Lecture 3:

Overview of Upstream
Processing
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Overview of Bioprocessing
1. Upstream 2. Harvest 3. Downstream

4. Fill Finish
Topics

Cell Culture and Cell Line Selection

Cell Banking

Cell Culture Facility

Subculture

Cell Culture Growth and Metabolism

Cell Culture and Cell Line Selection
Cell Culture

What does biologics refer to?

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Cell Culture

Which platforms can be used to produce biologics?

A. Bacterial Cells
B. Yeast Cells
C. Mammalian Cells
D. Plant Cells
E. All of the above
Why Do We Culture Cells?
Living cells are the chosen platform to Mammalian Cell
manufacture our drug substance.

Cell culture is the growth of cells

outside the body – in vitro.

More cells = More product.

Healthier cells = Better quality product.

Protein
Cell Culture & Scale up

Wright et al., 2015,

Bioprocessing International
Cell Culture
We can culture cells from many different organisms, including:

Bacterial Cell Yeast Cell

Ribosomes
Pilus Capsule Cell Wall Mitochondria
Plasma Membrane Cell Wall
Nucleoid (DNA) Cell
Cytoplasm Membrane
Vacuole
Cytoplasm
Ribosomes Nucleus
Flagellum
Cell Culture We can culture cells from many
different organisms, including:
Plant Cell Cell Wall
Cell Membrane
Golgi vesicles Golgi Apparatus
Ribosome
Smooth E R Chloroplast
(no ribosomes)
Nucleolus Vacuole Membrane
Nucleus
Rough E R Raphide Crystal
(endoplasmic reticulum)

Druse Crystal
Large Central Vacuole
Amyloplast Mitrochondrion
(starch grain)
Cytoplasm
Cell Culture Mammalian Cell
Nucleus
Nuclear Membrane
Genetic
Centriole Material (DNA)
Lysosome Nucleolus

Endoplasmic
We can culture cells Reticulum (ER)
from many different Ribosomes
organisms, including:

Plasma
Golgi Apparatus Membrane

Cytoplasm
Mitochondrion
Mammalian Cell Culture

What is the mammalian cell line most commonly used for

biopharmaceutical manufacturing?

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Cell Line Selection
Before manufacturing ever begins, some questions need to be asked;

Simple recombinant protein Simple recombinant protein

Product Growth &
Type? Monoclonal antibody Productivity Monoclonal antibody
Fusion Protein Fusion Protein

Potential for endogenous viral

Stability of Expression Levels
Genetic Safety contamination
Stability of transfection
Stability? Issues? Use of animal products in media
Long-term genetic stability
(potential contamination)
Why Mammalian Cells?
Characteristic Mammalian Cells Bacteria/Yeast

Correct protein folding ✓ ❌

Protein secretion ✓ ❌

PTMs ✓ ❌

Large, complex protein production ✓ ❌

Good regulatory track record ✓ ✓

Fast growth and cheap media ❌ ✓

Simple purification protocols ❌ ✓

Animal component free media ❌✓ ✓

Simple to characterize ❌ ✓
Development of an Industrial Bioprocess
Product required

Generation of producing cell line (GMO)

Cell Line
Screening/selection of high producers
Improvement
Formulation of media

Optimisation of media Product Purification

Preclinical Trials
Small-scale culture: choice of FDA Approval
Reactor/type culture Clinical Trials
No Marketing
Process control

Scale-up <100L
Are yields/ Yes
Process kinetics: productivity and yield productivity
Okay?
Cell Banking
Why Do We Freeze Cells?

Genetically modified cells contain the

information needed to make our drug

Growing cells indefinitely leads to mutations:

- protein or product is safe/efficacious?

Freezing stock confirms genetic similarity

Cell Banking
Stored in freezers or liquid nitrogen tanks/dewars.

Ultra-low temperatures - inhibit any biological activity

Temperature of:
liquid nitrogen -196°C
liquid nitrogen vapour up to -130°C
Cells can be a company’s most important asset!
Cell Freezing and Thawing
Role of Cryoprotectants
Establishing a Cell Bank
Gene for
Candidate clones
therapeutic Small Scale
protein Cell Pool
Studies

Transfection Selection

Host cell Master cell

selected

x 500 x 200 Culture

Expanded
Large Scale Working cell Master cell
Production bank (WCB) bank (MCB)
Walsh, Gary. Pharmaceutical biotechnology: concepts and applications. John Wiley & Sons, 2007.
Cell Culture Facility
Growth Kinetics

What sources of contamination can you think of?

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Aseptic Processing
“Handling sterile materials in a controlled environment, in which the air
supply, facility, materials, equipment and personnel are regulated to control
microbial and particulate contamination to acceptable levels” -PDA

Source of Contamination Preventative Measures

Gloves, Lab Coat, Scrubs,

Operator
Face Mask
Air quality control, restricted
Atmosphere
access to areas, EM
Work Surfaces and
Sterilization or Sanitization
Equipment

Sterile consumables (plastic,

Raw Materials
glass) and liquid materials
Gowning Requirements
Grade ISO Category Key Points
No sinks/drains
ISO 5 (rest &
A operational)
High-risk
operations

ISO 5 (rest) No sinks/drains

B ISO 7 (operational) Aseptic preparation

ISO 7 (rest) Prep work/to be

C ISO 8 (operational) sterilized

D ISO 8 (rest)

