

High conductivity Low Int High conductivity HIC

Hypothetical multimodal media

OH O
O
SO3-

Low conductivity IEX?

High conductivity HIC?

Fig 2.4. Multimodal media and chromatographic use. A combination of interaction modes can be expected
with multimodal media. The type of interaction(s) will depend on conditions and characteristics of the target
molecule. Int = interactions.

Bind/elute vs flowthrough mode in multimodal chromatography

In multimodal chromatography, the choice between bind/elute and flowthrough mode is more
complex than when using a single method, such as IEX, because multiple types of interactions
are occurring in multimodal chromatography, and the strength of these individual interactions
often depends on the overall process conditions. For example, the pH range for binding is
generally extended for multimodal media compared with traditional IEX media, which gives the
multimodal media unique selectivities and generally a wider operational window (Fig 2.5).

20 29-0548-08 AA
 Binding pH - Traditional AIEX Binding pH - Traditional CIEX
Binding pH - Multimodal AIEX Binding pH - Multimodal CIEX

pI
Net charge + +

Net charge
pH pH

- -

pI

Fig 2.5. Net charge of a protein vs pH. Schematic illustration of the extended pH binding range for
multimodal AIEX/CIEX (light green) compared with traditional AIEX/CIEX media (light blue).

Because the pI is generally not a good indicator for choosing the correct pH for binding and
elution with multimodal media, screening of conditions is paramount. This is preferably done
with high-throughput formats, such as microtiter plates or mini-columns (see the data files
for PreDictor and RoboColumn™ units listed in “Related literature”). Experimental setup for
screening studies is preferably done by using DoE, and typically the factors screened are pH
and conductivity. To help select the pH range to screen, a pH gradient elution experiment
can be performed where an analytical amount of sample is loaded on a small column. The
experiment will establish the elution pH of the sample. For a better understanding of the
multimodal behavior, a salt gradient or a combined salt and pH gradient can also be run.
An example is shown in Figure 2.6.

Column: Tricorn™ 5/100 packed with 2 mL Capto adhere; bed height 10.5 cm
Sample: Feed containing monoclonal IgG1, rProtein A elution pool, desalted
Sample load: 1 mg IgG1/mL medium
Buffer A: 20 mM sodium citrate + 20 mM sodium phosphate, pH 7.8
Buffer B: 20 mM sodium citrate + 20 mM sodium phosphate, pH 4.0
Flow velocity: 200 cm/h
System: ÄKTA™

160 9.0

140

120 8.0

100
A280 (mAU)

7.0
pH
Gradient start

80

60 6.0
Wash

40

20 5.0

0.0 10.0 20.0 30.0 40.0 mL

Fig 2.6. Establishing suitable conditions for DoE on Capto adhere in binding mode.

29-0548-08 AA 21
Salt types and additives
Different salt types and additives can modulate the interactions of target molecule with
multimodal chromatography media. Because hydrophobic interaction is one of the interaction
modes that is often involved, the choice of salt may play an important role.
Different salt types will affect the strength of interaction according to the Hofmeister series
(Fig 2.7). Typical ions used in HIC are found to the left in the series, while the chaotropic ions
to the right in the series, for example, iodine, reduce the hydrophobic interaction through the
salting-out effect.
Anion:
SO42- > HPO42- > acetate > Cl- > NO3- > Br- > ClO3- > I- > ClO4- > SCN-
Cation:
NH4+ > K+ > Na+ > Li+ > Mg2+ > Ca2+ > guanidinium
Fig 2.7. Hofmeister series.

Organic solvents, for example, ethanol and isopropyl alcohol, decrease the strength of
hydrophobic interactions and can, as such, affect the binding of biomolecules to multimodal
chromatography media. Detergents and antifoaming agents such as Tween™ 80 and
Triton™ X-100 can have a similar effect.
Hydrogen bond disruptors such as urea and guanidine hydrochloride also have the potential to
impact the strength of interaction on multimodal chromatography media. Some compounds
might influence several different interactions, for example, urea and guanidinium salt are
chaotropic as well as hydrogen bond disruptors. Studies on several other modifiers, for
example, amino acids or polyethylene glycol, have also been published. Examples of the
influence of salt types and additives for Capto adhere and Capto MMC are found in Chapter 3.

22 29-0548-08 AA
Chapter 3
Multimodal chromatography media
GE Healthcare offers multimodal media in a wide range of formats to meet users’ needs at all
stages of protein purification process development and manufacture. All of the multimodal
chromatography media from GE Healthcare are BioProcess™ media. BioProcess media are
developed and supported for production-scale chromatography. They are produced with
validated methods and are tested to meet manufacturing requirements. Secure ordering and
delivery routines give a reliable supply of media for production scale. Regulatory Support
Files (RSF) are available to assist process validation and submissions to regulatory authorities.
BioProcess media cover all purification steps from capture to polishing. The first multimodal
medium introduced by GE Healthcare was Capto MMC, shortly followed by Capto adhere.
As with all Capto media, the chosen ligands are coupled on a high-flow agarose base matrix,
which gives improved pressure-flow properties compared with older media (Fig 3.1).

2000 Capto

1800

1600
1
1400
Velocity (cm/h)

1200

Sepharose 6 2
1000
Fast Flow
Capto
800 ImpRes

600 Sepharose
High Performance 4
400
8
200

0
0 5 10 15 20 25 30 35 40 45 50
Bed height (cm)
Fig 3.1. Comparison of the window of operation (area below the curves) at large scale for different base
matrices. Gray lines give the residence time in the column in minutes. The particle size of Capto MMC/adhere
is 75 µm, and of Capto MMC/adhere ImpRes it is 40 µm. See Appendix 1 for characteristics of the various
multimodal media from GE Healthcare.

The highly cross-linked agarose base matrix gives the media high chemical and physical
stability. High flow velocities increase the productivity of large-scale bioprocessing operations
and allow large volumes to be processed in one working shift. The multimodal ligands in
Capto adhere and Capto MMC media were developed to offer novel selectivities compared
with anion and cation exchangers, respectively. The multimodal ligands are now also available
on the high-resolution base matrix Capto ImpRes (Fig 3.1), as Capto adhere ImpRes and
Capto MMC ImpRes. GE Healthcare has also recently introduced Capto Core 700, a product
that combines GF and multimodal anion exchange characteristics. Table 3.1 provides an
overview of available multimodal chromatography products from GE Healthcare.

29-0548-08 AA 23
Table 3.1. Characteristics, benefits, and uses of multimodal chromatography media

Medium Structure Main functionalities Advantages Uses

Capto adhere Ligand: Electrostatic High capacity and Intermediate purification
N-benzyl methyl interaction, productivity and polishing of MAbs
ethanolamine hydrogen bonding, Removal of impurities to after capture on protein A.
(see Fig 3.2) and hydrophobic formulation levels in post- Traditionally used in
Base matrix: interaction protein A purification flowthrough mode.
Capto Wide operational window Purification of other target
of pH and conductivity proteins from capture to
polishing steps.
Savings in time and
operating costs
with a two-step
chromatographic process
(see also Appendix 3)
Capto adhere Ligand: See Capto adhere Same as Capto adhere but Efficient MAb polishing,
ImpRes N-benzyl methyl with higher resolution and removal of aggregates and
ethanolamine lower elution volumes HCP, and separations of
(see Fig 3.2) charge variants. Polishing
Base matrix: resulting in smaller elution
Capto ImpRes volumes.
The properties of the small
Capto ImpRes particle are
best utilized in bind/elute
mode.
Capto MMC Ligand: Thiophilic interaction, Capto MMC gives high Capture and intermediate
N-benzoyl- hydrophobic productivity and reduced purification of proteins
homocysteine interaction, cost with: from large feed
(see Fig 3.6) hydrogen bonding, - High dynamic binding volumes by packed
Base matrix: and electrostatic capacity (DBC) at high bed chromatography.
Capto interaction conductivity Purification can be
performed at the
- High volume throughput conductivity of the feed
- Different selectivity material.
compared with
traditional ion
exchangers
Capto MMC Ligand: See Capto MMC Same as Capto MMC but Efficient MAb polishing,
ImpRes N-benzoyl- with higher resolution, removal of aggregates and
homocysteine lower elution volumes, HCP, and separations of
(see Fig 3.6) and increased possibility charge variants. Polishing
Base matrix: to elute with salt only. resulting in smaller elution
Capto ImpRes volumes.
The properties of the small
Capto ImpRes particle are
best utilized in bind/elute
mode.
Capto Core 700 Ligand: GF, AIEX, and HIC Significantly improved Purification of viruses
octylamine Core bead technology productivity compared and other large target
(see Fig 3.12) with ligand- with GF (100-fold) molecules.
Base matrix: activated core and Straightforward
high-flow agarose nonfunctionalized optimization and robust
shell allows performance
efficient capture of
contaminants while
target molecules
are collected in
flowthrough.
Custom Designed Wide selection Wide selection Tailored to the user’s Various
Media1 (CDM) needs
1
For challenging separation where standard multimodal media do not provide the desired results, CDM can provide libraries
of additional multimodal anion or cation exchangers. The libraries are provided in 96-well microtiter plate format for rapid
media screening. The multimodal cation and anion plates, respectively, contain 16 different multimodal media each.

24 29-0548-08 AA
Capto adhere
Capto adhere is a multimodal strong anion exchanger for BioProcess applications. It was
originally designed for post-protein A purification of MAbs at process scale in flowthrough
mode. However, Capto adhere can also be used in bind/elute mode and for applications other
than MAbs.
The strong multimodal ion exchange ligand (Fig 3.2) gives a different selectivity compared with
traditional ion exchangers. Capto adhere can remove key impurities in a single step, allowing the
design of a two-step process together with a protein A media (e.g., MabSelect™, MabSelect SuRe™,
or MabSelect SuRe LX). Capto adhere can also be used in combination with AIEX or CIEX for
polishing, as a second or third step in any MAb purification platform (see Fig A3.1 in Appendix 3).
Key performance benefits of Capto adhere include:
• high load and productivity
• impurity removal to formulation levels in post-protein A purification. Removal of:
- antibody dimers and aggregates
- HCP
- nucleic acids
- viruses
- leached protein A
- endotoxin
• wide operational window of pH and conductivity
• savings in time and operating costs with a two-step chromatographic process

(A)
(B)
OH OH
O O N+
OH (C)

Fig 3.2. The design of the Capto adhere ligand, N-benzyl-N-methyl ethanolamine. This ligand exhibits several
possibilities for interaction with proteins. The most pronounced are electrostatic interaction, hydrogen bonding,
and hydrophobic interaction, as shown by arrows: (A) for electrostatic interactions; (B) for hydrogen bonding;
and (C) for hydrophobic interactions.

As a first option, Capto adhere is recommended to be operated in flowthrough mode, because

this provides higher throughput. In flowthrough mode, the antibodies pass directly through
the column while contaminants (leached protein A, aggregates, HCP, nucleic acids, and viruses)
are adsorbed. Nevertheless, in cases where low molecular weight impurities, such as antibody
fragments, are present, Capto adhere in bind/elute mode might give higher purity.

pH operating window
As shown in Figure 3.3, the pH window of gradient elution of MAbs using Capto adhere differs
from that of traditional media and should be determined for each target protein. In the
experiment shown, five different antibodies with varying pI were run in bind/elute mode on
Capto adhere and Capto Q (see the discussion of bind/elute vs flowthrough mode in Chapter 2).

29-0548-08 AA 25
Analytical loads of the antibodies were used, and they were eluted from the chromatography
media with a pH gradient. The antibodies elute in the same order on both media, but they
elute much earlier—that is, at higher pH—on Capto Q. This result indicates that additional
interactions are involved on Capto adhere. Antibodies eluted below the isoelectric point on
Capto adhere.
With respect to pH, the operating window for Capto adhere is therefore at lower pH than for
traditional anion exchangers. If deamidation of the antibody is an issue, being able to run at
lower pH is of course beneficial.

(A)Capto
Capto adhere
adhere (B) Capto
Capto Q Q
Mab 4 Mab 3 Mab 5 Mab 2 Mab 1 Mab 4 Mab 3 Mab 5 Mab 2 Mab 1
Elution pH 6.2 5.3 5.1 5.1 4.9 Elution pH 9.9 9.6 9.4 8.4 6.2
pI 9-10 8.8-9.1 8.3-8.9 7.4 5.0-8.5 pI 9-10 8.8-9.1 8.3-8.9 7.4 5.0-8.5

8 11

10
7

9
pH

pH

8
5
7
4
6
10 20 30 40 0 10 20 30 40 50 60
Volume (mL) Volume (mL)

Fig 3.3. Different selectivity of Capto adhere compared with traditional ion exchangers. pH gradient elution
of five MAbs on Capto adhere and Capto Q. For the pH gradient (A) A-buffer (equilibration buffer) was 20 mM
Na-citrate + 20 mM Na-phosphate, pH 7.8, and B-buffer was buffer A at pH 4.0. (B) A-buffer was 20 mM
Na-phosphate, 20 mM Tris, 20 mM glycine, pH 11, and B-buffer was buffer A at pH 6.2 Gradient: 0 to 100%B,
10 column volumes (CV).

Removal of aggregates
High antibody titers tend to increase the generation of aggregates and impurities in the
feedstock. Capto adhere allows removal of aggregates to target values acceptable for
formulation. To achieve the best performance with Capto adhere operated in flowthrough
mode (i.e., to maximize the amount of impurities adsorbed to the medium while the monomeric
MAbs pass through the column), screening for optimal loading conditions is needed.
Optimization is preferably done with DoE. For details about how to set up a DoE, see Chapter
4, which includes an application example showing how Capto adhere effectively removes
aggregates. In this work, the sample was a cell culture supernatant (CSS) containing IgG1 that
was first purified on MabSelect SuRe. See also application notes 28-9078-89, “Optimization
of loading conditions on Capto adhere using design of experiments” and 28-9509-60, “High-
throughput screening and optimization of a multimodal polishing step in a monoclonal
antibody purification process.”

Viral clearance
An example of the use of Capto adhere for viral clearance is presented in Chapter 4. In this
work, Capto adhere was tested with two representative model viruses, and it was found that
even at high conductivity, where traditional ion exchangers do not work, the log reduction
factor was significant.

26 29-0548-08 AA
Removal of other impurities and contaminants
Removal of HCP and leached protein A is illustrated in Chapter 4. Negatively charged
impurities/contaminants such as nucleic acids and endotoxins are also effectively removed.

Salt type and additives

As previously discussed, separation of monoclonal monomer and aggregates is one of the
main challenges in MAb processes. In Figure 3.4, an experiment is presented in which the effect
of isopropyl alcohol and urea on monomer and aggregate static binding capacity (SBC) of
Capto adhere was investigated. With 20% isopropyl alcohol, the monomer capacity decreased
significantly with increased ionic strength, while the aggregate capacity remained essentially
unchanged. In the case of urea, the effect of ionic strength was similar for both the monomer
and the aggregates, leading to a decrease in binding capacity with increase in ionic strength.
At low ionic strength, the effect of urea on capacity of aggregates was minimal, while the
binding capacity for monomer decreased almost two-fold. These findings can be utilized to
optimize a Capto adhere step in flowthrough mode where low monomer binding and high
aggregate binding is desirable.

(A) Phosphate, pH 7 and isopropyl alcohol

Monomer capacity Aggregate capacity
60 20

50
15
40
SBC (g/L)

SBC (g/L)

30 10

20
No IPA 5 No IPA
10 IPA 10% IPA 10%
IPA 20% IPA 20%
0 0
0.11 0.21 0.31 0.41 0.11 0.21 0.31 0.41
Ionic strength (M) Ionic strength (M)

(B) Phosphate, pH 6 and urea

Monomer capacity Aggregate capacity
50 20

40
15
SBC (g/L)

SBC (g/L)

30
10
20

No urea 5 No urea
10
Urea 1 M Urea 1 M
Urea 2 M Urea 2 M
0 0
0.06 0.16 0.26 0.36 0.06 0.16 0.26 0.36
Ionic strength (M) Ionic strength (M)

Fig. 3.4. The influence of (A) isopropyl alcohol (IPA) and (B) urea on the SBC for monomeric and aggregate
forms of a MAb on Capto adhere. Phosphate was used as buffer, and ionic strength was adjusted using
NaCl. At high ionic strength, isopropyl alcohol can significantly lower monomer capacity while maintaining
most of the aggregate capacity (black circles). The effects are similar for urea at low ionic strength, although
not identical. These results illustrate that some additives could have multiple effects, i.e., both reducing
hydrophobic interactions and disrupting hydrogen bonds.

The type of salt chosen can also affect performance, as shown in Figure 3.5. In this experiment
the effect of different salt species on SBC was compared. With NaI the aggregate capacity
decreased significantly with increased ionic strength, while the monomer capacity was
essentially unchanged. The NaI effect suggests that chaotropic salts can be used to optimize a
bind/elute step, where high monomer capacity and low aggregate capacity is desirable.

29-0548-08 AA 27
 Monomer capacity Aggregate capacity
70 20

60
50 15
SBC (g/L)

SBC (g/L)
40
10
30
Na2SO4 Na2SO4
20 No salt 5 No salt
10 NaCl NaCl
Nal Nal
0 0
0.11 0.21 0.31 0.41 0.11 0.21 0.31 0.41
Ionic strength (M) Ionic strength (M)

Fig 3.5. The influence of different salt types on the SBC for MAb monomers and aggregates on Capto adhere.
Phosphate at pH 7.0 was used as buffer in all cases.

Regeneration
Due to its multimodal properties, regeneration of Capto adhere generally requires an acidic strip
prior to CIP (see Appendix 2).

Capto adhere ImpRes

Capto adhere ImpRes is a cost-effective and flexible chromatography medium designed for
high-resolution polishing of MAbs. Capto adhere ImpRes is a multimodal anion exchange
medium with the same ligand as used with Capto adhere (Fig 3.2). It displays high resolution
due to its small bead size (40 μm compared with 75 μm for Capto adhere). The high resolution
obtained with Capto adhere ImpRes enables reduced buffer consumption and improved product
yield when compared with Capto adhere. Impurities/contaminants such as DNA, HCP, leached
protein A, aggregates, endotoxin, and viruses are efficiently separated from the target MAb
in bind/elute or flowthrough modes in either two- or three-step purification schemes. The
influences of different salt types and additives are the same as for Capto adhere, as discussed
above. The medium can also be used for purification of other recombinant proteins and
biomolecules. For a MAb application example, see the Capto adhere ImpRes Example 1 in
Chapter 4.

Fast mass transfer

The small particle size of Capto adhere ImpRes generally results in a higher dynamic binding
capacity (DBC) and also less sensitivity to changes in residence time than is the case with
Capto adhere. These effects are exemplified in Figure 3.6.

60

50
DBC 10% (mg/mL)

40

30

20
Capto adhere ImpRes
10
Capto adhere
0
0 2 4 6 8 10
Residence time (min)
Fig 3.6. DBC of a MAb vs residence time. DBC at 10% breakthrough measured in 90 mM sodium phosphate,
pH 7.75.

28 29-0548-08 AA
 High resolution and small pool volumes
Another advantage of Capto adhere ImpRes, associated with the smaller particle size, is an
improved resolution that gives a better clearance of impurities. An example of this is shown in
Figure 3.7, where aggregate removal and yield of Capto adhere ImpRes and Capto adhere are
compared in bind/elute mode. The result shows that while a good separation was achieved
between monomer and aggregates using Capto adhere, separation was further improved using
Capto adhere ImpRes.
100

80
Cum monomer (%)

60

40
Capto ImpRes
Capto adhere
20

0
0 0.5 1.0 1.5 2.0
Cum aggregates (%)
Fig 3.7. Cumulated aggregates vs cumulated MAb monomer yield after linear gradient elution using
Capto adhere ImpRes and Capto adhere.

The high resolution of Capto adhere ImpRes is also maintained by using step elution instead of
linear gradient elution, as shown in Table 3.2. The sample load was 70% of DBC 10%. Fractions
were pooled and analyzed for yield, aggregate, HCP concentration, and pool volume. Despite
a 20% higher load, step elution from Capto adhere ImpRes resulted in higher yield and better
aggregate clearance compared with Capto adhere (Table 3.2). HCP levels were below the
detection limit for ELISA for both media. Pool volume was also significantly smaller with Capto
adhere ImpRes compared with Capto adhere.

Table 3.2. Results from step elution

Medium Sample load Yield Pool volume Aggregates HCP1

(mg/mL) (mg/mL) (%) (column volume) (%) (ng/mL)
Capto adhere ImpRes 30 91 4.4 0.5 Below detection limit
(< 20 ng/mL)
Capto adhere 25 79 6.1 0.8 Below detection limit
(< 20 ng/mL)
1
Measured with general ELISA from Cygnus Technologies.

Capto adhere ImpRes and Capto adhere were compared in a MAb flowthrough step (Table 3.3).
Using a residence time of 4 min, the aggregate and protein A clearance were equivalent for
both media, while Capto adhere ImpRes gave higher yield and improved HCP clearance.
At 2 min residence time, Capto adhere ImpRes showed equivalent yield and protein A and HCP
clearance, while a slight increase in aggregate level was observed.

29-0548-08 AA 29
Table 3.3. Results of flowthrough experiments with a MAb

Media and start Residence time Monomer yield Aggregates HCP reduction Protein A
material (min) (%) (%) (pool/load) (ppm)
Start material N/A N/A 3.4 N/A 3
Capto adhere 4 91 0.5 3 <1
Capto adhere ImpRes 4 94 0.5 4.5 <1
Capto adhere ImpRes 2* 94 0.7 4.5 <1

* Refer to Figure 3.1 for bed height limitations at short residence times.

Regeneration
Due to its multimodal properties, regeneration of Capto adhere ImpRes generally requires an
acidic strip prior to CIP. For maintenance of the medium, including strip, CIP, and storage, see
Appendix 2.

Capto MMC
Capto MMC is a multimodal cation exchanger with the properties of a weak cation exchanger.
In addition to electrostatic interactions, the ligand structure provides for additional interaction
modes such as hydrophobic interaction, hydrogen bonding, and thiophilic interaction (Fig 3.8).
The different possible interaction modes give the media novel selectivity and make it salt
tolerant, which in turn allows sample to be loaded without dilution or a buffer exchange step,
resulting in increased productivity.

(A)
(B)
O
OH OH
O O S NH (C)

O O- (D)

Fig 3.8. Capto MMC ligand. Interactions are shown by arrows: (A) for thiophilic; (B) for hydrophobic; (C) for
hydrogen bonding; and (D) for electrostatic interactions.