Source: Mecart Cleanrooms Source: Skillpad

Good Aseptic Technique
Biological Safety Cabinet
Know the source and direction of
laminar air flow

Clean, working and dirty zones

Minimize entries into the hood – buddy

system

Be aware of the location of your hands

at all times & sanitize often

Move slowly and deliberately – Disrupted

isokinetic
Avoid crossing over any open Moving too fast will disrupt the laminar air
containers flow and create turbulent air!
Healthy Cell Culture

Healthy cells grow and

divide

After 3 days cells have grown

to confluence i.e. filled the
available growth space
Contaminated Culture

Contaminated or unhealthy cells

will not grow as
desired/expected

Instead, contaminants will quickly

outnumber the cells and use up
available nutrients
Contaminated Culture

Source: Eppendorf
Subculture
Cell Counting - Manual
Mammalian cells have a cell membrane Live and dead cells under the microscope

If cells are healthy and membrane is intact,

trypan blue cannot enter cells

If cells are dying/dead, trypan blue enters

cells and stains them blue

We use this staining to count live and dead

cells

Trypan blue cell exclusion; live-dead stain

 Cell Counting
Automated
Automated cell counters are accurate,
reproducible and give further information on
cell size, morphology and viability.
Number of Cells

Stand-alone/Offline Instrumentation

Image analysis method

Measure cell concentration, size and viability

CytoSmart Exact FL
Cell Size (µm) (Axion Biosystems)
Cell Counting - Automated
In-line and integrated analysis
Machine automatically analyses live/dead cells and presents data

Cedex® (Roche) Cell culture image analysis Vi-CELL® (Beckman Coulter)

Subculture of Cells
Cell culture aims to keep cells:
Healthy
At maximal rate of division

Cells will grow until:

All nutrients have been depleted
Culture has reached maximal cell density

Cells must be subcultured/passaged to a

healthier environment before growth slows. In the correct environment
cells will grow and divide
Cell Culture Growth Equipment
The type of cell must be considered when designing cell culture equipment

Adherent cells (anchorage dependent):

require a surface to attach to
focus is on increased surface area

Suspension cells (anchorage independent):

grow freely suspended in the medium
focus is on increased volume
Adherent Cell Growth Equipment

Focus is on increased surface area to volume ratio

Tissue Culture Flasks Cell Factories Roller Bottles Microcarriers

Suspension Cell Growth Equipment

Focus is on increased volume

(Erlenmeyer) Shake Flasks Spinner Flasks

Bioreactors

Bench Top Autoclavable Disposable Stainless Steel Stirred Tank

Single Use
Equipment

Sartorius and Repligen partner to introduce next-generation perfusion-enabled bioreactors. (n.d.). Retrieved May
10, 2022, from [Link]
Cell Culture Growth and
Metabolism
Cell Culture Scale-Up

Grow cells in a controlled environment until we reach the

desired cell concentration at the appropriate volume

Volume/Concentration
Cell Culture Media Components
Carbon/ Energy source • Carbohydrate (usually glucose)

• Ammonia and amino acids (Amino acid and

Nitrogen source
protein synthesis)

Buffers • For pH control (often bicarbonate buffer)

Vitamins • Riboflavin, thiamine, ascorbic acid etc.

Growth Factors • Signalling hormones (IGF-1, IL-6, G-CSF etc.)

Inorganic salts/ Trace • Sodium chloride (osmolality)

metals • Iron, zinc, magnesium, potassium, calcium etc.
Media Optimization Culture
Conditions
• Cell type
• Density

Medium optimization depends on many factors:

• Batch
Feeding
Pattern • Fed batch
• Perfusion

Preparation • Powder
Format • Liquid

• Limitations of
Downstream purification
Purification
technology

• Yield
Business
Factors • Equipment
• Cost
Factors Affecting Growth
Temperature • Every cell line will have an optimal growth temperature

pH • Fluctuations in pH may affect growth rate

Osmolality • May damage plasma membrane or disrupt the water content of the cell

• Insufficient oxygen supply may become the rate limiting factor affecting
Dissolved Oxygen
growth rate

• Sufficient nutrient levels required for growth

Nutrients/Metabolites
• High levels of metabolites may negatively affect growth

Contamination • Contamination may affect cell growth and induce cell death
Cell Growth
The upstream
process needs to be
controlled to keep STATIONARY
cells in the Log phase
until max. cell DEATH
concentration is
reached

LOG
Log [X]

When max cell conc.

is reached, we need
to control the culture
in the stationary
phase until max.
LAG protein is produced.

Cell “Acclimatize” Optimum Conditions Limitations to Growth Cell Death

Time
Growth Kinetics

What parameters might cause a shock that

affects growth?

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Summary
Appropriate facility Process control (pH,
design and culture temp, O2, mixing, etc.)
materials used optimized

Optimal cell type Media

selected optimization
Consistently
high quality
drug
substance
manufacture
Topics Recap

Cell Culture and Cell Line Selection

Cell Banking
Cell Culture Facility
Subculture
Cell Culture Growth and Metabolism
Questions?
References
Sartorius and Repligen partner to introduce next-generation perfusion-enabled bioreactors. (n.d.). Retrieved May 10, 2022, from [Link]
[Link]
Walsh, Gary. Pharmaceutical biotechnology: concepts and applications. John Wiley & Sons, 2007.
Wikimedia Foundation. (2022, May 6). Glucose. Wikipedia. Retrieved May 10, 2022, from [Link]
Appendix