Capto MMC is based on a highly rigid agarose base matrix that allows high flow rates and low
back pressure at large scale, and is well-suited for fast, efficient and cost-effective protein
purification. See also Figure 3.1 and its accompanying discussion of pressure-flow properties.
Capto MMC gives increased productivity and reduced cost with:
• high DBC even at high conductivity (binding of proteins can be performed at the
conductivity of the feed material)
• high volume throughput
• new selectivity
• smaller unit operations (no dilution of feed material necessary, which leads to smaller tanks
and faster operation)

High salt tolerance

The various interactions of Capto MMC medium described above provide characteristics
different from traditional cation exchangers, including binding of proteins at high salt
concentration (Fig 3.9). Capto MMC can therefore be used for direct load of feedstocks, without
prior dilution or buffer exchange to reduce the conductivity of the starting material.

30 29-0548-08 AA
(A)
60

50

40
DBC (mg/mL)

30

20
Lysozyme
β-Lactoglobulin
10
BSA
0
0 10 20 30 40 50
Conductivity (mS/cm)

(B)
60
Capto MMC
Traditional cation exchanger
50

40
DBC (mg/mL)

30

20

10

0
0 5 10 15
Conductivity (mS/cm)

Fig 3.9. (A) DBC of Capto MMC at 1 min residence time for three different proteins at different conductivities.
(B) Capto MMC allows a much larger operating range in terms of conductivity of the starting material than
traditional cation exchangers.

Unique selectivity
The unique selectivity can also be used to solve specific purification problems, at high or low
conductivity. A comparison between a traditional cation exchanger (SP Sepharose Fast Flow)
and Capto MMC shows that the selectivity of the two media differs significantly (Fig 3.10). The
elution profile on SP Sepharose Fast Flow revealed one peak whereas the elution profile on
Capto MMC showed two, possibly three peaks. Native gel electrophoresis also showed that the
separation patterns differ between the media.

29-0548-08 AA 31
Column: Tricorn 5/100
Medium: A) Capto MMC; B) SP Sepharose Fast Flow
Sample: human blood plasma diluted 5 times, 10 CV
Buffer A: 100 mM acetic acid, 50 mM Na-phosphate, 20 mM Na-succinate, pH 5.0
Buffer B: 100 mM acetic acid, 50 mM Na-phosphate, 20 mM Na-succinate, pH 8.0 with 1 M NH4Cl
Flow: 150 cm/h
Gradient: linear gradient 0–100%B over 10 CV
System: ÄKTA

(A)
4000
A294 (mAU)

3000 Capto MMC

669 kD
2000 440 kD

232 kD
1000 140 kD

66 kD
0
HMW 3 2 1 FT Plasma
0 20 40 60 80
1 23
Elution volume (mL)

(B) 3000 SP Sepharose

Fast Flow
2500

2000
A294 (mAU)

1500
669 kD
440 kD
1000
232 kD
140 kD
500

66 kD
0
HMW 3 2 1 FT
0 20 40 60 80
12 3
Elution volume (mL)
Fig 3.10. The selectivity of (A) Capto MMC and (B) SP Sepharose Fast Flow was investigated using human
blood plasma, as described above. Fractions (indicated with arrows) and the flowthrough pool (FT) were
analyzed on native PhastGel™ gradient 8%-25% and Coomassie™ stained. High molecular weight marker
(HMW, GE Healthcare) and unfractionated plasma sample were also applied to the gels.

In contrast to traditional cation exchangers, Capto MMC may bind proteins above the pI of the
target protein (see Fig 2.5 in Chapter 2). Therefore, if pH is used for elution, higher pH is required
with Capto MMC than with traditional cation exchangers. This is illustrated in the elution
screening study shown in Figure 3.11.
The figure compares the results obtained in PreDictor plates with results obtained in columns.
The best recovery in the elution step is obtained with a simultaneous change in pH and salt
concentration. Identical results are not obtained in the two formats, but the trends as well as
the optimal conditions found are the same.

32 29-0548-08 AA
The best recovery is obtained at pH 6.75, which is approximately 2 pH units above the pI of BSA.
For further details see application note 28-9277-90, “High-throughput screening for elution
conditions on Capto MMC using PreDictor plates.”

(A) PreDictor plates

1.6 105
1.4
NH4Cl (M)

1.2 97 105
1.0
89 112
0.8 97
81
0.6 73 89 105
0.4 66 81 97
0.2

1.6
1.4
105
NaCl (M)

1.2
1.0 97 105
112
0.8 89
97
0.6 73
81
89
105
0.4 66 81 89 97
0.2
0.05 0.10 0.15 0.20 0.25 0.30 0.05 0.10 0.15 0.20 0.25 0.30 0.05 0.10 0.15 0.20 0.25 0.30
BIS (M) BIS (M) BIS (M)
pH = 5.75 pH = 6.25 pH = 6.75
(B) Tricorn columns
1.6
100
1.4
60 80 80
NH4Cl (M)

1.2 80
40 40
1.0
100 60
0.8 60 100
0.6
0.4 20 40
20
0.2

1.6
1.4 80 50
NaCl (M)

90 100
1.2 40 30
1.0 60
40
60
0.8 20 70
0.6 20 80
0.4 0 10

0.2
0.05 0.10 0.15 0.20 0.25 0.30 0.05 0.10 0.15 0.20 0.25 0.30 0.05 0.10 0.15 0.20 0.25 0.30
BIS (M) BIS (M) BIS (M)
pH = 5.75 pH = 6.25 pH = 6.75
Fig 3.11. Contour plots for the recovery in percent (see labels within each contour plot) of BSA in (A) PreDictor
plates and (B) Tricorn column. Recovery is plotted as a function of salt concentration (y axis) and buffer ionic
strength (BIS; x axis, running from 0.05 to 0.30 M) at three different pH values for the two salt types NH4Cl and
NaCl. The load was 70% of DBC at 10% breakthrough, and the loading buffer was 50 mM sodium acetate,
pH 4.75, 250 mM NaCl. Experimental data points are shown as black dots.

Salt type and additives

As previously discussed (Chapter 2), the choice of salt type or the use of additives will impact
the chromatographic behavior of multimodal media. For example, the recovery of the target
molecule is affected by the salt type used, as exemplified in Figure 3.11.
The effect of different additives on DBC on Capto MMC is shown in Fig 3.12. In this example,
detergents and antifoam agents did not have a significant impact on capacity, while organic
solvents and hydrogen bond disruptors had a larger impact.

29-0548-08 AA 33
 Hydrogen
Non-ionic detergents Organic solvents bond disruptors
110
100
80
90
80
DBC (% of ref)

70
60
50
40
30
20
10
0
Reference

Pluronic™ F68 + BHT

Triton X100

Breox™

Adekanol

Tween 80

Desmophen™

Struktol™

10% ethanol

20% ethanol

20% isopropyl alcohol

3 M urea

1 M guanidine HCl

Reference
Fig 3.12. Effect of additives on the DBC of BSA on Capto MMC. DBC is plotted as percent of reference run in
the absence of additives. BHT = butylated hydroxytoluene.

Binding capacity and recovery can also be influenced by different additives. The effect of
urea and organic solvents, ethanol, and isopropyl alcohol on DBC is shown in Figure 3.12. The
decreased capacity in the presence of urea and organic modifiers suggest that these can be
used to improve elution efficiency.

Regeneration
For maintenance of the medium, including CIP and storage, see Appendix 2.

Capto MMC ImpRes

Capto MMC ImpRes is based on the same ligand as Capto MMC (see Fig 3.6) and as such shows
overall similar properties. However, the ligand density is lower (25 to 39 µmol/mL compared with
70 to 90 µmol/mL for Capto MMC). This lower density is optimized for MAb polishing applications.
In addition, Capto MMC ImpRes is based on smaller beads (approximately 40 μm compared
with approximately 75 μm for Capto MMC). This smaller bead size and the optimized ligand
density gives an improved resolution in polishing applications as compared with Capto MMC.
The influences of different salt types and additives are the same as for Capto MMC, as
discussed above.

Fast mass transfer

The small particle size of Capto MMC ImpRes generally results in a higher DBC and less sensitivity
to changes in residence time than Capto MMC.

Salt tolerance
Capto MMC ImpRes has a higher salt tolerance than traditional cation exchangers, which enables
loading at higher levels of salt. This is exemplified in the PreDictor plate experiment presented
in Figure 3.13, which shows the capacity measured under different salt and pH conditions.
However, compared with Capto MMC (not shown), the low ligand density of Capto MMC ImpRes
gives a reduced salt tolerance that simplifies elution with salt, leading to higher yield and
smaller pool volumes.

34 29-0548-08 AA
 500 500 SBC (mg/mL)
450 450 65

60
400 400 Not studied 55
350 350 50

45
300 300
NaCl (mM)

NaCl (mM)
40
250 250 35

30
200 200
25
150 150 20

100 100 15

10
50 50 5

0 0 0

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
pH pH

Fig 3.13. Comparison of the effect of salt and pH on the SBC on Capto MMC ImpRes (left) and Capto SP
ImpRes (right).

High resolution and high capacity

The smaller particle size of Capto MMC ImpRes, compared with Capto MMC, provides increased
resolution. Capto MMC ImpRes also has a high DBC and efficiently removes impurities. In
Table 3.4, this is illustrated for MAb purification, and results are compared with those on Capto
MMC. At the same load, Capto MMC ImpRes gives better aggregate removal and higher yield.
Furthermore, this performance is also maintained at higher load.

Table 3.4. Comparison of the performance of Capto MMC ImpRes and Capto MMC in a MAb purification process
using a NaCl gradient for elution

Cumulated aggregate Yield at 1%

Prototype Load (mg) at 90% yield (%) aggregate (%)
Capto MMC 281 1.1 88.2
Capto MMC ImpRes 28 0.3 95.9
Capto MMC ImpRes 481 0.5 94.8
1
70% of DBC of the chromatographic media.

Regeneration
For maintenance of the medium, including CIP and storage, see Appendix 2.

Capto Core 700

Capto Core 700 is designed for intermediate purification and polishing of viruses and other
large biomolecules in flowthrough mode.

Designed for flowthrough chromatography

The Capto Core concept is based on a bead with an nonfunctionalized outer layer (without
ligand) and a functionalized core with an attached ligand (Fig 3.14). This design combines
properties of GF and adsorption chromatography. The bead’s pores in the outer layer, with
an approximate exclusion limit of 700 kD, have been specifically designed to exclude large
molecular entities such as viruses, DNA, large protein, or protein complexes from entering
the internal space and interacting with the ligand, thereby enabling an efficient flowthrough
purification step.

29-0548-08 AA 35
 Nonfunctionalized outer layer

Functionalized core

Fig 3.14. Schematic cross-sectional view of a Capto Core 700 particle with an average diameter of 85 µm.
Small protein impurities can enter the interior of the matrix particle and bind to the ligand. Large molecular
entities such as virus particles are hindered from entering the matrix.

The core of each bead is functionalized with octyl amine that is both hydrophobic and
positively charged (at pH < 10), resulting in a highly efficient multimodal binding of various
impurities over a wide range of pH and salt concentrations. This novel core bead technology
gives Capto Core 700 a dual functionality, combining size separation and multimodal binding.
These features make Capto Core 700 an excellent alternative to size exclusion media that are
typically employed in the final stages of virus purification in vaccine manufacture (see Heyward
et al. [1977]; Nayak et al. [2005]; and Kalbfuss et al. [2008]). Capto Core 700 offers a range of
performance advantages over GF, which is often regarded as a productivity bottleneck in
the polishing process due to low flow rates and limited sample loads. The characteristics of
Capto Core 700 are summarized in Appendix 1.
Key performance characteristics of Capto Core 700 include:
• significantly improved productivity enabled by up to 100-fold higher sample load and
significantly higher flow rates compared with GF
• core bead technology with ligand-activated core and inactive shell allowing efficient
capture of impurities while target molecules are collected in the flowthrough fraction
• straightforward optimization due to flowthrough chromatography and robust performance
allowing for a wide window of operation
For a more detailed description of the use of Capto Core 700, see Chapter 4.

Improved productivity
The core bead technology in Capto Core 700 enables high loading capacity during group
separation of molecules. The core bead technology also allows for short residence times
(sometimes as low as 1 min), and in combination with the large 85 μm high-flow agarose
matrix, flow velocities as high as 500 cm/h are possible. The short residence times, high flow
velocities, and high loading enable a larger operational window than traditional GF. The larger
operational window allows for increased volume throughput and smaller equipment with
reduced footprint. The large bead size also contributes to reducing back pressure during
purification of highly viscous samples. The improved window of operation provided by
Capto Core 700 allows greater freedom of process design.

36 29-0548-08 AA
 Figure 3.15 illustrates schematically the higher load capacity and flow velocities enabled with
Capto Core 700 relative to that of Sepharose 4 Fast Flow, which is a GF medium typically used
in large-scale polishing processes.

Load (mg/mL medium) 10

Capto Core 700

Gel filtration
0.3

100 500
Maximum flow velocity (cm/h)
Fig 3.15. Schematic illustration of the significantly greater sample load and flow velocity enabled with
Capto Core 700 in comparison with conventional GF media. Note that the schematic is not to scale.

Regeneration
Bound impurities are removed from the beads by CIP procedures (see Appendix 2 for
maintenance of the medium).

Multimodal libraries
Multimodal libraries from Custom Designed Media (CDM)
CDM is a collaborative service for industrial customers to develop tailor-made chromatography
media. CDM media can be produced for specific industrial process separations when suitable
media are not available. The CDM group at GE Healthcare and the customer work in close
collaboration to design, manufacture, test, and deliver media for large-scale purification.
The same approach used to develop Capto adhere and Capto MMC was used to design
diversified multimodal libraries, with the goal of producing a library as large and diverse as
possible in order to increase the chances of identifying the key parameters and the type of
media most appropriate for a given purification challenge. Use of such new libraries together
with good screening technology will favorably impact downstream process development.
The starting point of library design is the chemical and structural diversity of the ligands,
which should be chosen to encompass a large range of chromatographic behavior. With this in
mind, researchers at GE Healthcare developed two diversity-based multimodal ion exchange
libraries, one a multimodal anion exchanger and the other a multimodal cation exchanger, to
be used in an explorative phase when traditional media performance is not sufficient. Both
libraries are based on 16 unique multimodal constructions in an easy-to-screen 96-well-plate
format, and are available via the CDM group. The different constructs are not available as
standard products, but are supplied on request. For more information about these libraries,
contact your local GE Healthcare bioprocess representative. Examples of structures from the
libraries are shown in Figure 3.16.

29-0548-08 AA 37
 R1
O R1
OH
N+
NH R2

O O-

O O O
Spacer S O- Spacer N+
O
Spacer R1 Spacer R1
O O

Fig 3.16. Design principles of CDM libraries. Multimodal cation exchangers (left) and multimodal anion
exchangers (right) are illustrated.

Use of good screening tools is critical to successful use of multimodal libraries. Such screening
tools are important not only for the identification of the best media to use but also for the
optimization of operating conditions. Keep in mind that although the multimodal approach
has great potential to allow the development of unique chromatographic solutions, different
interactions have been added as compared with traditional media. As a result, the optimal
conditions can be quite different from those using traditional media. As with any medium, the
optimal conditions for use of a particular multimodal chromatography medium will need to be
determined for each target protein. See Chapter 2 for a discussion on optimizing conditions.

Custom Designed Products (CDP)

Custom-packed laboratory columns can be supplied by the Custom Products Group
at GE Healthcare. A wide range of columns ensures the highest performance from all
GE Healthcare purification media and meets the demands of modern pharmaceutical
manufacturing. Each column is packed, tested, and certified under stringent ISO 9001
standards. Contact your local representative for further details.

38 29-0548-08 AA
Formats of multimodal chromatography products
Multimodal media from GE Healthcare are available both in prepacked formats and as bulk
media to be packed in columns.
Prepacked formats range from PreDictor plates to ReadyToProcess™ columns (Table 3.5).

Table 3.5. Prepacked formats available for multimodal media

Formats Features
PreDictor 96-well plates 1
Supports HTPD by allowing parallel screening of chromatographic conditions
using a 96-well plate format. Allows fast and efficient evaluation of parameters
for binding/wash/elution conditions, and media selection. Can be used with
an automated robotic or manual setup.
Assist software supports the PreDictor workflow from setup of experimental
design to data evaluation.
PreDictor RoboColumn units Supports HTPD by allowing parallel screening of chromatographic conditions
using a miniaturized column format and robotic workstation.
HiTrap™ columns (1 and 5 mL) For easy screening and convenient process development.
Note: Capto adhere ImpRes, Capto MMC ImpRes, and Capto Core 700 are
available only in the 1 mL HiTrap column.
HiScreen™ columns (4.7 mL) Well-suited for parameter method optimization and parameter screening
due to the 10 cm bed height.
ReadyToProcess columns All wetted parts of the ReadyToProcess columns are of USP class VI, with all
(1, 2.5, 10, and 20 L) components traceable to their production batches. Prequalified (by efficiency
testing).
Prepacked in cleanroom (class ISO 8) environment. Presanitized and tested
for endotoxin as well as microbiological growth and released according to
specifications.
Custom packed columns A wide range of columns ensures high performance from all GE Healthcare
purification media.
Meet the demands of modern pharmaceutical manufacturing.
Each column is packed, tested, and certified under stringent ISO 9001
standards.
1
Not available for Capto Core.

The multimodal media can be used together with most equipment available for chromatography
from laboratory scale to production scale. At production scale, the preferred packing technique
for Capto media is axial compression. The optimal approach is to use AxiChrom™ columns with
Intelligent Packing and preset packing methods for all Capto media. Appropriate columns from
GE Healthcare are shown in Table 3.6.

29-0548-08 AA 39
Table 3.6. Appropriate columns for packing multimodal media

Format/columns Inner diameter Capto Capto adhere Capto MMC Capto MMC Capto
(mm) adhere ImpRes ImpRes Core 700

Laboratory scale1
Tricorn 5, 10 × × × ×
HiScale™ 16, 26, 50 × × × × ×

Pilot and production scale2

AxiChrom3 50–200 × × × × ×
AxiChrom3 300–1000 × × × × ×
BPG 4
100–300 × × × × ×
Chromaflow™ 400–8005 × ×6 × ×6
1
Visit [Link]/tricorn and [Link]/hiscale for the full range of Tricorn and
HiScale columns, respectively.
2
For other process-scale columns and diameters, please contact GE Healthcare or visit [Link]/bioprocess.
3
For details of Intelligent Packing methods, visit [Link]/axichrom.
4
The pressure rating of BPG 450 is too low to use with Capto media.
5
Larger pack stations and/or mechanical axial compression are required at larger diameters.
6
Chromaflow 400-600.

40 29-0548-08 AA
Chapter 4
Applications
This chapter describes numerous studies undertaken using the multimodal media products
from GE Healthcare. These media have found their place in many different applications, for
example, purification of recombinant proteins including insulin, albumin, MAb fragments, and
MAbs, as well as in virus purification. Most of the applications presented in this chapter are
for MAb processes, where multimodal media are becoming more and more established. For a
detailed discussion of MAb purification strategies, see Appendix 3.
The application examples in this chapter are presented based on the multimodal media used.
After each example, a reference (application note or data file) is provided for further reading.

Capto adhere applications

1. Optimization of loading conditions on Capto adhere using DoE
This study describes the optimization of loading conditions for a MAb polishing step to obtain
the window of operation for Capto adhere. In order to find the optimal conditions, a full
factorial DoE was used with three variables: pH, conductivity, and load. A brief discussion of the
basic principles of DoE precedes the experimental details and results (see also the discussion
in Chapter 3, page 13). The results demonstrate that it is possible to find a wide window of
operation in terms of pH and conductivity.
As previously discussed, DoE is a systematic approach to investigate how variations in factors
(X’s) affect the responses (Y’s) in a system (i.e., determining the mathematical relationship
between X and Y). DoE is used to plan experiments so that the maximum amount of information
can be extracted from the performed experiments. The factors in a DoE study are simultaneously
varied so that they are independent of each other in a statistical sense. This makes it possible
to evaluate the effect on the response of each factor separately (main effects). In addition,
interaction effects between factors can be evaluated. For optimizing purposes, the use of DoE
greatly increases the likelihood that the real optimum for a response is found.
A commonly used type of DoE is full factorial design, which is used both for screening and
optimization purposes. A great advantage with the full factorial design is that all main effects
and interaction effects are independent of each other, and therefore their effect on the
response can be resolved in the evaluation. A disadvantage with the full factorial design is that
the number of experiments increases as the number of factors studied increases—the number
of experiments is 2n, where n is the number of factors. A full factorial design with seven factors
would need 27 = 128 experiments. When many factors are included in the design, there are
other types of DoE that can be used that will significantly reduce the number of experiments,
with the trade-off being that some information is lost.
Center points are important for DoE. The center points are usually replicated and will give
information on experimental noise. The center points will also provide information on possible
curvature in the data.

Method design and optimization

Balancing product yield against product purity is the major consideration when optimizing a
method. When running in flowthrough mode, loading conditions will usually be a compromise
between conditions favoring yield and those favoring contaminant clearance. By adjusting
pH and conductivity of the sample as well as the sample load, conditions can be obtained

29-0548-08 AA 41
where most contaminants are adsorbed while the monomeric antibodies pass through the
column. Optimization of loading conditions is preferably performed by using DoE. A common
approach in DoE is to define a reference experiment (center point) and perform representative
experiments around that point. To be able to define the center point and the variable ranges,
some initial experiments are required.

Establish nonbinding conditions

To find conditions suitable for the DoE, initial experiments can be performed in binding mode,
using a pH gradient for elution (Fig 4.1). The elution position (i.e., pH at peak maximum) defines
the lower pH in the design. The upper pH in the design should normally be about two pH units
higher. Experiments can also be performed in flowthrough mode, keeping the conductivity
constant at a moderate level. A comparison of chromatograms is shown in Figure 4.2. At high
pH (i.e., close to pI for the antibodies) the breakthrough during sample load is delayed, the
breakthrough and wash curves are shallow, and significant amounts of MAb binds to the
adsorbent. A decrease in pH (i.e., further from the pI) results in weaker electrostatic interaction
between the antibodies and the adsorbent, steeper breakthrough and wash curves, and
increased yield.
An alternative approach to determine experimental conditions for the DoE is to screen conditions
using high-throughput formats (see following example, 2a to 2c). As large experimental spaces
can be explored with high-throughput formats, the use of these formats will greatly enhance
process understanding.

Column: Tricorn 5/100 packed with 2 mL Capto adhere; bed height 10.5 cm
Sample: Feed containing monoclonal IgG1, rProtein A elution pool, desalted
Sample load: 1 mg IgG1/mL medium
Loading buffer: 20 mM sodium citrate + 20 mM sodium phosphate, pH 7.8
Elution buffer: 20 mM sodium citrate + 20 mM sodium phosphate, pH 4.0
Flow velocity: 200 cm/h
System: ÄKTA

160 9.0

140

120 8.0

100
A280 (mAU)

7.0
Gradient start

80
pH

60 6.0
Wash

40

20 5.0

0.0 10.0 20.0 30.0 40.0 mL

Fig 4.1. Establishing suitable conditions for DoE on Capto adhere in binding mode.

42 29-0548-08 AA
Column: Tricorn 5/20 packed with 0.5 mL Capto adhere; bed height 2.5 cm
Sample: Feed containing monoclonal IgG1, rProtein A elution pool, desalted
Sample load: 75 mg IgG1/mL medium
Loading buffer: 25 mM Bis-Tris, pH 6.0 or 35 mM Tris, pH 8.0
Elution buffer: 100 mM acetic acid, pH 4.0
Flow rate: 0.25 mL/min (2 min residence time)
System: ÄKTA
4000

3500

3000

2500
mAU

2000

1500

1000

500

0
0.0 5.0 10.0 15.0 20.0 25.0 mL
Fig 4.2. Establishing suitable conditions for DoE on Capto adhere in flowthrough mode. Comparison of
chromatograms obtained at different pH: pH 8.0 (blue curve) and pH 6.0 (green curve).

In the DoE, pH, conductivity, and load must be included. It is important to include conditions at
the higher pH range resulting in lower yield and higher purity, as well as conditions at lower pH
range resulting in higher yield and lower purity.

Setup of a full factorial DoE with three parameters

Below is a stepwise description of how to set up a full factorial design.
1. Work prior to actual setup of the design
Perform initial loading experiments at varying pH, as described above. Choose parameters
to include and define parameter ranges and responses.
2. Choose design for screening or optimization
Full factorial design is commonly used in both screening and optimization. A full factorial
DoE in three parameters will give 23 = 8 experiments + center points. A graphical view of how
the experiments are organized is shown in Figure 4.3.
3. Choose center points for the design
Center points are important in DoE because they give an indication if there is curvature in
the data. Replicated center points are recommended. For example, a full factorial design in
three parameters with three center points gives a total of 11 experiments.
4. Systematic variation of the parameters
Limiting values, high and low, should be used for each parameter. The high and low values
should be combined in a way that makes the parameters independent (to be able to
separate effects).

29-0548-08 AA 43
Load

ity
tiv
uc
nd
Co

pH

Fig 4.3. Graphical representation of a full factorial design in three variables with center points.

DoE used for purification of an IgG1 MAb

DoE was applied for the optimization of loading conditions for an antibody, previously purified
on non-agarose based recombinant protein A chromatographic medium. The experiments
were designed and evaluated using Umetrics Modde™ 7.0 software ([Link]).
The feed contains a monoclonal IgG1 expressed in Chinese hamster ovary (CHO) cell
supernatant with pI of approximately 9. The impurity levels after protein A were determined:
leached protein A, 36 ppm; dimers and aggregates (D/A), 3.3%; and HCP, 210 ppm. The
experimental setup was a full factorial design with three variables: load, pH (based on Figs 4.1
and 4.2), and conductivity, with additional points to resolve curvature effects (Table 4.1). In total,
14 experiments were included in the model, and the measured responses were yield and
concentration of impurities ( protein A [ppm], D/A [%], and HCP [ppm]) in the flowthrough pool.
For each response a separate model was calculated. The models were fitted to multiple linear
regression (MLR) and are well explained and show good stability to cross validation. Response
surfaces were obtained for yield as well as for clearance of key contaminants.

44 29-0548-08 AA
Table 4.1. Design setup includes two center points (bold) and four additional points at pH 7 to resolve
curvature effects

Load (mg MAb/mL) pH Conductivity (mS/cm)

75 6 2
300 6 2
75 8 2
300 8 2
75 6 15
300 6 15
75 8 15
300 8 15
187.5 7 8.5
187.5 7 8.5
75 7 15
300 7 15
187.5 7 2
187.5 7 15

Results
Parameters affecting the yield
The parameters that affect the yield are shown in the coefficient plot1 (Fig 4.4). The plot shows
that high sample load, low pH, and high conductivity result in high yield. The interaction
effects (load × pH, load × conductivity) are also significant for the yield response. The response
surfaces (Fig 4.5) show that higher loads will give a larger pH window with yield > 90%.

1
The coefficient plot describes the impact of investigated parameters on the yield. In this experiment, load is positively
correlated to the yield, implying that a higher load will give a higher yield; pH is negatively correlated to the yield, meaning
that a lower pH will give a higher yield; and conductivity is positively correlated to yield, but to a smaller extent, meaning
that a higher conductivity will give higher yield. The interaction effects that are present in the coefficient plot (load × pH,
load × conductivity) mean that if pH is changed, the yield will not only change with the effect of pH but also with the effect
of load at that specific pH. The same discussion can be applied to the load × conductivity interaction effect.

6
4

0
%

-2

-4

-6
-8
Load pH Cond Load × Load ×
pH Cond

Fig 4.4. Coefficient plot for the yield model.

29-0548-08 AA 45
 300 300 95.7 300
95.6

250 250 250

94.7
90.4
Load (mg/mL)

Load (mg/mL)

Load (mg/mL)
200 200
91.1 200 90.5
85.2 86.5 86.3
150 150 150
80.0 81.9
82.1
100 100 100
74.8 77.3
6.0 6.5 7.0 7.5 pH 6.0 6.5 7.0 7.5 pH 6.0 6.5 7.0 7.5 pH

Cond = 2 mS/cm Cond = 8.5 mS/cm Cond = 15 mS/cm

Fig 4.5. Response surfaces for the yield model. Load versus pH at different conductivities, with yield
expressed in percent (labels).

Parameters affecting the protein A clearance

The coefficient plot shows that a high pH will give good protein A clearance (Fig 4.6). The
conductivity alone does not affect the response, but there is a significant interaction effect
for pH × conductivity. If this term is high, the protein A clearance will be low. Load was not a
significant factor for this response.
The response surfaces (Fig 4.7) show that high pH and low conductivity will give high
protein A clearance.

4
3
2
1
0
ppm

-1
-2
-3
-4
-5
-6
pH Cond pH ×
Cond
Fig 4.6. Coefficient plot for the Protein A clearance model.

14
6

12
4
2
Cond (mS/cm)

10
0
8

2
6.0 6.5 7.0 7.5 8.0
pH

Fig 4.7. Response surfaces for the Protein A clearance model, conductivity versus pH. Protein A
concentration expressed in ppm.

46 29-0548-08 AA
Parameters affecting D/A clearance
The coefficient plot shows that pH is the most important parameter and that high pH will give
a high D/A clearance in the flowthrough pool (Fig 4.8). The load parameter is also significant,
but very small. The load should be low to give high D/A clearance. There is a significant
curvature effect assigned to pH. If pH is too high or too low, the clearance will be less efficient.
The conductivity did not significantly affect D/A clearance.
The response curve (Fig 4.9) shows that the load has only a small effect on D/A clearance, so
only pH needs to be considered.

1.0
0.8
0.6
0.4
0.2
0.0
-0.2
%

-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
Load pH pH × pH

Fig 4.8. Coefficient plot for the D/A clearance model.

300

250
Load (mg/mL)

200
3

2
150 1
0
100

6.0 6.5 7.0 7.5 8.0

pH

Fig 4.9. Response curve for the D/A clearance model, load versus pH. D/A concentration expressed in percent.

Parameters affecting HCP clearance

The coefficient plot (Fig 4.10) and response curves (Fig 4.11) show that low sample load, low
conductivity, and high pH will give the best HCP clearance.

29-0548-08 AA 47
 5
4
3
2
1
ppm

0
-1
-2
-3
-4
-5
-6
pH
Load

Cond
Fig 4.10. Coefficient plot for the HCP clearance model.

14 14 14

16.1 18.2 14.1

12 12 12

13.1 15.2 11.1

Cond (mS/cm)

Cond (mS/cm)

Cond (mS/cm)
10 10 10

8 8 8
10.1 12.2 8.1
6 6 6

4 7.1 4 9.2 4 5.1

2 2 2
6.0 6.5 7.0 7.5 pH 6.0 6.5 7.0 7.5 pH 6.0 6.5 7.0 7.5 pH
Load = 187.5 mg/mL Load = 300 mg/mL Load = 75 mg/mL

Fig 4.11. Response surfaces for the HCP clearance model, conductivity versus pH at different loads.
HCP concentration is expressed in ppm.

Conclusions—optimal loading conditions and general trends

Each MAb is unique, and the level of contaminants varies between different cell lines and
differences in previous purification steps. This implies that it may be difficult to predict optimal
loading conditions. However, based on DoE performed with several different antibodies, some
general trends have been identified (Fig 4.12):
• For best yield, load should be high, the pH low, and conductivity high.
• For the best D/A clearance, the pH should be high, while load and conductivity should
be low. D/A clearance is typically less affected by conductivity than protein A and HCP
clearance.
• For the best protein A and HCP clearance, the pH should be high and conductivity low.
Loading conditions will therefore be a compromise between conditions favoring yield and
conditions favoring contaminant clearance. Optimal loading conditions will be a balance
between load, pH, and conductivity. Consequently, for optimization of the loading step, all three
parameters should be varied in the same experimental series.

Yield D/A PrA HCP

Conductivity

Conductivity
Conductivity

Conductivity

pH pH pH pH

Fig 4.12. General trends with respect to loading conditions for yield, D/A, and Protein A and HCP clearance.

48 29-0548-08 AA
Optimal loading conditions for five MAbs together with yield and contaminant clearance results
from a two-step process, including protein A medium and Capto adhere, are shown in Table 4.2.
pH should normally be well below the pI, while optimal conductivity is more difficult to predict.
The response surfaces above show the influence of sample load, pH, and conductivity on
four different responses (yield of monomeric MAb and clearance of protein A, D/A, and HCP,
respectively), and how to reach desired values for each of them. Even though the optimal
conditions for each response are not the same, there is a large area where acceptable values
can be obtained for all four responses. Suggested loading conditions for this MAb when purified
with Capto adhere are a sample load of 200 mg/mL, pH 7, and conductivity 8.5 mS/cm. The
expected outcome would be a yield of over 90%, leached protein A below the detection limit,
D/A < 0.5%, and HCP concentration < 15 ppm.

Table 4.2. Optimal loading conditions for different MAbs with regard to yield and clearance of HCP,
Protein A, and D/A

Conductivity Yield D/A Protein A HCP

MAb pl pH (mS/cm) % % ppm ppm
1 ~9 7 8 90 0.5 < LOQ < 15
2 8.3–8.9 5.5 3 95 0.6 < LOQ 2
3 7.5–8.4 6 2 95 0.8 < LOQ 9
4 7.7–8.0 7 20 91 0.2 < LOQ 30
5 6.5–9.0 7.5 20 92 < 0.1 < LOQ 7.5

For more information on this example, see application note 28-9078-89, “Optimization of
loading conditions on Capto adhere using design of experiments.”

2. Development of operational excellence in MAb process development and

manufacturing using Capto adhere
The following three application examples focus on the development of operational excellence
in MAb process development and manufacturing. They include: (2a) HTS and optimization of a
multimodal polishing step in a MAb purification process; (2b) Scale-up of a downstream MAb
purification process using HiScreen and AxiChrom column formats; and (2c) A flexible antibody
purification process based on ReadyToProcess products.

2a. HTS and optimization of a multimodal polishing step in a MAb purification

process using Capto adhere
This application describes the use of Capto adhere and MabSelect SuRe chromatography
media to significantly reduce the level of IgG antibody aggregates in a sample using an
efficient two-step method. The method resulted in high yields and purity. In addition, use of
a screening format employing the exceptional capabilities of PreDictor 96-well filter plates,
HiScreen prepacked columns, and a DoE approach allowed for effective and rapid screening
for optimal experimental conditions. Application of the optimized protocol led to a reduction
in aggregate levels from 12.6% to < 0.5% in a single step, with a monomer yield of 87%. HCP
and ligand leakage were reduced to negligible amounts. In total, 192 conditions (flowthrough
and selective elution experiments) were screened in approximately 4 h and analyzed in 48 h.
The use of a high-throughput method in the process described in this example led to a speedy
identification and subsequent optimization of the initial conditions.
An efficient approach to MAb purification involves a two-step process whereby Capto adhere,
with both hydrophobic and ion exchange interactions, is used to selectively remove antibody
aggregates from the monomeric forms. MabSelect SuRe is used for the preceding protein
A-mediated capture step.

29-0548-08 AA 49
Because the complexity of multimodal media requires a more thorough process optimization
study in order to take full advantage of the outstanding potential of this technology, the development
of efficient and rapid screening methods for optimal process conditions is critical. In the initial
part of this study, PreDictor 96-well filter plates prefilled with Capto adhere were used to
screen a large experimental space quickly. Promising results from the plate study were further
optimized with HiScreen columns and a DoE approach to establish the final process conditions.

Materials and methods

Liquid handling
All the experiments were performed with PreDictor plates containing 6 μL of Capto adhere in
each well. The buffers were prepared in an automated Tecan™ Freedom EVO™-2 200 Robotic
System, but procedures such as sample addition were performed manually. Liquid removal
during equilibration of the media was performed in a vacuum manifold, and sample collection
was performed by centrifugation (300 × g for 60 s).

Screening for initial conditions

The MabSelect SuRe elution pool was used as the sample after buffer exchange on a HiPrep™
Desalting column. The final IgG concentrations used were 0.53, 2.65, or 5.3 mg/mL depending
on the experiment. The antibody solution contained approximately 14% aggregates.
A 2× buffer stock solution was prepared for each experimental condition. The same volume of
sample and buffer stock solution was then mixed and dispensed into each well of the PreDictor
plate. The following parameters were tested in the initial screening phase: 50 mM sodium
citrate pH 5.5 or 6.5; 50 or 450 mM NaCl; three different IgG concentrations (0.53, 2.65, and
5.3 mg/mL); and four different incubation times (2.5, 10, 30, and 60 min).
The final plate layout is shown in Figure 4.13. The following protocol was used:
1. The medium was equilibrated with 3 × 200 μL of buffer, and excess liquid was removed by
vacuum.
2. The sample (200 μL) was added and incubated at four different incubation times (2.5, 10, 30,
and 60 min) at room temperature on an orbital shaker at 1100 rpm.
3. After incubation, the flowthrough fraction was collected by centrifugation (300 × g for 60 s
at room temperature) into PreDictor plates.

5.3 mg/mL 2.65 mg/mL 0.53 mg/mL

2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Time (min)
A 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Time (min)
B 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

10 10 10 10 10 10 10 10 10 10 10 10 Time (min)
C 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

10 10 10 10 10 10 10 10 10 10 10 10 Time (min)
D 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

30 30 30 30 30 30 30 30 30 30 30 30 Time (min)
E 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

30 30 30 30 30 30 30 30 30 30 30 30 Time (min)
F 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

60 60 60 60 60 60 60 60 60 60 60 60 Time (min)
G 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

60 60 60 60 60 60 60 60 60 60 60 60 Time (min)
H 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 5.5 5.5 6.5 6.5 pH
450 50 450 50 450 50 450 50 450 50 450 50 NaCl (mM)

Fig 4.13. Plate layout of the initial screening experiments.

The starting material and flowthrough fractions were analyzed by GF with two
Superdex™ 200 5/150 GL columns connected in series. Each sample was analyzed in 15 min.

50 29-0548-08 AA
Flowthrough experiments
Analysis of the initial screening conditions allowed selection of appropriate conditions for the
flowthrough experiments (Fig 4.14). The final IgG concentration was 5.3 mg/mL, and the sample
was incubated for 60 min. Sample and buffer handling were performed as described (see
“Screening for initial conditions”). In these experiments, 96 different conditions were studied in
one single plate as follows:
• 8 different pH levels with 50 mM sodium citrate (pH 4.0 to 6.0) or 50 mM sodium phosphate
(pH 6.5 to 7.5)
• 12 different concentrations of NaCl (0 to 550 mM)

NaCl
(mM) 0 50 100 150 200 250 300 350 400
0 450 500 550

4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0

4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5
CITRATE

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5

6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0

6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5
PHOSPHATE

7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0

7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5

Fig 4.14. Plate layout of the flowthrough experiments.

Apart from an incubation time of 60 min, the protocol for the flowthrough experiments was the
same as described (see “Screening for initial conditions”).

Selective elution study

An elution study (Fig 4.15) was performed to improve the proportion of monomer yield. Two
different binding conditions were investigated (500 mM NaCl, 50 mM sodium citrate, pH 4.5;
and 50 mM NaCl, 50 mM sodium phosphate, pH 7), for both the sample solution and the wash
buffer. Each elution step was performed with the same buffer species that was used in the
binding step. The elution conditions were:
• pH 4.0 to 6.0 with 50 mM sodium citrate
• pH 6.0 to 7.0 with 50 mM sodium phosphate
• 0 to 550 mM NaCl

29-0548-08 AA 51
NaCl
(mM) 0 50 100 150 200 250 300 350 400
0 450 500 550

4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0

4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5
CITRATE

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5

6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0

6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0
PHOSPHATE

6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5

7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0

Fig 4.15. Plate layout of the selective elution study.

Briefly, the following protocol was used:

1. The medium was equilibrated with 3 × 200 μL of buffer, and excess liquid was removed by
vacuum filtration.
2. The sample (200 μL) was added to each well. The plate was incubated at room temperature
on an orbital shaker at 1100 rpm for 60 min followed by centrifugation (300 × g for 60 s at
room temperature) into an empty 96-well plate.
3. Each well was washed with 2 × 200 μL of equilibration buffer.
4. Elution was performed with 3 × 200 μL of elution buffer.

Column optimization with a factorial design

The MODDE software v8 (Umetrics) was used to set up a Central Composite Face (CCF)
design with a response surface modeling (RSM) objective. This resulted in 26 design runs plus
replicated center points. The factors investigated are summarized in Table 4.3.

Table 4.3. Factors investigated in the optimization study

Aggregates 9% to 14%
Concentration 5 to 15 mg/mL
Load 60 to 100 mg/mL
Elution pH 6.1 to 6.5
NaCl for elution 150 to 450 mM

Loading was carried out according to the optimal conditions discovered in the screening
phase. The pH and NaCl concentrations in Table 4.3 refer to the elution conditions from the
column. The residence time was 5 min throughout the entire study. The starting IgG sample for
this study consisted of two MabSelect SuRe elution pools containing 9% and 14% aggregates,
respectively. The center points were created by mixing equal amounts of the two samples to
produce a final sample containing 11.5% aggregates. A HiScreen Capto adhere column (4.7 mL)
was used for this run. A freshly produced IgG sample containing 12.6% aggregates was used
for the column verification experiment on a 1 mL HiTrap Capto adhere column.

52 29-0548-08 AA
GF analysis
The flowthrough fractions were analyzed by GF using two interconnected Superdex 200 5/150
GL columns. An aliquot (10 μL) of each sample was applied to the column and run in phosphate
buffered saline (PBS) at a flow rate of 0.35 mL/min for 15 min. Yield and purity were calculated
from the GF results as follows:

Areamonomer (eluted)
= Areamonomer
Yield =
Yield
(eluted) Equation 1
Area
Areamonomer
monomer (loaded)
(loaded)

Areamonomer
Area
Purity
Purity =
= Area monomer
Areamonomer
monomer +
+ aggregates
aggregates Equation 2
(in the
(in the elution
elution or
or fl
flowthrough)
owthrough)

HCP and ligand leakage analyses

HCP levels were measured using commercial anti-CHO HCP antibodies (Cygnus Technologies).
Essentially, an ELISA methodology was adapted to a Gyrolab™ Workstation LIF (Gyros AB) using
Gyrolab Bioaffy 20 HC microlaboratory discs. Ligand leakage measurements were performed
using a commercial ELISA kit (Repligen Corporation) with a slightly modified protocol compared
with the one supplied by the manufacturer.

Column prediction
The data obtained from the PreDictor plate experiments was used to predict the column
conditions as follows: assuming that monomer plate capacities equaled dynamic binding
capacities (most likely valid for longer residence times), then purity and yield can be calculated
based on the following equations:

Vsample
Qm = (Cini, m - CFT, m ) Equation 3
Vmedium

Vload × Cini, m - CV × Qm
Yield =
Vload × Cini, m Equation 4

Vload × Cini, m - CV × Qm
Purity =
Vload × (Cini, m + Cini, a ) - CV × (Qm + Qa ) Equation 5

where Qm is the binding capacity for monomers, Cini, m is the initial monomer concentration in the
flowthrough, CFT, m is the monomer concentration, Vload is volume loaded, CV is column volume,
Vmedium is volume of chromatographic medium, Cini, a is the initial aggregate concentration, and
Qa is the binding capacity for aggregates.

Results and discussion

The overall two-step process was based on MabSelect SuRe as the capture step and
Capto adhere as the second step in the aggregate removal. A monoclonal IgG antibody
feed, containing approximately 14% aggregates, was used as a representative sample for
the second step after the initial MabSelect SuRe capture step. Screening and optimization of
the process conditions were performed with the goal of obtaining less than 1% aggregates
(approximately 99% monomer purity) in the final sample with acceptable yields (> 85%) over
the final step. A secondary goal was to explore new formats such as PreDictor plates and
HiScreen columns—in combination with a DoE approach—to produce rapid screening and
reduce the number of experiments required to establish optimal process conditions.

29-0548-08 AA 53
Screening for initial conditions with PreDictor plates
One of the goals of the initial screening phase was to determine the incubation time required
for all the components to reach a state of equilibrium so that the binding properties of both
monomers and aggregates can be estimated (Fig 4.16). Adsorption was completed after
approximately 10 min and 30 min for the monomer and aggregate species, respectively. The
aggregates produced slower kinetics, so in order to ascertain complete binding, an incubation
time of 60 min was chosen for the remaining experiments.

(A) 100
90
Remaining monomer in solution (%)

80
70
60
50
40
30
20
10

0
0 10 20 30 40 50 60 70
Incubation time (min)

(B) 100
90
80
Remaining dimer in solution (%)

70
60
50
40
30
20
10

0
0 10 20 30 40 50 60 70
Incubation time (min)

Fig 4.16. Adsorption curves of (A) monomer and (B) aggregates. This shows the remaining monomer
and aggregate concentrations in the flowthrough fractions under the investigated conditions of antibody
amounts, NaCl concentrations, and pH. Legends correspond to protein concentration (mg/mL), pH, and
NaCl concentration (mM).

54 29-0548-08 AA
Flowthrough experiments with PreDictor plates
An IgG sample containing 14% aggregates was used. After applying the sample, the flowthrough
fractions were subjected to GF analysis. The capacities for monomer and aggregate IgG (Fig 4.17)
were calculated (Equation 3). The capacity for IgG monomers exceeded that of aggregates
under all the conditions tested, which implied that the removal of aggregates would result in
the inevitable loss of some monomer IgG.

(A) 40–45
35–40
45 30–35
40 25–30
Monomer capacity (mg/mL)

35 20–25
30
15–20
25
10–15
20
5–10
15
10 0–5
5
7.0
0
0 50 6.0
100 150
200 250 5.0 pH
300 350
400 450 500 4.0
NaCl concentration (mM) 550

14–16
(B)
12–14
16 10–12
14 8–10
Aggregates capacity (mg/mL)

12 6–8
10 4–6
8 2–4
6 0–2
4

2
7.0
0
0 50 6.0
100 150
200 250 5.0 pH
300 350
400 450
500 4.0
NaCl concentration (mM) 550

Fig 4.17. (A) Monomer and (B) aggregate capacities determined from the PreDictor plate experiments.

Column prediction
Data from the flowthrough experiments and the application of Equations 3, 4, and 5 were used
to predict column performance. In the example shown in Figure 4.18, a prediction based on a
CV of 10 mL and a sample load of 130 mg/mL produced > 98% monomer and a yield of 60% to
65%. A yield as low as that is not feasible for a large-scale process, so a selective elution study
was used instead.

29-0548-08 AA 55
 Highest purity
Load = 130 mg/mL
7.5
96 95
7.0 98 97 94 93

6.5
92

6.0
91
pH

Yield
5.5
> 60%
> 65%
5.0
> 70%
> 75%
4.5
> 80%
4.0 > 85%
0 50 100 150 200 250 300 350 400 450 500 550
NaCl concentration (mM)

Fig 4.18. Column prediction of purity (iso-lines) and yield (color map) at a sample load of 130 mg/mL.

Selective elution experiments with PreDictor plates

An elution profile (Fig 4.19) was created from the difference in adjacent peak areas (e.g., the area
for a peak at 450 mM was subtracted from that at 500 mM NaCl). The greatest IgG monomer
peak area occurred at the lowest pH of 6.0 and a NaCl concentration of 250 to 300 mM. This
elution pool contained negligible amounts of IgG aggregates.

(A) 120

100
Area (mAU × mL)

80

60

40

20

0
50 100 150 7.0
200 250 6.5
300 350
400 450 6.0 pH
500 550
NaCl concentration (mM)

(B) 120

100
Area (mAU × mL)

80

60

40

20

0
50 100 150 7.0
200 250 6.5
300 350
400 450 6.0 pH
500 550
NaCl concentration (mM)

Fig 4.19. (A) Monomer and (B) aggregate elution profiles based on the data from PreDictor plates.

56 29-0548-08 AA
The raw data was also plotted as a function of purity against yield for all the elution conditions
with the aim of optimizing both the yield and purity (Fig 4.20). The optimum spot in such a plot is
expected to produce the highest purity and yield at the same time. The peak values were found
at an approximate pH of 6 and 250 mM NaCl.
7.0
6.9
0.4 0.5 0.6
6.8
0.3
6.7
0.2
6.6
pH

6.5
6.4
6.3
0.7
6.2 Optimum
conditions
6.1
0.1
6.0
0 50 100 150 200 250 300 350 400 450 500 550
NaCl concentration (mM)

Fig 4.20. Effect of NaCl concentration and buffer pH on a normalized objective function purity × yield (shown
in labels within image).

Optimization study with HiScreen columns

The following factors were investigated:
• protein concentration
• aggregate content of the start sample
• aggregate content of the load sample
• elution pH
• elution NaCl concentration
The experiments were performed to find the best conditions for monomer purity (> 99% in the
final sample) and acceptable monomer yield (> 85%). The purity of the monomer IgG (Fig 4.21)
was adversely affected by an:
• increase in start aggregate level (Aggr)
• increase in start protein concentration (Conc)
• increase in load (Load)
• increase in NaCl concentration (NaCl)

0.6
0.4
0.2
-0.0
-0.2
%

-0.4
-0.6
-0.8
-1.0
Aggr Conc Load NaCl Load × Aggr × Aggr × Conc ×
Load Load NaCl Load

Fig 4.21. Coefficients plot for monomer purity.

29-0548-08 AA 57
The yield of monomer IgG was found to be adversely affected by an increase in the amount of
aggregate IgG in the starting sample and also by an increase in the pH of the elution buffer.
On the other hand, the yield of monomer IgG was enhanced by an increase in the sample
load and also by an increase in the amount of NaCl in the elution buffer. Although the effect
of the sample load concentration was not significant, it was left in Figure 4.22 because one of
the interactions contained this factor. For this model, quadratic terms and other interactions
were present.

5
4
3
2
1
%

-0
-1
-2
-3
-4
Aggr Conc Load pH NaCl NaCl × Aggr × Aggr × Load Load ×
NaCl Conc Load × pH NaCl

Fig 4.22. Coefficients plot for monomer yield.

The models for purity and yield can be combined to produce a sweet spot for a particular set
of user-defined criteria (Fig 4.23). In this case, the set criteria were: > 85% monomer yield and
> 99% monomer purity (which is equivalent to less than 1% of aggregated IgG). The load was
set to 60 mg/mL, and the NaCl concentration for elution was 300 mM. A broad zone within
the investigated pH interval was observed where both criteria were fulfilled. The broadest
operational area was discovered at the most acidic elution pH of 6.1. Because there was a
good correlation between the data from the optimization study and that from the PreDictor
plate experiments, a column verification study was set up with a 1 mL HiTrap Capto adhere
column using similar run conditions to those from the sweet spot analysis:
• The sample load was 60 mg/mL.
• The concentration of IgG aggregates in the starting sample was 12.6%.
• The starting concentration of the IgG sample was adjusted to 5 mg/mL.
• The elution buffer had a pH of 6.1 and a NaCl concentration of 250 mM.

58 29-0548-08 AA
(A) 15
14 2 of 2 criteria met (sweet spot)
13 1 of 2 criteria met (sweet spot)

Concentration (mg/mL)
12
11
10
9
8
7
6
5
9 10 11 12 13 14
Aggregates (%)
15
(B)
14
2 of 2 criteria met (sweet spot)
13
Concentration (mg/mL)

1 of 2 criteria met (sweet spot)

12
11
10
9
8
7
6
5
9 10 11 12 13 14
Aggregates (%)
15
(C)
14 2 of 2 criteria met (sweet spot)
13 1 of 2 criteria met (sweet spot)
Concentration (mg/mL)

12
11
10
9
8
7
6
5
9 10 11 12 13 14
Aggregates (%)

Fig 4.23. A sweet spot plot for IgG monomer yield and purity. The conditions for these plots were a sample load
of 60 mg/mL media and elution with 300 mM of NaCl at the three different pH levels of: (A) 6.1; (B) 6.3; and (C) 6.5.

The column verification study (Fig 4.24) produced an eluted IgG monomer yield of 87%, which
was a significant improvement on the 60% to 65% yield obtained from the PreDictor plate
experiments in which only the flowthrough was included in the process step. The purity level
(99.5%) of the eluted IgG monomer met the sweet spot analysis criteria of > 99.0% (Fig 4.25). In
addition, the HCP content of the eluted IgG monomer was reduced from 131 ng/mL (26 ppm)
to a negligible amount of < 4.6 ng/mL. MabSelect SuRe ligand leakage was also reduced from
10 ng/mL (2 ppm) to a negligible amount of < 3 ng/mL.

29-0548-08 AA 59
Column: HiTrap Capto adhere 1 mL
Sample: Diafiltered elution pool from MabSelect SuRe, 5 mg/mL
Load: 60 mg/mL
Binding buffer: 50 mM sodium phosphate, 50 mM NaCl, pH 7.0
Elution buffer: 50 mM sodium phosphate, 250 mM NaCl, pH 6.0
Flow rate: 0.2 mL/min
System: ÄKTA

3000 Collected volume

12.0

2500
10.0
2000
A280 (mAU)

pH
8.0
1500

6.0
1000

500 4.0

0 0.0

0.0 10.0 20.0 30.0 40.0 50.0 mL

Fig 4.24. Chromatogram from the column verification. Blue: Absorbance at 280 nm. Red: pH.
Purple: Conductivity (mS/cm).

600

500

400
A280 (mAU)

300

200

100

0
2.5 3.0 3.5 4.0 4.5 5.0 5.5 mL

Fig 4.25. GF analysis of the start material (purple), flowthrough/elution fraction (red), and strip fraction (blue).
All the curves were normalized against the flowthrough/elution fraction.

Conclusions
Using Capto adhere (as the polishing step) with MabSelect SuRe (capture step) reduces high
levels of IgG antibody aggregates in an efficient two-step method that produced high yields
and purity. In addition, this application of new screening formats employing the exceptional
capabilities of PreDictor 96-well plates, HiScreen prepacked columns, and a DoE approach
for effective and rapid screening for optimal conditions. The plate format is suitable for initial
screening whereas the more refined screening, based on the findings from the plate results,
should be performed with the column formats for optimal results. The optimized process was
able to reduce aggregates levels from 12.6% to < 0.5% in a single step with a monomer yield
of 87%. Furthermore, HCP and ligand leakage were reduced to negligible values. In total, 192
conditions (flowthrough and selective elution experiments) were screened in approximately 4 h
and analyzed in 48 h. The high-throughput workflow produced a high-level knowledge of the
process and allowed for a rapid identification of the conditions for optimization.
For more information on this example, see application note 28-9509-60, “High-throughput
screening and optimization of a multimodal polishing step in a monoclonal antibody
purification process.”

60 29-0548-08 AA
2b. Scale-up of a downstream MAb purification process using
HiScreen and AxiChrom columns with Capto adhere
The main challenge from the MAb purification process described here was the high incidence
of aggregation (12%) in the starting sample. This antibody feed stream was successfully scaled
up more than 10 times while maintaining preset criteria for purity and yield from a two-step
chromatography process based on MabSelect SuRe and Capto adhere media. The optimal
process conditions worked out from small-scale studies were further improved and tested for
robustness using a workflow comprising DoE and Monte Carlo simulation in silico. The DoE
studies performed at small scale using 4.7 mL HiScreen columns (diameter 7.7 mm) generated
sweet spot analyses for the capture and polishing steps where the predefined criteria
regarding yield and purity were met. The results from the DoE studies then served as input
for Monte Carlo simulations to test the robustness of the optimal conditions obtained from
the two chromatographic steps. The workflow (Fig 4.26) allowed for a rapid screening of both
chromatographic conditions and process robustness prior to scale-up.

Cell culture

Harvest (centrifugation & filtration)

Capture (MabSelect SuRe)

Virus inactivation

Buffer exchange (UF/DF)

Polishing (Capto adhere)

Formulation (UF/DF) & sterile filtration

Final product

Fig 4.26. Flow scheme of the purification process in which steps involving in-process filtration of the sample
to reduce bioburden are indicated with asterisks (*). UF/DF = ultrafiltration/diafiltration.

AxiChrom 70/300 columns (diameter 70 mm) were packed automatically with an ÄKTApilot™
system to give a bed height of 20.5 cm and 14.1 cm for the MabSelect SuRe and Capto adhere
columns, respectively. CHO cells expressing the target IgG were cultured in a 120 L stirred tank
bioreactor with a working volume of 100 L. Culture duration was 20 d with a peak cell density
of 4.5 × 106 viable cells/mL and a final viability of 28%. The capture step was performed with
MabSelect SuRe AxiChrom column at an approximate load of 31 g/L and a residence time
of 4 min. The load for the Capto adhere AxiChrom column was approximately 60 g/L with a
residence time of 5 min. The loading sample concentration was 5 g/L.
A representative chromatogram from the Capto adhere step (second cycle) is presented in
Figure 4.27.

29-0548-08 AA 61
Column: AxiChrom 70/300 (14.1 cm bed height)
Sample: 60 g/L of diafiltrated elution pool from the MabSelect SuRe step
Binding buffer: 50 mM sodium phosphate, 50 mM NaCl, pH 7.0
Elution buffer: 50 mM sodium phosphate, 250 mM NaCl, pH 6.1
Flow rate: 109 mL/min
System: ÄKTApilot

Collected fraction
3500

3000

2500
A280 (mAU)

2000

1500

1000

500 Elution Strip Cl

Wash
0
5000 10 000 15 000
Volume (mL)

Fig 4.27. Chromatogram from the Capto adhere step. A280 trace is shown in blue, pH in green, and
conductivity in purple.

In the scaled-up process, the starting aggregate concentration of 12% was reduced to 0.6%
in a single step (data not shown). The monomer yield of 86% was relatively high for a sample
containing such a high level of aggregates.
The results for the overall scaled-up purification are shown in Table 4.3.

Table 4.3. Summary of monomer yield, aggregate content, HCP reduction, and ligand leakage in the
scale-up process

Process step HCP (ppm) Ligand (ppm) Aggregate Yield (%)

content (%)
Fermentation 37 000 N/A3 12
Harvest 37 000 N/A 12 100
MabSelect SuRe (4 cycles) 24 1
1.9 121
96.21
Buffer exchange 25 1.9 12 97.8
Capto adhere < LOQ 2
< LOQ 2
0.6 4
86.03
Formulation and sterile filtration 1.0 < LOQ2 0.6 102
Total yield: 80.8
1
Average of 4 cycles.
2
LOQ = level of quantitiation (4.6 ng/mL for HCP, 3 ng/mL for ligand).
3
Not applicable.
4
Average of 2 cycles.

For more information on this example, see application note 28-9403-49, “Scale-up of a
downstream monoclonal antibody purification process using HiScreen and AxiChrom columns.”

62 29-0548-08 AA
2c. A flexible antibody purification process based on ReadyToProcess products with
Capto adhere
In this study, a series of experiments was undertaken to determine whether shorter time-to-
clinic and cost savings could be achieved using ReadyToProcess products. The work involved
scaling up a two-step chromatography purification process from 4.7 mL HiScreen columns
(diameter 7.7 mm) to pilot scale using 1 L ReadyToProcess columns (diameter 80 mm).
Chromatography was run using ReadyToProcess columns on an ÄKTA ready system, and
filtration was performed using ReadyToProcess filters and a fully disposable cross-flow filtration
system for ReadyToProcess hollow fiber cartridges. The chromatography steps were performed
on the same ÄKTA ready system; only the flow kit was changed between the runs. The process
consisted of a capture step on MabSelect SuRe and a polishing step on Capto adhere with a
buffer exchange step in between and a formulation step at the end. The buffer-exchanged
sample was loaded in one cycle onto a 1 L ReadyToProcess Capto adhere column. The load
was 60 g/L. The flowthrough, wash, and elution fractions were collected in one pool. The
starting aggregate concentration of 10% was reduced to 0.4% in this single step (Fig 4.28). The
monomer yield was 89%, which was judged to be good considering the high aggregate content
at the start.
The full series of experiments was able to reduce the HCP concentration from 37 500 ppm
to 1.0 ppm (Table 4.4). In addition, the Capto adhere step removed aggregates from a
concentration of 10% down to 0.4%, and the protein A ligand leakage was reduced to below
the limit of quantitation (LOQ) from 9 ppm. The total yield of the downstream process, including
all filtration steps, was 81%.

Column: Two Superdex 200 5/150 GL connected in series

Sample: 10 μL of IgG
Mobile phase: PBS, pH 7.0
Flow rate: 0.35 mL/min
System: ÄKTA

1000

800
A 280 (mAU)

600

400

200

2.0 2.5 3.0 3.5 4.0

Volume (mL)

Fig 4.28. GF analysis of the MAb in the Capto adhere step—sample before purification (green), purified
fraction (purple), and strip fraction (blue). The curves were normalized with respect to the monomer
peak of the purified fraction.

29-0548-08 AA 63
Table 4. 4. Summary of monomer yield, aggregate content, and HCP reduction in the scale-up

Aggregate
Process step HCP (ppm) Ligand (ppm) content (%) Yield (%)
Fermentation 37 500 Not done 10
Harvest 37 500 Not done 10 100
Capture, MabSelect SuRe (2 cycles) 19 8.8 10 96.01
UF/DF 1 12 9.1 10 97.7
Polishing, Capto adhere < LOQ 2
< LOQ 2
0.4 89.0
UF/DF 2 & sterile filtration 1.0 0.1 0.4 97.4
Total yield: 81.3
1
Average of 2 cycles.
2
LOQ = level of quantification (4.6 ng/mL for HCP, 3 ng/mL for ligand).

For more information on this example, see application note 28-9403-48, “A flexible antibody
purification process based on ReadyToProcess products.”

3. Viral clearance using Capto adhere

Viral clearance using Capto adhere was tested with two representative model viruses, Minute
Virus of Mice (MVM) and Murine Leukemia Virus (MuLV). Monoclonal IgG1 was purified from
CHO cell supernatant on MabSelect SuRe. Buffer concentration and pH of the elution pool
were adjusted to typical process conditions. The conductivity was adjusted to 10 and
30 mS/ cm by addition of NaCl. The samples were spiked with virus stock solution and were
then applied in flowthrough mode on Capto adhere. The log10 reduction factor at 10 mS/cm
was 5.8 logs for MVM and 4.5 logs for MuLV. Even at high conductivity (30 mS/cm), where
traditional ion exchangers do not work, the log reduction factor was 5.9 logs for MVM and
3.6 logs for MuLV (Table 4.5).

Table 4.5. Capto adhere viral clearance1

Virus Conductivity (mS/cm) Log10 Reduction Factor

± 95% confidence Iimit
MVM 10 5.8 ± 0.3
MVM 30 5.9 ± 0.3
MuLV 10 4.5 ± 0.4
MuLV 30 3.6 ± 0.4
1
Study performed at NewLab BioQuality AG, Germany. Test conditions were: pH 6.75, temperature 22°C, experiments
performed in duplicates.

For more information on this example, see data file 28-9078-88, “Capto adhere.”

64 29-0548-08 AA
Capto adhere ImpRes applications
1. Polishing of MAbs using Capto adhere ImpRes in bind/elute mode
In these studies, the binding capacity for MAbs and the efficiency in the clearance of impurities
using Capto adhere ImpRes in bind/elute mode was evaluated. The studies present results
from optimization of the loading conditions using the DoE approach. The effects of buffer, pH,
conductivity, and sample load were investigated. The studies include measurement of static
and dynamic binding capacities (SBC and DBC, respectively) at various binding conditions, as
well as screening and optimization of gradient- and step-elution conditions.
Two different MAbs were studied. The results showed high yields of monomeric MAb, as well as
good clearance of aggregates, HCP, and leached protein A.

Materials and methods

Start material
The two MAbs used in this study were initially purified from CHO cell supernatant by protein A
affinity chromatography. Some characteristics of the MAbs are shown in Table 4.6.

Table 4.6. Characteristics of the two antibodies used in the study

DBC 10% (mg/mL)1

Antibody pI Aggregate content (%) Capto adhere ImpRes Capto adhere
MAb A 7.3 2.5 71 56
MAb B > 7.0 2.4 44 36
1
DBC at 10% breakthrough (DBC 10%) for various antibodies measured at 4 min residence time.

Determination of SBC
SBC was determined in 6 μL PreDictor 96-well filter plates. Equilibration of wells in the filter
plates was performed by addition of 200 μL of loading buffer per well followed by agitation
at 1100 rpm for 1 min, after which the buffer was removed by vacuum extraction. The
equilibration step was performed three times. MAb solution (200 μL volume, 4 mg/mL sample
load, corresponding to 133 mg MAb/mL chromatography medium) was added to each well
followed by agitation for 90 min. Unbound material (flowthrough fraction) was removed
by centrifugation for 3 min, and MAb concentration was determined by measurement of
absorbance at 280 nm. SBC was calculated according to:

Vload
SBC = (C - C ) Equation 6
Vmedium ini FT

where Vload = load volume, Vmedium = medium volume in each well, Cini = MAb concentration in the
sample, CO (VCX% -=VMAb
O)
DBC X% =and FT
concentration in the flowthrough fraction.
VC
Determination of DBC
DBC was determined by+frontal
V eluate (C eluate1 Celuate2 +analysis
Celuate3 ) using an ÄKTA chromatography system. The UV
Yield (%) = × 100
absorbanceVload at 280 nm Vloadwas
× Ciniused for determination of breakthrough. Before frontal analysis,
SBCMAb
the = solution (C - CFT ) injected bypassing the column to obtain a maximum absorbance value.
Vmedium ini was
DBC was then calculated according to:

CO (VX% - VO )
DBC X% = Equation 7
VC

where C0 = MAb concentration in the sample (mg/mL), VX% = load volume (mL) at x% breakthrough,
VYield
= void V eluate (mL),
(C eluate1 + CVeluate2 + Celuate3 ) bed volume (mL).
0 (%) volume
= and c
= volumetric × 100
Vload × Cini
29-0548-08 AA 65
Screening of elution conditions
Measurement of yield at different elution conditions was performed in PreDictor 96-well
filter plates. Equilibration of wells in the filter plates was performed by addition of 200 μL of
loading buffer per well followed by agitation at 1100 rpm for 1 min, after which the buffer was
removed by centrifugation. The equilibration step was performed three times. MAb solution
(200 μL, 2.8 mg/mL, corresponding to 93 mg MAb/mL medium) was added to each well followed
Vload
bySBC =
agitation (C 60- Cmin.
for ) Unbound material was removed by centrifugation. Elution of bound
Vmedium ini FT
material was then performed by addition of 200 μL of elution buffer/well; the elution step was
performed three times. MAb concentration was determined by measurement of absorbance at
280 nm. CO (VX% - VO )
DBC X% =
VC
Yield was calculated according to:

V eluate (C eluate1 + Celuate2 + Celuate3 )

Yield (%) = × 100 Equation 8
Vload × Cini

where Veluate = eluate volume, Celuate 1, 2, 3 = MAb concentration in eluates 1 to 3, Vload = load
volume, and Cini = MAb concentration in MAb solution.

Optimization of step elution conditions

Conditions for step elution were investigated in a packed column using ÄKTA pure
chromatography system, DoE, and scouting functionalities included in UNICORN™ 6.3.

Determination of aggregates and aggregate clearance

Fractions from the chromatographic runs were collected and analyzed by analytical GF on
a Superdex 200 5/150 GL column. The peaks were integrated, and the D/A concentrations
(in percent) were estimated. Cumulated yield of monomers was plotted against cumulated
aggregates (Fig 4.29).

100
Cumulated monomer yield (%)

80

60

40

20

0
0 2 4 6 8
Cumulated aggregates (%)

Fig 4.29. Evaluation of gradient elution was performed by GF. The figure shows an example of the resulting
plot of cumulated yield of monomers vs cumulated aggregates derived from the GF analysis.

Protein A and HCP ELISA

The protein A concentration in the start materials and flowthrough fractions was determined by
Protein A ELISA kit (Repligen). HCP concentration was determined by HCP ELISA (Cygnus Technologies).

66 29-0548-08 AA
Results and discussion
Case study, MAb A
The case study with MAb A shows a suggested workflow for method development including
screening of conditions for SBC and DBC, screening of elution conditions, and optimization of
conditions for step elution.

SBC
To find optimal binding capacity for MAb A, SBC was determined in 6 μL PreDictor 96-well filter
plates. Binding pH was varied between pH 4.0 and 8.02,3 and the salt concentration from 0 to
500 mM NaCl. All samples and buffers were prepared automatically using a Tecan robot. The
results show that the highest SBC was obtained at high pH and low salt concentration (Fig 4.30,
orange region). Based on these results, a narrower range of pH and NaCl concentration was
used for further investigation of conditions for DBC.

2
Binding buffers were citrate, pH 4; acetate, pH 4.6 and 5.7; phosphate, pH 5.7, 6.3, and 6.9; and Tris, pH 7.4 and 8.0.
The ionic strength from the buffer salts was kept constant at 40 mM.
3
To avoid deamidation of the MAb, pH should normally be maintained below pH 8.0.

500 SBC (mg/mL)

450 75

400
70

350
65
300
NaCl (mM)

250 60

200 55

150
50
100
45
50

0 40

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

pH
Fig 4.30. Contour map showing screening of SBC for Capto adhere ImpRes.

DBC
The influence of pH and salt concentration on DBC was measured by DoE using Capto adhere
ImpRes packed in a Tricorn 5/50 column. Based on the results for SBC, binding pH was varied
between pH 6.0 and 7.84 and salt concentration from 0 to 200 mM NaCl. In addition, the
residence time was varied from 2 to 8 min.
The results from the DoE are shown in Figure 4.31. Modeling of data was performed using
MODDE v9.0 software, resulting in a good model fit and predictive power (data not shown). In
accordance with the trend for SBC, an increase in pH and decrease in salt concentration resulted
in higher DBC, while lower capacity was obtained at short residence time. Further experiments
described below were performed using binding with 40 mM sodium phosphate, pH 7.8.

4
Binding buffers: Sodium phosphate, 0 to 200 mM NaCl, pH 6 to 7.8. The ionic strength from the buffer salts was kept
constant at 110 mM.

29-0548-08 AA 67
 54 58
180 180 180 58

160 160 160

62
140 140 140
NaCl (mM)

NaCl (mM)

NaCl (mM)
58 62
120 120 120
100 100 100
80 80 66 80
66
60 62 60 60
40 40 70
40 70
20 66 20 20
0 0 0
6.6 6.8 7.0 7.2 7.4 7.6 6.6 6.8 7.0 7.2 7.4 7.6 6.6 6.8 7.0 7.2 7.4 7.6
pH pH pH
Residence time = 2 min Residence time = 5 min Residence time = 8 min

Fig 4.31. Contour maps for DBC at 10% breakthrough and different residence times on Capto adhere ImpRes.

Screening of elution conditions

Measurement of yield at different elution conditions was performed in 96-well filter plates as
described in “Materials and methods.” Binding was performed in 40 mM sodium phosphate,
pH 7.8. Elution pH was varied between 4.5 and 8.0 and salt concentration between 0 and
1 M NaCl. The result, Figure 4.32, shows that the highest yield was obtained at low pH and low
salt concentration. Based on this result, further studies of elution conditions were performed
by gradient elution in packed columns.

1000 Yield (%)

100
900
90
800
80

700 70
NaCl (mM)

60
600
50
500
40

400
30

300 20

10
200
0
0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
pH

Fig 4.32. Screening of elution conditions in PreDictor plate. Yield obtained by varying elution pH between 4.5 and
8.0 and NaCl concentration between 0 and 1 M. Evaluation performed by Assist software for PreDictor plates.

Gradient elution
Gradient elution was performed from 40 mM sodium phosphate, pH 7.8 to 20 mM sodium
phosphate, 20 mM citrate, pH 4.0 with or without addition of 100 mM NaCl5. Chromatograms
are shown in Figure 4.33. Fractions were collected and analyzed by GF. Cumulated concentration
of aggregates (%) vs cumulated yield of monomeric MAb (%) was calculated according to
“Materials and methods.” The results showed that addition of 100 mM NaCl in the elution buffer
resulted in slightly lower elution pH, lower aggregate content, and a broader elution peak than
elution buffer without NaCl (Table 4.7).
5
A mixed buffer with ionic strength that is too high might result in elution of MAb during the wash step or early in the gradient.

68 29-0548-08 AA
Column: Tricorn 5/50, CV ~ 1 mL
Medium: Capto adhere ImpRes
Sample: MAb A, partially purified by protein A chromatography
Sample load: 43.4 mg MAb/mL chromatography medium
Start buffer: 40 mM sodium phosphate, pH 7.8
Elution buffer: 20 mM sodium phosphate, 20 mM citrate, pH 4.0 (blue curve);
20 mM sodium phosphate, 20 mM citrate, 100 mM NaCl, pH 4.0 (green curve)
Gradient: 0% to 100% elution buffer in 20 CV
Residence time: 4 min
System: ÄKTA

8.0

2000
7.0

1500
6.0
A280 (mAU)

pH
1000 5.0

4.0
500

3.0
0
0 5 10 15 20 25 30 35
Volume (mL)
Fig 4.33. Gradient elution on Capto adhere ImpRes using elution buffer with NaCl (green curve) and without
NaCl (blue curve) of MAb A, which was partially purified by protein A affinity chromatography.

Table 4.7. Results from gradient elution on Capto adhere ImpRes using elution buffer with and without NaCl

NaCl (mM) Elution pH (peak maximum) Aggregate at 90% yield (%) Elution volume (CV)
0 4.87 0.5 8.9
100 4.77 0.4 9.8

Step elution
Based on results from screening in 96-well filter plates and gradient elution, conditions for step
elution were further investigated in a packed column using DoE, varying sample load between
approximately 50% and 70% of DBC (37.2 to 49.6 mg MAb/mL chromatography medium).
Elution pH was varied between 3.5 and 4.5, and salt concentration between 0 and 100 mM
NaCl. The responses from the design were yield, aggregate concentration, pool volume, HCP,
and protein A concentration. The results from the design are shown in Table 4.8.
Modeling of the experimental data was performed with MODDE v9.0 software. Good models
were obtained for all responses except for protein A6. The model showed that the only
significant factor was elution pH. Thus, a higher elution pH resulted in lower yield, lower
aggregate concentration, higher pool volume, and lower HCP concentration (Fig 4.34).
6
As the values and the variation of protein A concentration in the elution pools were very low, no model could be obtained
for this response.

29-0548-08 AA 69
Table 4.8. Results from DoE evaluation of step elution on Capto adhere ImpRes

pH NaCl Sample load Yield Aggregates Pool HCP Protein A

(mM) (mg/mL) (%) (%) (CV) (ppm) (ppm)
3.5 0 37.2 95.2 3.33 1.88 1233 1
4.5 0 37.2 83.6 0.61 5.24 319 Below LOQ2
3.5 100 37.2 92.9 4.11 1.90 973 2
4.5 100 37.2 85.4 0.74 5.59 405 Below LOQ
3.5 0 49.6 94.9 3.35 1.96 713 2
4.5 0 49.6 85.4 0.80 5.48 306 Below LOQ
3.5 100 49.6 93.4 4.01 2.50 1103 3
4.5 100 49.6 87.1 0.55 5.86 661 Below LOQ
4.0 50 43.4 93.6 2.26 3. 45 684 1
4.0 50 43.4 92.5 1.83 3.54 577 1
4.0 50 43.4 93.5 1.99 3.52 666 1
3.3 50 43.4 92.6 4.94 1.66 ND 1
ND
4.7 50 43.4 83.4 0.29 6.89 ND ND
4.0 50 43.4 92.6 2.11 3.53 ND ND
1
ND = Not determined.
2
LOQ = Limit of quantitation.

Yield predicted for pH Aggregate predicted for pH

Aggregate

95 4
Yield

90 2
85 0
3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7
pH pH

Pool predicted for pH HCP predicted for pH

7 1000
Pool

HCP

5 500
3
0
3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7
pH pH
Fig 4.34. Response plots for yield, pool volume, aggregate, and HCP concentrations.

Verification of the design

The model suggested an elution pH of 4.5 (0 M NaCl) and a sample load of 70% of DBC (≈ 50 mg/mL).
Column verification of the method was performed on a Tricorn 5/50 column. The obtained
result was in good agreement with the expected result for yield, pool volume, and aggregate
and HCP clearance (Table 4.9). The relatively high initial HCP level in the sample used accounts
for the high HCP level after polishing. HCP levels could be further reduced, either by including
a wash step before elution of the MAb or by addition of a third purification step.

Table 4.9. Verification of the suggested design

Result Yield of monomer (%) Pool volume (CV) Aggregates (%) HCP (ppm)
Expected result 86 5.6 0.7 400
Experimental result 85 5.5 0.8 400

70 29-0548-08 AA
Case study, MAb B
The related multimodal anion exchanger, Capto adhere, has been successful for MAb polishing
in flowthrough mode. However, Capto adhere has also found use in bind/elute mode, even
though the particle size is not optimal. In a case study using MAb B, the performance of
Capto adhere in bind/elute mode was compared with that of Capto adhere ImpRes, considering
DBC at various residence times and gradient and step-elution conditions.

SBC and DBC

SBC and DBC for MAb B were determined using the same methodology as shown in the first
case study. Highest SBC and DBC were obtained at high pH and low ionic strength (i.e., 20 mM
sodium phosphate, pH 7.87).
7
To avoid deamidation of the MAb, pH should normally be maintained below pH 8.0.

DBC vs residence time

DBC at 10% breakthrough for Capto adhere ImpRes and Capto adhere was measured at
different residence times (linear flow rates) in the range of 1 to 10 min. As seen in Figure 4.35,
DBC for Capto adhere ImpRes is higher and less sensitive to residence time than is the case
for Capto adhere. Capto adhere ImpRes can therefore be operated at shorter residence times
(i.e., higher flow rates) while maintaining process robustness with regard to capacity8.
8
Due to pressure-flow limitations, a maximum bed height of 10 cm is recommended at 2 min residence time.

50

40
DBC 10% (mg/mL)

30

20
Capto adhere ImpRes
Capto adhere
10

0
0 2 4 6 8 10 12
Residence time (min)

Fig 4.35. DBC vs residence time. DBC at 10% breakthrough measured in 28 mM sodium phosphate, pH 7.75.

Gradient elution
Gradient elution by pH was performed on Capto adhere ImpRes. Unlike the example with MAb
A, addition of NaCl to the elution buffer resulted in a narrower elution peak (Fig 4.36, green curve).
Collected fractions were analyzed by GF and cumulated yield of monomer was plotted against
cumulated concentration of aggregates. The result shows good separation between monomer
and aggregates, and that separation was improved on Capto adhere ImpRes compared with
Capto adhere (Fig 4.37).

29-0548-08 AA 71
 6.5
500

A254 (mAU)

pH
6.0
Column:
400 Tricorn 5/50, CV ~ 1 mL
Medium: Capto adhere ImpRes 5.5
Sample:
300 MAb B, partially purified by protein A affinity chromatography
Sample load: 30 mg/mL 5.0
Start buffer: 28 mM sodium phosphate, pH 7.75
200
Elution buffer: 30 mM sodium phosphate, 25 mM citrate, pH 4.5 4.1 (blue curve)
30 mM sodium phosphate, 25 mM citrate, pH 4.1 + 250 mM NaCl (green curve)
0
Gradient: 0% to 100% elution buffer in 20 CV 4.0
Residence0time: 5 4 min
10 15 20 25 30 35
System: ÄKTA Volume (ml)

100
2000

80
1500

Elution buffer (%)

A280 (mAU)

60

1000
40

500
20

0 0
0 5 10 15 20 25 30
Volume (mL)

Fig 4.36. Gradient elution of MAb B from Capto adhere ImpRes with (green curve) and without (blue curve)
NaCl in elution buffer.

100
Cumulated monomer yield (%)

80

60

40
Capto adhere ImpRes
Capto adhere
20

0
0 0.5 1.0 1.5 2.0
Cumulated aggregates (%)
Fig 4.37. Cumulated aggregates vs cumulated MAb monomer yield after gradient elution using
Capto adhere ImpRes and Capto adhere.

72 29-0548-08 AA
Step elution
From the gradient elution results above, step elution from Capto adhere ImpRes and Capto adhere
was performed at pH 6.5 and 62.5 mM NaCl (i.e., 25% of elution buffer, Fig 4.38). The sample
load was 70% of DBC 10%. Fractions were pooled and analyzed for yield, aggregate, and HCP
concentration. Despite 20% higher load, step elution from Capto adhere ImpRes resulted in
higher yield and improved aggregate clearance compared with Capto adhere (Table 4.10).
HCP levels were below the detection limit for ELISA.

Column: Tricorn 5/50, CV ~ 1 mL

Sample: MAb B, partially purified by protein A affinity chromatography
Sample load: 30 mg/mL
Start buffer: 28 mM sodium phosphate, pH 7.75
Elution buffer: 30 mM sodium phosphate, 25 mM citrate, 250 mM NaCl, pH 4.1
Step elution: 25% elution buffer (pH 6.5, 11.3 mS/cm)
Residence time: 4 min
System: ÄKTA pure

8.0
3000
7.5

2500 7.0

2000 6.5
A280 (mAU)

pH
6.0
1500
5.5
1000
5.0

500 4.5

0 4.0
0 5 10 15 20 25
Volume (mL)

Fig 4.38. Step elution of MAb B using Capto adhere ImpRes.

Table 4.10. Results from step elution

Medium Sample load Yield Pool volume Aggregates HCP1

(mg/mL) (mg/mL) (%) (CV) (%) (ng/mL)
Capto adhere ImpRes 30 91 4.4 0.5 Below detection limit (< 20 ng/mL)
Capto adhere 25 79 6.1 0.8 Below detection limit (< 20 ng/mL)
1
Measured with general ELISA from Cygnus Technologies.

Conclusions
In this work, results have been presented from two case studies using Capto adhere ImpRes,
a multimodal anion exchanger designed for polishing. Two different MAbs were purified in bind/
elute mode. The results show high yields of MAb monomers and good clearance of aggregates,
HCP, and leached protein A.
For more information on this example, see application note 29-0273-38, “Polishing of monoclonal
antibodies using Capto adhere ImpRes in bind and elute mode.”

29-0548-08 AA 73
2. Viral clearance using Capto adhere ImpRes
The capability of Capto adhere ImpRes for viral clearance from MAb was tested with two model
viruses: the enveloped RNA retrovirus, MuLV, and non-enveloped DNA parvovirus, MVM. MAb
samples partially purified by protein A affinity chromatography were spiked with virus stock
solution and were then applied to Capto adhere ImpRes in bind/elute and flowthrough mode.
Eluted fractions were analyzed for virus titer by endpoint titration and large-volume plating.
Capto adhere ImpRes showed efficient viral clearance in both bind/elute and flowthrough
mode (Table 4.11). The log10 virus reduction factor was approximately 5.0 in bind/elute mode for
both MuLV and MVM. In flowthrough mode, log10 virus reduction factor was > 4.0 for both MuLV
and MVM.

Table 4.11. Viral reduction factor (log10) of MuLV and MVM purified using Capto adhere ImpRes in bind/elute
and flowthrough mode

Minimal log10 viral reduction factor

Process purification mode MuLV MVM
Bind/elute1 4.98 4.95
Flowthrough 2
> 5.0 4.0
1
Bind/elute conditions: phosphate/citrate, pH 7.9 (binding); phosphate/citrate + 45 mM NaCl, pH 5.4 (elution).
2
Flowthrough conditions: pH 5.5, 19 mS/cm conductivity.

Capto MMC applications

1. HTS and process development for capture of recombinant pro-insulin from
E. coli using Capto MMC
This study describes the development of a robust capture method for recombinant pro-insulin.
PreDictor plates and Assist software were used to determine a chromatographic medium
for capture and identify promising binding and elution conditions. Based on the screening
results, Capto MMC was selected as the most promising medium due to its ability to bind
sample without prior dilution. With the binding and elution conditions found in the screening
experiments as the starting point, the capture step was optimized on a Tricorn 5/50 column
packed with 1 mL of medium. Once a robust capture protocol had been established, the
process was successfully scaled up from Tricorn to HiScreen prepacked columns, HiScale 16/40
column (20 cm bed height) packed with Capto MMC, and finally to an AxiChrom 50/300 column
(19.5 cm bed height, 400 mL packed bed volume).

Materials and methods

Screening with PreDictor plates
Whenever possible, experiments with PreDictor plates were performed with fully automated
protocols on a Tecan Freedom EVO-2 200 Robotic System. More complex protocols such as
sample handling were carried out manually. Liquid removal was performed by vacuum or
centrifugation throughout the study.
The pro-insulin used in all experiments originated from E. coli. It was supplied by BIOMM S.A.,
Belo Horizonte, Brazil. The pro-insulin solution was subjected to sulfitolysis to hinder the
formation of disulfide bridges. The suspension that contained 8 M urea was approximately
10 mg/mL in recombinant pro-insulin and 18 mg/mL in total protein. The conductivity of the
sample was approximately 14 mS/cm. PreDictor experiments followed the illustration shown
in Figure 4.39. Conditions studied are presented in “Results and discussion.”

74 29-0548-08 AA
Medium Wash/ Sample Wash Elution
in well equilibration addition 1–3 times 1–3 times

Incubation

Mixing Mixing Mixing

Vacuum filtration or centrifugation Vacuum filtration or centrifugation

Waste

Analysis

Fig 4.39. Schematic illustration of the workflow of a batch experiment in the wells of a PreDictor plate.
The same steps would be employed in a column experiment, that is, equilibration, sample addition, wash,
and elution. The gray color in the wells represents the chromatography medium; red shades (red and pink)
represent different concentrations of protein solution. Brown represents the medium with bound sample.

Analysis
In the PreDictor binding studies, capacities were measured from analyses of the flowthrough
fraction. In the elution studies, the first elution fraction was evaluated. Start samples were
analyzed in all studies. All analyses were performed by AIEX on a Mono Q™ 5/50 GL column .
The pro-insulin sample concentration was determined by integrating the area of the peak
eluting at a retention time of 9 to 10 min and relating its surface area to that in the crude sample:

Concentrationcrude sample × Peak areasample

Concentrationsample = Equation 9
Peak areacrude sample

The resulting pro-insulin concentration in the flowthrough or first elution fraction for each
condition was used as in-data in Assist software where the response surfaces for experimental
evaluation were generated.

Column experiments
Column experiments comprising optimization, DBC experiments, a robustness study, and
scale-up, were performed with the Capto MMC multimodal medium on chromatography
systems suitable for the column dimensions. Table 4.12 summarizes the columns, systems,
samples, and purification conditions for these experiments.
All eluent buffers were prepared in 8 M urea and all experiments were concluded with 1 M NaOH
CIP followed by storage in 20% ethanol. Detection was performed at 280, 405, and 260 nm.
In the preliminary elution experiments, salt/pH gradients were used while optimization,
robustness, and scale-up experiments were performed as step elutions. As in the PreDictor
experiments, sample analyses were performed by the previously described Mono Q method.

29-0548-08 AA 75
Table 4.12. Summary of the Capto MMC column experiments in process development

Vc Sample load Flow rate

Study Column (mL) (mL) System (mL/min)
DBC Tricorn 5/50 1 10 ÄKTAmicro™ 0.2
Elution optimization Tricorn 5/50 1 2.5 ÄKTA avant 25 0.2
Robustness study Tricorn 5/50 1 2.5 ÄKTA avant 25 0.2
Scale-up 2 × HiScreen 4.7/10 9.4 24 ÄKTA avant 25 1.9
HiScale 16/40 40 100 ÄKTA avant 150 8
AxiChrom 50/300 400 960 ÄKTA avant 150 80

Results and discussion

Screening experiments for binding
Binding experiments were performed on a selection of ion exchange and multimodal media;
the PreDictor plates contained 2 μL or 6 μL of SP Sepharose Fast Flow, Capto S, or Capto MMC.
The small media volumes (2 and 6 μL) enabled binding experiments by overloading the media
with the buffered sample (200 μL solution 2.5 mg/mL in respect to pro-insulin per well) in 8 M urea
without consuming more than 15 mL of crude sample for the binding study. The binding with
respect to both the initial salt concentration and the pH value of the binding buffer was examined.
Table 4.13 summarizes the test conditions for each medium.

Table 4.13. Summary of media and parameters in the binding experiments conducted on PreDictor plates

Experiment pH NaCl (mM)

Binding study—CIEX screening plate 3.4–5.0 0–300
Capto S, 2 µL
Capto MMC, 6 µL
SP Sepharose Fast Flow, 6 µL
Binding study—Capto MMC, 6 µL 3.0–7.0 0–300

In all binding experiments, the flowthrough fraction was collected and analyzed with respect
to nonbound pro-insulin as compared with the start sample, which gives an indication of the
binding capacity at each condition. The resulting response surfaces for all media, generated
using Assist software, are shown in Figure 4.40.
Capto S and SP Sepharose Fast Flow indicate high binding capacities at the lowest pH tested
(i.e., 3.4) and no salt. Capto MMC binds at 150 mM salt and higher pH compared with these
two media. Because the starting sample of the fusion protein has an ionic strength close to
150 mM NaCl, high binding capacity at this concentration is an advantage.
A second binding study was thus performed with Capto MMC and a broader parameter interval
intended to reveal the optimum binding for this medium. As Figure 4.41 shows, the highest
binding capacities (red/orange zone) for pro-insulin binding to Capto MMC are obtained at pH 5
(or just above) and 0 to 160 mM NaCl. It was decided to continue with Capto MMC and to study
conditions for elution.

76 29-0548-08 AA
 SP Sepharose Fast Flow Capto S Capto MMC
5.0 80.89 5.0 54.20 5.0
75.47 50.55
72.76 48.73
4.8 4.8 46.91
4.8
67.34
64.63 45.09
43.26
4.6 61.92 4.6 42.44
4.6
59.21
39.62
56.49
37.80
4.4 51.07 4.4 34.15
4.4
pH

pH

pH
48.36 32.33
45.65 30.51
4.2 42.94
4.2 4.2
28.69
40.23 26.87
4.0 37.52 4.0 25.05 4.0
34.10 23.22
29.39 21.40
3.8 23.97 3.8 19.58 3.8
21.26 15.94
14.11
18.55
3.6 15.84 3.6 12.29 3.6
10.47
13.13 8.65
3.4 10.42 3.4 6.83 3.4
7.71 5.00
0 50 100 150 200 250 300 5.00
0 50 100 150 200 250 300 0 50 100 150 200 25
3.18
NaCl concentration (mM) 2.29 NaCl concentration (mM) 1.36 NaCl concentration (mM

Capto MMC
54.20 5.0 31.63
50.55 29.75
48.73 28.81
46.91
4.8 27.87
45.09 26.93
43.26 25.99
42.44
4.6 25.05
39.62 24.12
37.80 23.18
34.15
4.4 21.30
pH

32.33 20.36
30.51 4.2 19.42
28.69 18.48
26.87 17.54
25.05 4.0 16.60
23.22 15.66
21.40 14.72
19.58 3.8 13.78
15.94 11.90
14.11 10.96
12.29 3.6 10.02
10.47 9.08
8.65 8.14
6.83 3.4 7.20
0 250 300 5.00 0 50 100 150 200 250 300 6.26
3.18 5.32
n (mM) 1.36 NaCl concentration (mM) 4.38

Fig 4.40. Response surfaces generated by Assist software for pro-insulin binding (g/L) as a function of NaCl
concentration (x-axis) and buffer pH (y-axis) for SP Sepharose Fast Flow, Capto S, and Capto MMC, respectively.
The range of binding capacities achieved is shown to the right of each surface. Black crosses represent
actual data between results that have been interpolated.

23.95
22.40
7.5 21.63
20.86
20.09
7.0 19.32
18.55
17.78
6.5 17.01
15.47
14.70
6.0 13.92
pH

13.15
12.38
5.5 11.61
10.84
10.07
5.0 9.30
7.76
6.99
4.5 6.21
5.44
4.67
4.0
3.90
0 50 100 150 200 250 300 3.13
2.36
NaCl concentration (mM) 1.59

Fig 4.41. Response surface for binding (g/L) of pro-insulin on Capto MMC as a function of NaCl concentration
(0 to 300 mM) and buffer pH (4 to 7.5). Assist software was used in visualizing this data.

29-0548-08 AA 77
Screening experiments for elution
PreDictor plates with 50 μL of Capto MMC media volume were used for elution studies. This
ensured sufficient loading to detect the target molecule without overloading the medium. The
amount of protein applied in the loading step corresponded to 70% of the binding capacity that
was estimated in the binding study, that is, 180 μL of sample, 5 mg/mL in respect to pro-insulin.
The elution study was performed using a range of eluent compositions: pH 3.7 to 7.6 and 150 to
1000 mM NaCl. The evaluation procedure was the same as for the binding study, but now the
first elution fraction was analyzed. This showed the conditions required to obtain elution in the
column verification work that followed.
As only the first elution fraction was analyzed, one may not expect full yield in this step.
The highest yield achieved was 70% and was found at pH 7.5 and a NaCl concentration above
600 mM (Fig 4.42).
7.5 70.01
65.27
62.90
7.0 60.53
58.16
53.42
6.5 51.05
48.68

6.0 46.31
43.94
39.20
pH

5.5 36.83
34.46
32.09
5.0 29.72
24.98
22.61
4.5 20.24
17.87
15.50
4.0 10.76
8.39
6.02
200 400 600 800 1000 3.65
1.28
NaCl concentration (mM)

Fig 4.42. Pro-insulin yield (%) (first elution fraction) on Capto MMC as a function of NaCl concentration (150 to
1000 mM) and buffer pH (4 to 7.5). Assist software was used in obtaining these data.

Optimization in Tricorn columns on ÄKTA avant 25

The HTS experiments on PreDictor plates suggested that the best conditions for pro-insulin
capture would be binding at around pH 5 and a NaCl concentration of 50 to 150 mM on
Capto MMC followed by eluting at a pH greater than 7 and a NaCl concentration above 600 mM.
With these parameters added as factors in a DoE protocol, the capture step was optimized on
Capto MMC packed in a 1 mL Tricorn column (diameter 5 mm). As 150 mM NaCl corresponds
to the isotonic salt concentration found in the start sample, this salt concentration was an
obvious starting point for binding because it eliminated the need to dilute sample prior to
loading. Binding buffer pH was set at 5.2, and the pH of the start sample was set accordingly.
In the first column experiment, elution with a salt gradient was tested by loading 20 mg of pro-
insulin (2 mL sample) in 50 mM sodium acetate buffer, pH 5.2 in 8 M urea on the 1 mL column
and eluting with a linear salt gradient of 150 to 1000 mM NaCl for 7 CV.
Figure 4.43 shows the results. The fraction collected at the maximum height of the elution peak
was analyzed on Mono Q and the resulting chromatogram compared with that of the crude
sample and one flowthrough fraction.
Analysis of the flowthrough fraction (Fig 4.43B) showed good binding of the target molecule
with no pro-insulin detected in the flowthrough. The nonprotein impurity seemed to be low
binding as it appeared in the flowthrough fraction while the corresponding peak in the elution
fraction was significantly smaller. This indicated good capture and purification of pro-insulin.

78 29-0548-08 AA
However, a large peak was seen during CIP (Fig 4.43A), suggesting that high salt concentration
alone was not adequate to recover all of the pro-insulin. Experience with several other target
proteins indicates that multimodal media frequently require more than just high ionic strength
for efficient elution.

(A) (B)
Flowthrough CIP
100
5000 1000

80
4000 800

Conductivity (mS/cm)
Pro-insulin

A280 (mAU)
A280 (mAU)

60 600
3000 Elution

2000 40 400
Crude sample
Flowthrough
1000 20 200

Elution
0 0 0
0 5 10 15 20 0 5 10 15 20
Time (min) Time (min)

Fig 4.43. (A) A 2 mL crude sample, pH 5.2 in 8 M urea, loaded on a Tricorn 1 mL 5/50 column packed with
Capto MMC and eluted by a linear salt gradient from 150 to 1000 mM NaCl for 7 CV. (B) Corresponding
Mono Q analysis of crude sample, flowthrough, and one elution fraction (collected at the main elution peak
maximum). In both A) and B), detection was at 280 nm.

Figure 4.44 shows the capture and analysis results where the salt gradient was supplemented
with a pH 5.2 to 7.5 gradient.

(A) (B)
Flowthrough
Elution 100
5000 3000

80 2500
4000
Conductivity (mS/cm)
A280 (mAU)

A280 (mAU)

2000
60
3000
Wash
1500

2000 40
1000

1000 CIP 20
500 Flowthrough
Wash
Elution
0 0 0
0 5 10 15 20 0 5 10 15 20
Time (min) Time (min)

Fig 4.44. (A) A 2 mL crude sample, pH 5.2 in 8 M urea, loaded on a Tricorn 1 mL column packed with
Capto MMC and eluted with a linear combined salt and pH gradient from 150 to 1000 mM NaCl and pH 5.2
to 7.5 for 7 CV. (B) Corresponding Mono Q analysis of the flowthrough, wash, and pooled fractions in the main
elution peak. In both A) and B), detection was at 280 nm.

Comparing chromatograms for the constant pH (Fig 4.43A) and the pH gradient (Fig 4.44A)
capture experiments revealed that a combined pH and salt gradient gave both a narrow
elution peak and a high yield, neither of which was achieved when salt gradient elution alone
was employed.

29-0548-08 AA 79
DBC experiments
Once promising conditions for binding and eluting pro-insulin had been established, attention
was turned to DBC. This was determined by overloading the column with crude sample and collecting
and analyzing fractions to determine the point at which pro-insulin breakthrough occurred.
Based on DBC experiments, the loading in the experimental work was set to 25 mg pro-insulin
(2.5 mL crude sample, approx. 80% of DBC) to secure complete binding.

Elution optimization
Aiming at a step elution mode, elution conditions were optimized using the buffer prep and
DoE tools of ÄKTA avant 25. A full factorial design with three center points based on two
variables (pH and NaCl concentration) each at three levels was set up to determine the salt
concentration and the pH needed to obtain sufficient purity and yield (above 80% and 95%,
respectively). The area of the pro-insulin peak as well as the area percent of pro-insulin in the
analysis chromatogram (purity) were set as responses. See Table 4.14 for details.

Table 4.14. Design variables, values for the elution optimization, and purity data of pro-insulin in the eluted peak

Run NaCl (mM) Elution pH read2 Area (mAU × mL) Purity (%)
1 1
450 7.1 196 76
2 150 7.1 181 79
31 450 7.1 196 81
4 150 8 246 83
51 450 7.1 187 82
6 750 8 251 84
7 750 7.1 241 82
8 450 6.2 69 71
9 450 8 250 84
10 150 6.2 37 53
11 750 6.2 116 78
1
Center points.
2
8 M urea influences the pH reading; settings in ÄKTA avant were approximately 1 pH unit lower.

Figure 4.45 shows the pro-insulin peak area in the collected elution peak as a function of pH and
NaCl concentration. This clearly demonstrates that the optimal elution for pro-insulin is found at
high pH, whereas an increase in the concentration of NaCl above 150 mM has only a minor effect.
The purities achieved were also highest at high pH. It was decided to perform the elution at pH 8
and 150 mM NaCl.

Peak area
7.8
230
7.6
210
7.4
190
7.2
170
pH

7.0
150
6.8
130
6.6 110
6.4 90
6.2 70

50
200 300 400 500 600 800
NaCl concentration (mM)
Fig 4.45. Response surface for the elution peak area of pro-insulin as a function of pH and NaCl
concentration in mM. R2 (explained variation) = 0.989, Q2 (predicted variation) = 0.736. ÄKTA avant was
used in obtaining these data.

80 29-0548-08 AA
Robustness study
To conclude process development, a robustness study was performed on 1 mL Tricorn 5/50
columns packed with Capto MMC using the optimized elution conditions of pH 8 and
150 mM NaCl. The robustness study was designed using a Plackett–Burman DoE based on four
variables (two chromatography media batches, two crude sample batches, elution pH 7.8 to
8.2 and load volume 2.3 to 2.7 mL) with 150 mM NaCl in all eluent buffers. Figure 4.46 shows
the scaled and centered coefficients for the purity data (all above 80% purity) from the eluted
peaks as a function of the variable parameters. It is clear that no significant model terms can
be detected. The yield was approximately 95% for all conditions in this study.

Scaled and centered coefficients for purity (extended)

2

1
%

-1

-2
Ca

Ca

Sa

Sa

pH

Lo
ad
m

m
pt

pt

pl

pl
o

vo
e

e
M

lu
1

2
M

m
C

e
(lo

(lo
t1

t1
00

00
07

34
12

22
4)

4)

Fig 4.46. Scaled and centered coefficients for purity as a function of four variable parameters: chromatographic
medium lot (×2), sample (×2), pH, and load volume.

Scale-up experiments
Columns with 20 cm bed heights were used for 9-, 40-, and 400-fold scale-up by increasing
column diameter while keeping other parameters such as residence time and sample load/
mL chromatographic medium constant. Other conditions were similar to those found in the
optimization study on the Tricorn 5/50 column packed with Capto MMC (loading at pH 5.2 and
elution at pH 8, both in the presence of 150 mM NaCl).
Two HiScreen Capto MMC columns were connected in series to give 20 cm bed height. In
addition, a HiScale 16/40 column (diameter 16 mm) and an AxiChrom 50/300 column (diameter
50 mm) were packed with Capto MMC to bed heights of 20 and 19.5 cm, respectively. The
capture experiment was performed at 240 cm/h at all three extended scales (5 min residence
time). Fractions from the flowthrough and the eluted peaks were analyzed on the Mono Q
column. Results and purity data (Table 4.15 and Fig 4.47) show that the capture step of the
pro-insulin purification was successfully transferred from the 1 mL Tricorn 5/50 column to the
400 mL AxiChrom 50 column. The resulting pro-insulin purity was 82%, and the yield was 96%
measured at the 400 mL scale.

29-0548-08 AA 81
Table 4.15. Purity data after four column steps

Column1 Scale-up factor Crude sample load (mL) Pro-insulin purity (%)
Tricorn 5/50 1 2.5 83
HiScreen Capto MMC × 2 9.4 23.5 86
HiScale 16/40 40 100 84
AxiChrom 50/300 400 960 82
1
Total packed bed heights were 20 cm, except for AxiChrom 50, which was 19.5 cm.

6000 8
7
5000
6
A280 (mAU)

4000 5

pH
3000 4
3
2000
2
1000
1
0 0
0 20 40 60
Time (min)
Fig 4.47. Chromatogram from the final (AxiChrom column) step after preceding columns. The pro-insulin
crude sample was loaded on Capto MMC at pH 5.2 and 150 mM NaCl in a HiScreen (9 mL), HiScale (40 mL),
and AxiChrom 50/300 (bed height 19.5 cm; 400 mL) column. An ÄKTA avant 150 system was used with the
AxiChrom column.

Conclusions
HTS with PreDictor plates and the Assist software allowed quick selection of most suitable
chromatography medium and identification of promising binding and elution conditions for the
capture of recombinant pro-insulin expressed in E. coli. This gave a fast and confident start to
the purification process development.
Based on these screening experiments, Capto MMC was the medium of choice for further
work due to its ability to bind sample without prior dilution. The capture step was further
optimized in a Tricorn 1 mL column packed with Capto MMC, again based on the binding and
elution conditions determined by the screening experiments. Once the optimized protocol had
been confirmed to be robust, the process was successfully scaled up from a 1 mL Tricorn 5/50
column to a 400 mL AxiChrom 50/300 column. The resulting purity for the capture step was
82% with a yield of 96%.
The overall outcome demonstrates the value of introducing high-throughput methods into
process development workflows. In this PreDictor plate screening example, media and
condition selection was completed in 1 wk using 30 mL of crude sample (300 mg of the target
molecule). When UV absorbance in a plate reader is sufficient for evaluation, media screening
can be finalized within two days. The screening described here enabled fast development of a
pilot-scale process (400 mL AxiChrom column) within 4 wk.
For more information on this example, see application note 28-9966-22, “High-throughput
screening and process development for capture of recombinant pro-insulin from E. coli.”

82 29-0548-08 AA
2. Evaluation of three media for capture of recombinant human serum albumin
from Pichia pastoris and scale-up using Capto MMC
Three media (Capto MMC, SP Sepharose Fast Flow, and SP Sepharose XL) were evaluated for their
ability to capture recombinant human serum albumin (rHSA, pI 5.5) from cell culture supernatant
(CCS) of Pichia pastoris. The CCS was clarified by centrifugation and used directly as a feed
material (conductivity 15 mS/cm) to measure dynamic binding capacities of the three media.
The calculated results (Table 4.16) show that Capto MMC gives a productivity of 19 kg/m3/h,
which is approximately 3 times higher than that obtained with SP Sepharose Fast Flow and
SP Sepharose XL when the diluted feed is used and more than 13 times higher than when the
undiluted feed is used. The latter result is expected because the SP ligand is not salt tolerant.
This example shows how high DBC at high conductivity combined with high flow velocities can
improve the overall productivity of a capture step.
Scale-up with Capto MMC is straightforward (Fig 4.48).

Table 4.16. Productivity calculations for purification of rHSA from P. pastoris CCS. Flow velocities and residence
times are based on the restrictions of the respective media in a large-scale column at 20 cm bed height.
In all cases, 93% recovery and 70% loading safety factor were used1

Residence Max flow

Dilution Capacity time velocity Productivity Productivity
factor (g/L) (min) (cm/h) (kg/m3/h) (kg/24 h)1
Capto MMC 15 mS/cm no dil 44 2 600 19 102
SP Sepharose XL 3 mS/cm 6.4 195 6 200 6.4 35
SP Sepharose XL 15 mS/cm no dil 0 6 200 0 0
SP Sepharose Fast Flow 3 mS/cm 6.4 135 6 200 6.0 33
SP Sepharose Fast Flow 15 mS/cm no dil 6 6 200 1.4 7.4
1
Assuming column dimensions of 120 cm diameter, 20 cm bed height (CV ≈ 225 L).

Column: (A) Tricorn 5/100, 10 cm bed height (CV 2 mL); (B) AxiChrom 50, 10 cm bed height (CV 208 mL)
Medium: Capto MMC
Sample: rHSA in P. pastoris CCS
Buffer A: 25 mM sodium acetate, pH 4.5
Buffer B: 50 mM sodium phosphate, pH 7.2 + 1 M NH4Cl
Flow velocity: 600 cm/h
Gradient: 100% B, 10 CV
System: (A) ÄKTAexplorer 100; (B) ÄKTApilot

(A) 5000 (B) 3500

3000
4000
2500
3000
A294 (mAU)

A294 (mAU)

2000

2000 1500

1000
1000
500

0 0
0 10 20 30 40 50 0 1000 2000 3000 4000 5000
Elution volume (mL) Elution volume (mL)

Fig 4.48. Straightforward scale-up from A) Tricorn 5/100 to B) AxiChrom 50 (100 times). In both cases the
purification factor was 4 and the recovery was 93%.

For more information on this example, see data file 11-0035-45, “Capto MMC.”

29-0548-08 AA 83
Capto MMC ImpRes applications
1. Polishing of MAbs using Capto MMC ImpRes in bind/elute mode
This study describes a fast and efficient method to separate monomeric MAb from aggregates,
HCP, and protein A remnants. The method described includes screening for optimal binding
conditions in 96-well plate format followed by verification in packed column format, and
optimization of elution conditions using DoE. The running conditions were then validated in
larger scale with satisfactory correspondence to the DoE model prediction. All preparative
chromatography experiments were performed in bind/elute mode.
A summary of the steps in the study is shown in Figure 4.49.

Confirm binding DoE model for fine- Validation of running

conditions in columns tuning of elution conditions in columns

Find optimal binding Investigation of Monte Carlo simulation based

conditions in plates elution conditions on DoE model to investigate
protocol robustness

Fig 4.49. The steps used in this performance evaluation of Capto MMC ImpRes in removing contaminants
from monomeric MAb.

Materials and methods

Start material
The MAb used in this study was initially purified from CHO cell supernatant by protein A affinity
chromatography. Some characteristics of the antibody are shown in Table 4.17.

Table 4.17. Characteristics of the antibody used in the study

Antibody pI Aggregate content (%) DBC 10% (mg/mL)1 Capto MMC ImpRes
MAb A 7.3 2.5 71
1
DBC at 10% breakthrough (DBC 10%) measured at 4 min residence time.

Determination of SBC
SBC was determined in 6 μL PreDictor Capto MMC ImpRes 96-well plates. Equilibration of
wells in the plates was performed by addition of 200 μL of loading buffer per well followed
by agitation at 1100 rpm for 1 min, after which the buffer was removed by vacuum suction.
The equilibration step was performed three times. MAb, partially purified by protein A affinity
chromatography (200 μL volume, 4 mg/mL sample load, corresponding to 133 mg MAb/mL
chromatography medium) was added to each well followed by agitation for 90 min.
Unbound material (flowthrough fraction) was removed by centrifugation for 1 min, and MAb
concentration was determined by measurement of absorbance at 280 nm.

84 29-0548-08 AA
SBC was calculated according to:
Vload
SBC = (Cini – CFT) Equation 10
Vmedium

where Vload = load volume, Vmedium = medium volume in each well, Cini = MAb concentration in the
CO(VX% – VO)
DBCX% = and CFT = MAb concentration in the flowthrough fraction.
sample,
VC
Determination of DBC
DBC was Vload
determined by frontal analysis using an ÄKTA chromatography system. The UV
SBC =
absorbance (Ciniat– C FT)
280 nm was used for determination of breakthrough. DBC was then calculated
Vmedium
according to:

CO(VX% – VO)
DBCX% = Equation 11
VC

where C0 = MAb concentration in the sample (mg/mL), VX% = load volume (mL) at x%
breakthrough, V0 = void volume (mL), and Vc = volumetric bed volume (mL).

Screening of elution conditions

Conditions for optimizing elution were investigated in a Tricorn 5/50 packed column with
Capto MMC ImpRes at a bed height of 4.7 cm. Optimization was performed using ÄKTA avant 25
chromatography system, DoE, and scouting functionalities included in UNICORN 6.0 software.
The factors considered in the design were load volume, gradient length, and flow velocity. The
responses were resolution of monomer/aggregates and pool volume. The method used for the
DoE runs was the following:
Column: Tricorn 5/50, bed height 4.7 cm
Medium: Capto MMC ImpRes
Sample: MAb (8 mg/mL) equilibrated in start buffer
Start buffer: 25 mM sodium phosphate, 25 mM sodium citrate, 100 mM NaCl, pH 6.0
Elution buffer: Start buffer + 1 M NaCl
Wash: Start buffer (5 CV)
CIP: 1 M NaOH

Determination of MAb aggregates and aggregate clearance

Fractions from the chromatographic runs were collected and analyzed by analytical GF on
a Superdex 200 5/150 GL column. The peaks were integrated and the D/A content (in percent)
were estimated. Cumulated yield of monomers was plotted against cumulated aggregates as
exemplified in Figure 4.29.

Protein A and HCP ELISA

The protein A concentration in the start materials and flowthrough fractions was determined by
Protein A ELISA kit (Repligen). HCP concentration was determined by HCP ELISA (Cygnus Technologies).

Results and discussion

This case study with MAb shows a suggested workflow for method development including
screening of binding conditions in 96-well format, verification of dynamic binding capacities in
column format, screening, optimization of elution conditions, and validation of the DoE model
prediction in a HiScreen column. It also includes a Monte Carlo simulation that addresses the
protocol robustness.

29-0548-08 AA 85
SBC
To find optimal binding capacity for the MAb, SBC was determined in 6 μL PreDictor 96-well
filter plates. Binding pH was varied between pH 4.0 and 8.09,10 and salt concentration from
0 to 500 mM NaCl. All samples and buffers were prepared automatically using an automatic
liquid handling system for the preparation of buffers. The results from the SBC study display
an area of conditions with high binding capacities between pH 5.0 and 7.0 and NaCl between
0 and 150 mM. The highest SBC was obtained at approximately pH 6.0 and salt concentration
of 0 to 150 mM (Fig 4.50). The binding capacity appeared to be more salt tolerant at lower pH
and could represent an alternative binding condition. This is important to take into account
because the choice of binding conditions affects the elution strategy. When binding at pH
between 6.0 and 6.5, elution can be performed merely using a salt gradient whereas binding at
pH between 5.0 and 5.5 is likely to require salt and pH gradient elution.
9
Start buffers were sodium acetate, pH 4.0 and 5.3; sodium phosphate, pH 6.3; Tris, pH 8.0.
10
To avoid deamidation of the MAb, pH should normally be maintained below pH 8.0.

500 SBC (mg/mL)

450 65

60
400
55
350 50

45
300
NaCl (mM)

40
250 35

30
200
25
150 20

100 15

10
50 5

0 0

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

pH

Fig 4.50. Contour map showing screening of SBC for Capto MMC ImpRes. The lower right corner is excluded
since MAb tended to precipitate at pH > 6.7 and low salt concentration.

DBC
The area with high SBC in the PreDictor plate studies—between pH 5.0 and 6.0 and NaCl
content between 0 and 200 mM—were chosen for further investigation in column format. This
particular MAb showed a tendency to precipitate at pH > 6.7 under certain conditions, which
explains the choice of pH values for verification. The trends seen in the PreDictor plate SBC
experiment correlated well with the DBC studies (Fig 4.51). The conditions with highest DBC
(100 mM NaCl, pH 6.0) were chosen for further investigation in which the influence of residence
time was studied (Fig 4.52). The DBC was found to be relatively independent of residence time
in the investigated interval.

86 29-0548-08 AA
 70 pH 5.0
60 pH 6.0

DBC 10% (mg/mL)

50
40
30
20
10
0
0 100 200
NaCl concentration (mM)

Fig 4.51. DBC of Capto MMC ImpRes at 4 min residence time in different salt concentrations and two
different buffer pH.

pH 6.0 and 100 mM NaCl

60
DBC 10% (mg/mL)

40

20

0
0 2 4 6 8 10
Residence time (min)

Fig 4.52. Influence of residence time on DBC measured at 10% breakthrough.

29-0548-08 AA 87
Investigating elution conditions for selectivity
As high binding capacities were found at pH 5.0 to 7.0, aggregate removal and yield were
investigated by linear NaCl gradient elution at pH 5.0, 6.0, and 7.0 (Fig 4.53).

Column: Tricorn 5/50, bed height 4.7 cm

Medium: Capto MMC ImpRes
Sample: 4 mL of MAb (6.3 mg/mL)
Start buffer stock BufferPro CIEX 2–7 (sodium phosphate, sodium
solution: formate, sodium acetate buffer, various pH)
Start buffer A: pH 5.0
Start buffer B: pH 6.0
Start buffer C: pH 7.0
Elution buffer: Start buffer + 1 M NaCl
Wash: Start buffer, 5 CV
Gradient: 0% to 100% elution buffer in 20 CV
Residence time: 4 min
CIP: 1 M NaOH
System: ÄKTA avant 25

(A) (B)
100 2000 100
1200
pH pH
1000 80 80
1500

Elution buffer (%)

Elution buffer (%)

800
60 60
A280 (mAU)
A280 (mAU)

600 1000

40 40
400
500
20 20
200

0 0 0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50
Volume (mL) Volume (mL)
(C)
100
2000
pH
80
1500
Elution buffer (%)

60
A280 (mAU)

1000
40

500
20

0 0
0 10 20 30 40 50
Volume (mL)

Fig 4.53. Elution of the MAb in a salt gradient at three different pH: (A) pH 5.0, (B) pH 6.0, and (C) pH 7.0.

Fractions were collected and analyzed by GF, and fractions containing 90% of the MAb were
pooled and analyzed for HCP and protein A content. A summary of the results is found in
Table 4.18. As can be seen, efficient aggregate removal at 90% yield was obtained for all three
binding pH values. However, at pH 5.0, larger pool volumes were observed, and precipitation
tendencies were seen for pH 7.0. Therefore, the conditions chosen were binding at pH 6.0 and
elution with an NaCl gradient. If higher purity levels or higher yield at maintained purity had
been required than the performance observed, a pH closer to 5.0 for binding and NaCl gradient
elution would be a suitable alternative.

88 29-0548-08 AA
Table 4.18. Summary of the results of the chromatography at different pH; start concentrations for HCP and
protein A are shown in brackets

Aggregate at
pH 90% yield (%) Pool volume (CV) HCP (ng/mL) Protein A (ng/mL)
5.0 0.04 14.1 16 (245) Below LOQ1 (16)
6.0 0.2 5.4 56 (245) Below LOQ (16)
7.0 0.2 6.5 44 (245) Below LOQ (16)
1
LOQ = Limit of quantitation.

Optimization of the purification performance

The binding study showed that binding at pH 6.0 with an addition of 100 mM NaCl resulted in
high binding capacities. Also, high purity and yields were obtained at pH 6.0 using a linear salt
gradient for elution. It would be possible at this point to stop further evaluation, but in order
to build understanding and to optimize the purification performance, a DoE model was set up
for the influence of three factors on aggregate content and pool volume. The factors that were
varied and responses are displayed in Table 4.19. The reduction of HCP and protein A was not
included as a response in the design but was measured. The start concentration of HCP was
164 ng/mL and the start concentration of protein A was 26 ng/mL. Other factors that affect the
purification performance and could be of interest to study from a robustness perspective using
this methodology are, for example, aggregate content or HCP levels.
The rationale behind the high and low levels of the parameters in the DoE model was as
follows: flow velocity was chosen to ensure that high and low flow velocities corresponded to
a residence time of 2 and 8 min, respectively. For many processes, it is not possible to have
shorter residence time than 2 min due to limitations in the pumps and other equipment. The
low flow velocity gives a longer residence time but is still acceptable.
Gradient length was between 5 and 15 CV. The short gradient length of 5 CV challenged the
performance of Capto MMC ImpRes because this gradient is shorter than most gradients used
in purification processes today. A gradient length of 15 CV is closer to that typically used by
process developers and represents an average, normal gradient length.
In this study, loads of 42 and 30 mg sample/mL were used. These represent loading of 70%
and 50%, respectively of the DBC 10%. A loading of 70% of DBC 10% is usually regarded as the
upper limit for loading without risking any leakage of target molecules. A loading of 50% of DBC
10% is substantially lower, but represents a plausible loading for a process.

Table 4.19. Factors and levels studied in by DoE

Factors Low High

Load (mg/mL MAb) 30 42
Gradient length (CV) 5 15
Flow velocity (cm/h) 37 140

Aggregate removal
Flow velocity and gradient length were found to significantly affect aggregate removal while the
influence of load was insignificant. Lower flow velocities and longer gradients resulted in lower
aggregate amounts. The significant factors in the model are shown in the coefficient plot (Fig 4.54A).
The summary plot in Figure 4.54B shows different model characteristics such as model fit
(R2), an estimate of the precision of future predictions, model validity, and information on the
reproducibility. The summary plot indicates that the model is valid.
29-0548-08 AA 89
Table 4.20. Summary of the factors and responses used in the three-factor screening design

Factors Responses
Flow velocity Gradient Sample load Pool volume Aggregate HCP Protein A
(cm/h) length (CV) (mg/mL) (CV) at 90% yield (%) (ng/mL) (ng/mL)
37 5 30 2.7 0.4 93 Below LOQ
37 15 30 4.9 0.23 39 Below LOQ
37 15 42 6.0 0.26 71 Below LOQ
89 10 36 4.3 0.39 41 Below LOQ
89 10 36 4.3 0.39 60 Below LOQ
89 10 36 3.8 0.37 27 Below LOQ
140 5 30 3.3 0.48 58 Below LOQ
140 15 30 5.4 0.37 74 Below LOQ
140 5 42 3.3 0.42 62 Below LOQ
140 15 42 6.0 0.37 79 Below LOQ

(A) 0.06
(B) 1.0

0.04 0.8
Aggregates (%)

0.02
0.6
0
0.4
-0.02
-0.04 0.2
-0.06
0
-0.08 Model fit (R2) Model validity
Flow Gradient Sample
velocity length load Precision of Reproducibility
future predictions

Fig 4.54. (A) Coefficient plot showing factors affecting the aggregate removal. (B) Summary plot showing
different model characteristics for the aggregate removal response.

Pool volume
Gradient length and sample load were significant factors affecting pool volume (Fig 4.55).
The summary plot in Figure 4.55B describes the characteristics and validity of the model.

(A) 1.4 (B) 1.0

1.2
0.8
Pool volume (CV)

1.0
0.8 0.6

0.6 0.4
0.4
0.2
0.2
0 0
Gradient length Sample load Model fit (R2) Model validity
Precision of Reproducibility
future predictions
Fig 4.55. (A) Coefficient plot showing factors affecting pool volume. (B) Summary plot showing different
model characteristics for the response pool volume.

90 29-0548-08 AA
Prediction of aggregate removal and pool volumes using DoE and Monte Carlo simulation
To find optimal parameters for a purification protocol and investigate the robustness of that
protocol, a Monte Carlo simulation based on the DoE model was used. The investigated design
space for the DoE model and the target purification performance are shown in Table 4.21.
The suggested chromatographic protocol and the allowed variation in each factor (triangular
distribution) are shown in Table 4.22.
A Monte Carlo simulation was used in order to assess the design space with probabilities of
failing to meet the target purification performance. The resulting design space defined by the
Monte Carlo simulation is shown in Figure 4.56.

Table 4.21. Factors, responses, and target values for optimization in the DoE model

Factors Low High

Flow velocity (cm/h) 37 140
Gradient length (CV) 5 15
Load (mg/mL) 30 42
Response Criterion Target Max.
Aggregate content (%) Minimize 0.2 0.4
Pool volume (CV) Minimize 2.0 4.5

Table 4.22. Factors, variation, and distribution of the factors of the final purification protocol used in the
Monte Carlo simulation

Factors Low Optimum High Distribution

Flow velocity (cm/h) 46 49 52 Triangular
Gradient length (CV) 10 10.5 11 Triangular
Load (mg/mL) 31 34 36 Triangular

(A) (B) (C)

4.6 4.6 4.6

0.1
4.4 1 4.4 1 4.4
0.1 1

50 50
4.2 50 4.2 4.2
Load volume (mL)

0.05
Load volume (mL)

Load volume (mL)

10 0.05
10 0.1
10
4.0 0.05 5 4.0 5 4.0
5

3.8 3.8 3.8

3.6 3.6 3.6

3.4 3.4 3.4

7 8 9 10 11 12 13 7 8 9 10 11 12 13 7 8 9 10 11 12 13
Gradient length (CV) Gradient length (CV) Gradient length (CV)

Fig 4.56. Contour plots from the Monte Carlo analysis showing risk of failing to meet the set criteria for
aggregate level and pool volume in percentage, at flow velocities of (A) 46, (B) 49, and (C) 52 cm/h.

29-0548-08 AA 91
Validation of the DoE model
To validate the model, running conditions that would fulfill the desired purification performance
(Fig 4.56, green area) were chosen and applied to a 4.7 mL HiScreen Capto MMC ImpRes column
on ÄKTA avant 25 chromatography system. Flow velocity and load volume were recalculated
according to the size of the HiScreen column (see “Materials and methods”). The chosen
running conditions are summarized below, and the purification performance was predicted
using UNICORN 6.0 software. The factor settings selected for validation of the model are shown
in Table 4.23.

Column: HiScreen Capto MMC ImpRes, 4.7 mL

Sample: 20.25 mL MAb A (34 mg/mL) in 25 mM sodium citrate +100 mM NaCl, pH 6.0
Start buffer: 25 mM sodium citrate + 100 mM NaCl, pH 6.0
Elution buffer: Start buffer + 1 M NaCl
Flow velocity: 49 cm/h
Gradient: 0% to 100% in 10.5 CV
System: ÄKTA avant 25

Table 4.23. Factors selected for validation of the model

Flow velocity (cm/h) Gradient length (CV) Sample load (mg/mL)
49 cm/h 10.5 34

Table 4.24. Comparison of the responses between the predicted value from the model and the validation
run using a HiScreen Capto MMC ImpRes column with the predicted settings

Aggregate at Yield at 1% Pool volume

Identity 90% yield (%) aggregate (%) (CV)
Predicted value 0.34 NA 4.1
HiScreen Capto MMC ImpRes 0.39 > 95 4.0

Conclusions
This work describes a rapid procedure to establish a robust second step in bind/elute mode
for the purification of a MAb using Capto MMC ImpRes. The medium provides high yield of
monomeric MAb, as well as good clearance of aggregate, HCP, and leached protein A. A model
approach to the choice of running parameters defined by the desired purification performance
was also shown.
For more information on this example, see application note 29-0273-49, “Polishing of monoclonal
antibodies using Capto MMC ImpRes in bind and elute mode.”

2. Effective polishing of domain antibodies (DAbs) using Capto MMC ImpRes

Antibody fragments (e.g., Fab, scFv, and DAbs, Fig 4.57) are becoming an important class of
protein-based products. The structure and smaller size give antibody fragments properties to
suit a range of applications (e.g., easier tissue penetration), and their effective purification is
therefore of great interest for manufacturers of biopharmaceuticals.

92 29-0548-08 AA
Fig 4.57. Structure of a recombinant DAb.

The performance of Capto MMC ImpRes was evaluated in a study where the medium was
used after an initial DAb capture step using Capto L. The recombinant DAb, expressed in E. coli,
included the kappa light chain (VL). Binding and elution conditions for Capto MMC ImpRes
and Capto SP ImpRes were screened using PreDictor 96-well plates using a HTPD approach.
The binding capacity calculated using Assist software revealed information on binding and
elution conditions (Fig 4.58), with the red areas on the contour maps showing optimal binding
conditions while blue areas show optimal elution conditions.
Figure 4.58 shows the DAb binding capacities for Capto MMC ImpRes and Capto SP ImpRes.
Both media showed a large pH range for binding. However, Capto MMC ImpRes had a larger
window of operation with respect to NaCl concentration.

96-well plates: PreDictor Capto MMC ImpRes and PreDictor Capto SP ImpRes
Sample: DAb (Mr 12 900; pI 9.2)
Sample load: 100 mg/mL chromatography medium
Binding buffers: 25 mM sodium citrate (pH 4.1–5.1), sodium phosphate (pH 6.1–7.1), and
Tris-HCl (pH 8.1–9.1); NaCl 0–350 mM

(A) (B)
350 350 SBC (mg/mL)
50
300 300

45
250 250

40
NaCl (mM)
NaCl (mM)

200 200

30
150 150

25
100 100

50 50 20

0 0 15

5.0 6.0 7.0 8.0 9.0 5.0 6.0 7.0 8.0 9.0
pH pH

Fig 4.58. Contour maps showing SBC of DAb on (A) Capto MMC ImpRes and (B) Capto SP ImpRes at different
pH and NaCl concentrations.

29-0548-08 AA 93
Capto MMC ImpRes is effective in removing E. coli protein (ECP) contaminants in the polishing
step of DAb purification processes. To study ECP removal using Capto MMC ImpRes, DAb sample
was applied to Capto MMC ImpRes at a load of 20 mg/mL, pH 5.0. As shown in Figure 4.58, the
salt tolerance at pH 5.0 is high. Three different wash conditions were investigated—0, 100, and
125 mM NaCl. DAb was eluted with 500 mM NaCl and the ECP content in the elution pool and DAb
yield are shown in Figure 4.59. The results showed improved ECP clearance at 125 mM NaCl
without major impact on yield.

Column: Tricorn 5/50, 1 mL

Medium: Capto MMC ImpRes
Sample: Capto L purified DAb
Sample loads: 20 mg/mL chromatography medium
Start buffers: 20 mM sodium citrate, pH 5.0
Wash buffers: Start buffer including 0, 100, and 125 mM NaCl
Elution buffer: Start buffer + 500 mM NaCl
Residence time: 4 min
System: ÄKTA
(A) (B)
97.4
250 232 100 91.8 90.2
200 185 80
ECP content (ppm)

150 60
Yield (%)

100 40

50 47 20

0 0
0 100 125 0 100 125
NaCl in wash (mM) NaCl in wash (mM)

Fig 4.59. Purification of a recombinant DAb using Capto MMC ImpRes. (A) ECP contaminants in the elution
pool and (B) DAb yield using different NaCl concentrations in the binding and wash buffers.

For more information on this example, see data file 29-0356-74, “Capto MMC ImpRes.”

Capto Core 700 application

Purification of influenza A/H1N1 using Capto Core 700
This example shows results from a process stream with the following steps: clarification using
ULTA™ Prime GF microfiltration (MF), capture, and polishing using Capto Core 700.
Madin-Darby canine kidney (MDCK) cells (inoculation concentration of 500 000 cells/mL) were
grown on Cytodex™ 3 microcarriers for 48 h in an Applikon™ Bioreactor (Applikon Biotechnology).
The final cell density was approximately 2 500 000 cells/mL at which point cells were infected
with influenza A/Solomon Islands/3/2006 (H1N1) and harvested at 72 h post infection.
After sample clarification by MF, the virus was captured, and eluted fractions from this step
were applied to an XK column packed with Capto Core 700 for final purification (Fig 4.60).
Because of the robust binding performance of Capto Core 700, equilibration of the medium
was achieved using the buffer used for elution in the capture step. The need for buffer
exchange or dilution between steps was thereby eliminated, contributing to speeding up the
chromatography process. This demonstrates the advantages of the large window of operation
that is enabled by Capto Core 700.

94 29-0548-08 AA
Table 4.25 shows the results in terms of hemagglutinin (HA, e.g., total virus titer) recovery,
infectious virus titer (Tissue Culture Infectious Dose (TCID50), DNA and protein removal at each
step of the process. In this case, good yield of virus HA as well as significant removal of HCP
and DNA were observed. In the capture step, DNA was reduced 2.8 log and proteins 5- to 7-fold.
Capto Core 700 further reduced protein levels by 3- to 5-fold. The infectivity of the virus was
retained throughout the process, as indicated by the titer measured with TCID50 (data not shown).

Column: XK 16/20 packed with 25 mL of Capto Core 700

Sample: Eluted fractions from capture step
Sample load: 8 CV of eluate, 250 cm/h (3 min residence time)
Equilibration/wash: 20 mM sodium phosphate, 500 mM NaCl, pH 7.2
Flow velocity during loading: 250 cm/h
CIP: 1 M NaOH, 27% 1-propanol (total contact time 60 min)
System: ÄKTA

Flowthrough (virus) CIP pH

140

2000 120

100

Conductivity (mS/cm)
1500
80
A280 (mAU)

1000 60

40
500
20

0 0
0 100 200 300 400 500 600 700
Volume (mL)
Fig 4.61. Two-step purification of influenza A/H1N1 virus after MF. After capture of the virus, final purification
was achieved using Capto Core 700.

Table 4.25. Virus HA yield, TCID50, DNA, total protein, and HCP/HA quotient in a purification scheme
incorporating MF, DNA reduction step using Benzonase™ endonuclease, and final chromatography step
using Capto Core 700

HA yield Titer DNA/HA Total protein/ HCP/HA

Step (%) (TCID50/mL) (ng/μg) HA (μg/μg) (μg/μg)
Microfiltration 64 9.7 2672 22.0 32.3
Chromatography—capture 94 4.0 3.1 6.1
Chromatography—polishing (Capto Core 700) 94 9.3 5.0 1.1 1.1

For more information on this example, see application note 29-0003-34, “Purification of
influenza A/H1N1 using Capto Core 700.”

29-0548-08 AA 95
96 29-0548-08 AA
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Appendix 1
Characteristics of multimodal
chromatography media
Table A1.1. Characteristics of Capto adhere

Matrix highly cross-linked agarose

Functional group multimodal strong anion exchanger
Particle size (d50v)1 75 μm
Total ionic capacity 0.09 to 0.12 mmol Cl-/mL medium
Flow velocity2 at least 600 cm/h in a 1 m diameter column with 20 cm bed height at 20°C
using process buffers with the same viscosity as water at < 3 bar (0.3 MPa)
pH stability3
working range 3 to 12
cleaning-in-place (CIP) 2 to 14
Working temperature 4
4°C to 30°C
Chemical stability 5
all commonly used aqueous buffers, 1 M acetic acid, 1 M sodium hydroxide
Avoid oxidizing agents, anionic detergents
1
d50v is the median particle size of the cumulative volume distribution.
2
The capacity for selective removal of some key contaminants may decrease at high flow velocity
3
Working range: pH interval where the medium can be operated without significant change in function.
CIP: pH stability where the medium can be subjected to cleaning- or sanitization-in-place without significant change in
function.
4
Capto adhere can be used under cold-room conditions, but the capacity for some key contaminants may decrease.
5
No significant change in ionic binding capacity and carbon content after 1 wk storage in 1M NaOH at 40°C.

Table A1.2. Characteristics of Capto adhere ImpRes

Matrix highly cross-linked agarose

Functional group multimodal strong anion exchanger
Particle size (d50v)1
36 to 44 μm
Total ionic capacity 0.08 to 0.11 mmol Cl-/mL medium
Maximum flow velocity 2
at least 220 cm/h in a 1 m diameter column with 20 cm bed height at 20°C
using process buffers with the same viscosity as water at < 3 bar (0.3 MPa)
pH stability3
working range 3 to 12
CIP 2 to 14
Working temperature4 4°C to 30°C
Chemical stability 5
all commonly used aqueous buffers, 1 M acetic acid, 1 M sodium hydroxide
Storage 4°C to 30°C in 20% ethanol
Regulatory support Regulatory support file is available
1
d50v is the average particle size of the cumulative volume distribution.
2
The capacity for selective removal of some key contaminants may decrease at high flow velocity.
3
Working range: pH interval where the medium can be operated without significant change in function.
CIP: pH stability where the medium can be subjected to CIP without significant change in function.
4
Capto adhere ImpRes can be used under cold-room conditions, but the capacity for some key contaminants may decrease.
5
No significant change in nitrogen and carbon content after 1 wk storage in 1 M NaOH at 40°C.

100 29-0548-08 AA
Table A1.3. Characteristics of Capto MMC

Matrix highly cross-linked agarose

Functional group multimodal weak cation exchanger
Particle size (d50v)1
75 μm
Total ionic capacity 0.07 to 0.09 mmol H+/mL medium
Flow velocity at least 600 cm/h in a 1 m diameter column with 20 cm bed height at 20°C
using process buffers with the same viscosity as water at < 3 bar (0.3 MPa).
DBC2 > 45 mg BSA/mL medium at 30 mS/cm
pH stability3
short term 2 to 14
long term 2 to 12
Working temperature4 4°C to 30°C
Chemical stability all commonly used aqueous buffers, 1 M acetic acid, 1 M sodium hydroxide5,
8 M urea, 6 M guanidine hydrochloride, and 70% ethanol
Avoid oxidizing agents, cationic detergents
1
d50v is the median particle size of the cumulative volume distribution.
2
DBC at 10% breakthrough as measured at a residence time of 2 min, 300 cm/h in a Tricorn 5/100 column with 10 cm bed
height in 50 mM Na-acetate, pH 4.75, 250 mM NaCl.
3
Short term pH: pH interval that the medium can be subjected to, for cleaning- or sanitization-in-place (accumulated 90 to
300 h at room temperature) without significant change in function.
Long term pH: pH interval where the medium can be operated without significant change in function.
4
Capto MMC can be used under cold-room conditions, but for some proteins the capacity may decrease.
5
No significant change in ionic binding capacity and carbon content after 1 wk storage in 1 M NaOH at 40°C.

Table A1.4. Characteristics of Capto MMC ImpRes

Matrix highly cross-linked agarose

Functional group multimodal weak cation exchanger
Particle size (d50v)1 36 to 44 μm
Ligand density 25 to 39 μmol ligand/mL medium
Maximum flow velocity2 at least 220 cm/h in a 1 m diameter column with 20 cm bed height at 20°C
using process buffers with the same viscosity as water at < 3 bar (0.3 MPa)
pH stability3
working range 3 to 12
CIP 2 to 14
Working temperature 4
4°C to 30°C
Chemical stability5 all commonly used aqueous buffers, 1 M acetic acid, 1 M sodium hydroxide
Storage 4°C to 30°C in 20% ethanol, 0.2 M sodium acetate
Regulatory support Regulatory support file is available.
1
d50v is the average particle size of the cumulative volume distribution.
2
The capacity for selective removal of some key contaminants may decrease at high flow velocity.
3
Working range: pH interval where the medium can be operated without significant change in function.
CIP: pH stability where the medium can be subjected to CIP without significant change in function.
4
Capto MMC ImpRes can be used under cold-room conditions, but the capacity for some key contaminants may decrease.
5
No significant change in nitrogen, sulfur, and carbon content after 1 wk storage in 1 M NaOH at 40°C.

29-0548-08 AA 101
Table A1.5. Characteristics of Capto Core 700

Matrix High flow agarose

Size cut-off of outer layer Mr ≈ 700 000
pKa of protonated octylamine 1
10.65
Functional group in the core CH3CH2CH2CH2CH2CH2CH2CH2NH-
Ionic capacity/mL 40 to 85 μmol Cl-/mL medium
Particle size (d50v)2 85 μm
Maximum operational flow velocity3 500 cm/h in columns with 20 cm bed height at < 2 bar (0.2 MPa)
Binding capacity /mL
4
13 mg ovalbumin/mL medium
pH stability
CIP 2 to 14
working range 3 to 13
Working temperature 4ºC to 30ºC
Chemical stability all commonly used aqueous buffers, 1 M sodium hydroxide5,
6 M guanidine hydrochloride, 30% isopropyl alcohol, and 70% ethanol
Avoid oxidizing agents, anionic detergents
Storage 20% ethanol
1
pKa of protonated octylamine before attachment to the medium. After attachment the pKa may be slightly different.
2
d50v is the average particle size of the cumulative volume distribution.
3
Maximum flow velocity that has been verified for long-term use.
4
DBC was measured at 10% breakthrough with a residence time of 3 min (1.6 mL/min = 200 cm/h) in HiScreen columns. The
mobile phase was 20 mM Tris/HCl with 0.15 M NaCl at pH 7.5.
5
No significant change in ionic capacity and carbon content after 1 wk storage in 1 M NaOH at 40°C.

102 29-0548-08 AA
Appendix 2
Maintenance of media and storage conditions
For best performance of multimodal chromatography media over a long working lifetime,
follow the procedures described below.

Equilibration
After packing, and before a chromatographic run, equilibrate with loading buffer by washing with
at least 5 bed volumes or until the column effluent shows stable conductivity and pH values.

Regeneration/strip
After each step, elute any reversibly bound material with
• Capto adhere/Capto adhere ImpRes: low pH (e.g., 0.1 to 0.5 M acetic acid)
• Capto MMC/Capto MMC ImpRes: high ionic strength solution (e.g., 2 M NaCl in buffer) and at
the same time increase pH to > 9 (e.g., Tris buffer with 2 M NaCl)
Regenerate the medium by washing until the column effluent shows stable conductivity and
pH values.

Cleaning-in-place (CIP)
CIP is a procedure for removal of contaminants such as lipids, endotoxins, nucleic acids, and
precipitated or denatured proteins that remain in the packed column after regeneration.
Regular CIP prevents the build-up of contaminants in the medium and helps to maintain
capacity, flow properties, and general performance. The frequency of CIP depends on the
nature and the condition of the feedstock.
However, for capture steps CIP is normally recommended after each cycle. A specific CIP
protocol should be designed for each process according to the type of contaminants present.
CIP protocols should always be applied in reverse flow because contaminants are usually found
in the first part of the column.
Typically, it is recommended to perform a CIP:
• when an increase in back pressure is seen
• if reduced column performance is observed
• before first-time use or after long-term storage
• between runs when the same column is used for purification of different batches of protein
to prevent possible cross-contamination
• after every run if media is used for capture

CIP protocol—Capto adhere/Capto adhere ImpRes and Capto MMC/Capto MMC ImpRes
The nature of the sample will ultimately determine the final CIP protocol so the CIP procedure
below may require optimization. NaOH concentration, contact time, and frequency are typically
the main parameters to vary during the optimization of the CIP.
The CIP procedure that follows removes common contaminants.

29-0548-08 AA 103
For increased contact time and due to the viscosity of the CIP solutions it is recommended to
use a lower flow rate than during the purification.
1. Wash with at least 2 CV of 2 M NaCl at a pH > 9 (Capto MMC/Capto MMC ImpRes) or
0.5 M acetic acid (Capto adhere/Capto adhere ImpRes).
2. Wash with at least 4 CV of 1 M NaOH.
3. Wash with 5 CV of start buffer or until eluent pH and conductivity have reached the
required values.

CIP protocol—Capto Core 700

Regular CIP is necessary to remove captured contaminants and allow re-use of Capto Core 700
with maintained capacity. Use of 1 M NaOH in 27% 1-propanol is recommended for effective
CIP and sanitization of the medium after every cycle. Due to the strong binding of a wide range
of contaminants to the ligand, an organic solvent will be needed for CIP with most samples.
However, this will be sample dependent, and it may be possible to use CIP solutions without
organic solvents. CIP protocols are dependent on the feed material and running conditions, and
optimization is therefore recommended for the chosen application.

Sanitization
To reduce microbial contamination in the packed column, sanitization using 0.5 to 1 M NaOH
with a contact time of at least 1 h is recommended (Table A2.1). For spore-forming bacteria
(e.g., Bacillus spp.), including 20% ethanol will improve the efficiency of the sanitization
significantly (Table A2.2). Including propanol instead of ethanol will also improve the sanitization
efficiency (Table A2.3).

Table A2.1. Inactivation of microorganisms by NaOH

Organism NaOH (M) Time1 Temp. (ºC)

E. coli 0.01 2h 4 or 22
S. aureus 0.1 1h 4 or 22
C. albicans 0.5 1h 4 or 22
A. niger 0.5 1h 4 or 22
B. subtilis spores 1.0 48 h2 22
B. subtilis spores 1.0 8 d3 4
P. aeruginosa 0.5 1h 22
1
for reduction to below detection limit of < 3 organisms/mL
2
for reduction to below detection limit of 10 organisms/mL
3
for reduction to below detection limit of 100 organisms/mL

Table A2.2. Antimicrobial effect (log10 reduction) of NaOH with the addition of 20% ethanol on Bacillus
subtilis spores

0.5 M NaOH with 0.1 M NaOH with

Time 0.5 M NaOH 20% ethanol 0.1 M NaOH 20% ethanol
24 h 3 log 7 log – –
300 h – – 2 log 4 log

104 29-0548-08 AA
Table A2.3. Sanitization effect of solutions containing 1-propanol or 2-propanol on Bacillus subtilis spores

Remaining B. subtilis (ATCC 6633) spores after

treatment with propanol, Log10 values
Solutions Initial 0h 1h 2h 5h 10 h
0.5 M NaOH, 20% 1-Propanol 5.8 5.1 4.4 3.6 1 0
0.5 M NaOH, 40% 1-Propanol 5.8 4.9 3.2 1.2 0 0
0.5 M NaOH, 20% 2-Propanol 5.8 5.1 4.2 3.4 1 0
0.6 M NaOH, 20% 2-Propanol 5.8 5.2 4 2.4 0.6 0
0.7 M NaOH, 20% 2-Propanol 5.8 5.2 3.7 2.6 0 0
0.8 M NaOH, 20% 2-Propanol 5.8 5.1 3.1 1.3 0 0

Storage
Store used medium in the container at a temperature of 4°C to 30°C. Recommended storage
solutions is:
• 20% ethanol in 0.2 M sodium acetate for Capto MMC ImpRes
• 20% ethanol in water for Capto MMC, Capto adhere, Capto adhere ImpRes, and Capto Core 700
• Do not freeze.

29-0548-08 AA 105
Appendix 3
Use of multimodal media for MAb purification
Capture step
Typical MAb purification processes consist of capture by protein A followed by one or two
polishing steps (Fig A3.1). MabSelect SuRe is based on a protein A derivative with higher alkali
stability compared with recombinant protein A. MabSelect SuRe LX, which is based on the
same alkali-tolerant ligand as MabSelect SuRe, is also optimized for high capacity. MabSelect
SuRe is recommended to be used for feed titers up to 3 g/L whereas MabSelect SuRe LX is most
cost effective for high-titer (> 3 g/L) feedstocks. The polishing steps can include, for example,
ion exchange and multimodal chromatography.

MabSelect SuRe/SuRe LX MabSelect SuRe/SuRe LX

Capto adhere/ImpRes (FT or BE) Capto MMC ImpRes

Capto adhere ImpRes (FT or BE)

Fig A3.1. Strategy for use of multimodal media in two- and three-step processes for MAb purification.
FT = flowthrough; BE = bind/elute.

Two-step processes
In a two-step process, the multimodal AIEX medium Capto adhere or Capto adhere ImpRes
removes the vast majority of impurities (aggregates, HCP, protein A, DNA, and viruses)
remaining after the protein A capture step. In the most cost-effective process, Capto adhere or
Capto adhere ImpRes is used in flowthrough mode where the load can be as high as 250 mg
MAb/mL medium. For some MAbs, the load on Capto adhere ImpRes is higher compared with
Capto adhere at the required purity. The selection between the two media can then be based
on process economy calculations. Capto adhere can, compared with Capto adhere ImpRes,
be operated at higher flow rates. For example, at a column bed height of 20 cm, Capto adhere
can be operated at a flow rate corresponding to a residence time of 2 min, whereas for Capto
adhere ImpRes at the same bed height, the flow rate needs to be decreased, corresponding to
a residence time of > 4 min. However, if the column dimensions can be modified to a shorter,
wider column, Capto adhere ImpRes can advantageously be used at higher flow rates and
lower residence times utilizing the fast mass transfer in the small bead.
To remove MAb fragments and/or charge isoforms, Capto adhere ImpRes operated in bind/
elute mode is recommended. The smaller particle size of Capto adhere ImpRes will result in
improved resolution between target protein and impurities compared with Capto adhere.
Typically, an elution from Capto adhere ImpRes is performed by a decrease of pH, sometimes
combined with change in conductivity.

Three-step processes
A common three-step process begins with a protein A capture step, followed by polishing
steps that employ traditional IEX techniques (e.g., Capto SP ImpRes followed by Capto Q in
flowthrough mode). The CIEX purification step reduces HCP, protein A, fragments, aggregates,
and other MAb isoforms. The AIEX step efficiently reduces the amount of remaining impurities
such as DNA, viruses, HCP, and leached protein A.
106 29-0548-08 AA
An alternative three-step process could contain multimodal media: Capto MMC ImpRes and
Capto adhere or Capto adhere ImpRes. The multimodal functionality of Capto MMC ImpRes
results in different selectivity as well as a larger window of operation in terms of pH and
conductivity compared with traditional ion exchangers. This allows the use of Capto MMC ImpRes
in a variety of process conditions to solve challenging purification problems. The possibility
to bind at higher pH is beneficial for MAbs that are sensitive to low pH. The last polishing step
could include Capto adhere (flowthrough) or Capto adhere ImpRes (flowthrough or bind/elute)
to achieve the final reduction of impurities. The aggregate reduction and viral clearance have
been shown to be efficient and robust under a wide variety of conditions. Other negatively
charged impurities like DNA and endotoxins are also efficiently removed.

29-0548-08 AA 107
Product index1
Capto adhere
Capto adhere 19, 21, 23, 24, 25-28, 29-30, 39, 40, 41-49, 49-64, 65,
71-72, 73, 100, 103-105, 106-107
PreDictor Capto adhere 39, 49, 50, 53, 54, 55, 56, 60
PreDictor RoboColumn Capto adhere 39
HiTrap Capto adhere 39, 52, 58, 60
HiScreen Capto adhere 39, 49-50, 52, 57, 60
ReadyToProcess Capto adhere 39, 63

Capto adhere ImpRes

Capto adhere ImpRes 23, 24, 28-30, 39, 40, 65-74, 100, 103-105, 106-107
Predictor Capto adhere ImpRes 39
PreDictor RoboColumn Capto adhere ImpRes 39
HiTrap Capto adhere ImpRes 39
HiScreen Capto adhere ImpRes 39

Capto MMC
Capto MMC 19, 23, 24, 30-34, 34-35, 39, 40, 74-83, 101, 103-105,
106-107
PreDictor Capto MMC 33, 39, 74-82
PreDictor RoboColumn Capto MMC 39
HiTrap Capto MMC 39
HiScreen Capto MMC 39, 74-82
ReadyToProcess Capto MMC 39

Capto MMC ImpRes

Capto MMC ImpRes 23, 24, 34-35, 39, 40, 84-94, 101, 103-105, 106-107
PreDictor Capto MMC ImpRes 39, 84, 93
PreDictor RoboColumn Capto MMC ImpRes 39
HiTrap Capto MMC ImpRes 39
HiScreen Capto MMC ImpRes 39

Capto Core 700

Capto Core 700 18, 19, 24, 35-37, 39, 40, 94-95, 102, 104-105
HiTrap Capto Core 700 39
HiScreen Capto Core 700 39

1
Bold page numbers indicate main entry for product.

108 29-0548-08 AA
Related literature

Code number

Data files
Capto adhere 28-9078-88
Capto adhere ImpRes 29-0344-97
Capto MMC 11-0035-45
Capto MMC ImpRes 29-0356-74
Capto Core 700 28-9983-07
PreDictor 96-well filter plates and Assist software 28-9258-39
PreDictor RoboColumn 28-9886-34
MabSelect SuRe 11-0011-65
MabSelect SuRe LX 28-9870-62
HiScale columns 28-9755-23
HiScreen prepacked columns 28-9305-81
ReadyToProcess columns 28-9159-87
Tricorn empty high performance columns 18-1147-36
AxiChrom columns 28-9290-41
BPG columns 18-1115-23

Application notes
A flexible antibody purification process based on ReadyToProcess products 28-9403-48
High-throughput screening and process development for capture of 28-9966-22
recombinant pro-insulin from E. coli
High-throughput screening and optimization of a multimodal polishing step 28-9509-60
in a monoclonal antibody purification process
High-throughput screening for elution conditions on Capto MMC using PreDictor plates 28-9277-90
Methods for packing Capto MMC in production scale columns 28-9259-33
Optimization of loading conditions on Capto adhere using design of experiments 28-9078-89
Optimizing elution conditions on Capto MMC using design of experiments 11-0035-48
Polishing of monoclonal antibodies using Capto adhere ImpRes in bind and elute mode 29-0273-38
Polishing of monoclonal antibodies using Capto MMC ImpRes in bind and elute mode 29-0373-49
Purification of influenza A/H1N1 using Capto Core 700 29-0003-34
Purification of a monoclonal antibody using ReadyToProcess columns 28-9198-56
Rapid process development for purification of a MAb using ÄKTA avant 25 28-9573-47
Scale-up of a downstream monoclonal antibody purification process using 28-9403-49
HiScreen and AxiChrom columns
Selective removal of aggregates with Capto adhere 28-9078-93
Two-step purification of monoclonal IgG1 from CHO cell culture supernatant 28-9078-92

Selection guides
Comparison guide for process development tools 28-9951-64
Prepacked chromatography columns for ÄKTA systems 28-9317-78

29-0548-08 AA 109
Ordering information

Product Quantity Code number

Capto adhere 1
25 mL 17-5444-10
100 mL 17-5444-01
1L 17-5444-03
5L 17-5444-04
10 L 17-5444-05
60 L 17-5444-60
PreDictor Capto adhere, 6 μL 4 × 96-well filter plates 28-9258-17
PreDictor Capto adhere, 20 μL 4 × 96-well filter plates 28-9258-18
PreDictor Capto adhere, 50 μL 4 × 96-well filter plates 28-9258-19
PreDictor Capto adhere Isotherm 4 × 96-well filter plates 28-9432-82
PreDictor RoboColumn Capto adhere, 200 μL one row of eight columns 28-9860-85
PreDictor RoboColumn Capto adhere, 600 μL one row of eight columns 28-9861-79
HiTrap Capto adhere 5 × 1 mL 28-4058-44
5 × 5 mL 28-4058-46
HiScreen Capto adhere 1 × 4.7 mL 28-9269-81
ReadyToProcess Capto adhere 1L 28-9511-09
2.5 L 28-9017-14
10 L 28-9017-15
20 L 28-9017-16
Capto adhere ImpRes1 25 mL 17-3715-01
100 mL 17-3715-02
1L 17-3715-03
5L 17-3715-04
10 L 17-3715-05
Predictor Capto adhere ImpRes, 6 μL 4 × 96-well filter plates 17-3715-30
PreDictor Capto adhere ImpRes, 20 μL 4 × 96-well filter plates 17-3715-31
PreDictor RoboColumn Capto adhere ImpRes, 200 μL one row of eight columns 17-3715-40
PreDictor RoboColumn Capto adhere ImpRes, 600 μL one row of eight columns 17-3715-41
HiTrap Capto adhere ImpRes 5 × 1 mL 17-3715-10
HiScreen Capto adhere ImpRes 1 × 4.7 mL 17-3715-20
Capto MMC 1
25 mL 17-5317-10
100 mL 17-5317-02
1L 17-5317-03
5L 17-5317-04
10 L 17-5317-05
60 L 17-5317-60
PreDictor Capto MMC, 6 μL 4 × 96-well filter plates 28-9258-14
PreDictor Capto MMC, 20 μL 4 × 96-well filter plates 28-9258-15
PreDictor Capto MMC, 50 μL 4 × 96-well filter plates 28-9258-16
PreDictor Capto MMC Isotherm 4 × 96-well filter plates 28-9432-81
PreDictor RoboColumn Capto MMC, 200 μL one row of eight columns 28-9860-84
PreDictor RoboColumn Capto MMC, 600 μL one row of eight columns 28-9861-78
1
All bulk media products are supplied in suspension in 20% ethanol. For additional information, including data file, please
contact your local GE Healthcare representative.

110 29-0548-08 AA
Product Quantity Code number
HiTrap Capto MMC 5 × 1 mL 11-0032-73
5 × 5 mL 11-0032-75
HiScreen Capto MMC 1 × 4.7 mL 28-9269-80
ReadyToProcess Capto MMC 1L 28-9511-18
2.5 L 28-9291-20
10 L 28-9291-21
20 L 28-9291-22
Capto MMC ImpRes 1
25 mL 17-3716-01
100 mL 17-3716-02
1L 17-3716-03
5L 17-3716-04
10 L 17-3716-05
PreDictor Capto MMC ImpRes, 6 μL 4 × 96-well filter plates 17-3716-30
PreDictor Capto MMC ImpRes, 20 μL 4 × 96-well filter plates 17-3716-31
PreDictor RoboColumn Capto MMC ImpRes, 200 μL one row of 8 columns 17-3716-40
PreDictor RoboColumn Capto MMC ImpRes, 600 μL one row of 8 columns 17-3716-41
HiTrap Capto MMC ImpRes 5 × 1 mL 17-3716-10
HiScreen Capto MMC ImpRes 1 × 4.7 mL 17-3716-20
Capto Core 7001 25 mL 17-5481-01
100 mL 17-5481-02
1L 17-5481-03
5L 17-5481-04
HiTrap Capto Core 700 5 × 1 mL 17-5481-51
HiScreen Capto Core 700 1 × 4.7 mL 17-5481-15

Related products—columns/plates, software

PreDictor MabSelect SuRe, 20 μL 4 × 96-well filter plates 28-9258-24
PreDictor MabSelect SuRe, 6 μL 4 × 96-well filter plates 28-9258-23
HiTrap MabSelect SuRe 5 × 1 mL 11-0034-93
5 × 5 mL 11-0034-95
HiScreen MabSelect SuRe 1 × 4.7 mL 28-9269-77
ReadyToProcess MabSelect SuRe 1L 28-9511-10
2.5 L 28-9017-17
10 L 28-9017-18
20 L 28-9017-19
PreDictor MabSelect SuRe LX, 6 μl 4 × 96-well plates 17-5474-30
PreDictor MabSelect SuRe LX, 20 μl 4 × 96-well plates 17-5474-31
PreDictor MabSelect SuRe LX, 50 μl 4 × 96-well plates 17-5474-32
HiScreen MabSelect SuRe LX 1 × 4.7 ml 17-5474-15
ReadyToProcess MabSelect SuRe LX 1L 29-0269-27
HiPrep 26/10 Desalting 1 × 53 mL 17-5087-01
4 × 53 mL 17-5087-02
1
All bulk media products are supplied in suspension in 20% ethanol. For additional information, including data file, please
contact your local GE Healthcare representative.

29-0548-08 AA 111
Product Quantity Code number
Tricorn 5/100 column 1 28-4064-10
Tricorn 10/100 column 1 28-4064-15
Tricorn 5/20 column 1 28-4064-08
Tricorn 5/50 column 1 28-4064-09
Tricorn 10/600 column 1 28-4064-19
HiScale 16/20 1 28-9644-41
HiScale 16/40 1 28-9644-24
HiScale 26/20 1 28-9645-14
HiScale 26/40 1 28-9645-13
HiScale 50/20 1 28-9644-45
HiScale 50/40 1 28-9644-44
Assist 1.2 Software package 1 28-9969-17

112 29-0548-08 AA
 GE Healthcare
Life Sciences

Multimodal Chromatography – Handbook

GE, imagination at work, and GE monogram are trademarks of General Electric Company.
ÄKTA, ÄKTAmicro, ÄKTApilot, AxiChrom, BioProcess, Capto, Chromaflow, Cytodex,
HiTrap, HiPrep, HiScale, HiScreen, HiTrap, MabSelect, MabSelect SuRe, Mono Q,
PhastGel, PreDictor, ReadyToProcess, Sepharose, Superdex, Tricorn, ULTA, UNICORN,
and Drop design are trademarks of GE Healthcare companies.
UNICORN software: Any use of UNICORN software is subject to GE Healthcare Standard
Software End-User License Agreement for Life Sciences Software Products. A copy of
this Standard Software End-User License Agreement is available on request.
Capto Q chromatography media: Separating viral particles with Capto Q
chromatography media may require a license under United States Patent 6,537,793 B2
and foreign equivalents owned by Centelion SAS.

Multimodal
Applikon is a trademark of Applikon Biotechnology BV. Benzonase is a trademark of
Merck KGaA. Breox and Pluronic are trademarks of BASF Corp. Coomassie and Tween
are trademarks of Imperial Chemical Industries, PLC. Desmophen is a trademark of
the Bayer Group. Freedom EVO and Tecan are trademarks of Tecan Group Ltd. MODDE
is a trademark of Umetrics AB. RoboColumn is a trademark of Atoll GmbH. Struktol is a
For local office contact information, trademark of Schill + Seilacher “Struktol” GmbH. Triton is a trademark of Dow Chemical Co.

Chromatography
© 2013 General Electric Company – All rights reserved.
please visit [Link]/contact First published Nov. 2013.
All goods and services are sold subject to the terms and conditions of sale of the
company within GE Healthcare which supplies them. A copy of these terms and
[Link]/bioprocess conditions is available on request. Contact your local GE Healthcare representative
for the most current information.

GE Healthcare Bio-Sciences AB
GE Healthcare UK Limited
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Handbook
Little Chalfont
Björkgatan 30 Buckinghamshire, HP7 9NA
751 84 Uppsala UK
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Piscataway, NJ 08855-1327
USA
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imagination at work Sanken Bldg., 3-25-1, Hyakunincho
Shinjuku-ku, Tokyo 169-0073
imagination at work
Japan
imagination at work
29-0548-08 AA 11/2013