Chapter 8

Microbiological
Control

©2016 Montgomery County Community College

Objectives
This chapter provides an overview of microbiological control in the biomanufacturing industry.
After completing this chapter the student will be able to:
 Explain why microbiological control is important in a biomanufacturing facility and
provide a number of examples as to how it is achieved and maintained.

 Describe the various sources of microbial contamination within a biomanufacturing

facility/process and name specific microbial contaminants and their possible sources.

 Explain the different microbiological cleanliness standards required for the manufacture
of biopharmaceutical drug substances and drug products.

 Define aseptic processing and provide examples of aseptic processing practices.

 Identify measures taken in controlled and classified environments within cleanrooms to

prevent microbial contamination.

 Describe the components of an effective environmental monitoring program along with

specific environmental monitoring testing methods.

 Explain the importance of information derived from environmental monitoring and

describe how this information is utilized in investigations.

 List the quality control practices that are essential in the Microbiology QC Laboratory.

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Terms
Action level: an established microbial or airborne particulate level that, when exceeded,
should trigger appropriate investigation and corrective action based on that investigation
Alert level: an established microbial or airborne particle level giving early warning of
potential drift from normal operating conditions and triggering appropriate scrutiny and
follow-up to address the potential problem. Alert levels are always lower than action levels.
Aseptic: the absence of pathogenic (disease-causing) microorganisms
Aseptic processing: biomanufacturing methods for those axenic products that cannot be
subjected to terminal sterilization. Typically utilized for those products that are heat-labile
(products that are damaged by heat-sterilization methods)
Aseptic techniques: techniques that prevent contamination by unwanted microorganisms.
Used not only in biomanufacturing methods but also with medical procedures
Cleanroom: a room or interconnected rooms maintained and controlled to prevent particle
and microbiological contamination of drug products. Cleanrooms are assigned and
reproducibly meet an appropriate air cleanliness classification.
Contamination: the presence of any unwanted substance that may affect the purity, safety,
identity, or strength of a drug product
Disinfection: the elimination of most recognized disease-causing or harmful microorganisms
but not necessarily all microbial forms. It is a less lethal process than sterilization
Pyrogen: A substance which causes fever when present in the blood of an organism.
Sterile: the complete absence of viable (living) microorganisms
Sanitization: the general reduction of the number of microorganisms on a surface
Sterilization: the act or process, either physical or chemical, that destroys, inactivates, or
eliminates all forms of life, including bacterial endospores (the most resistant form of
microorganism)
Terminal sterilization: the application of a lethal agent to sealed, finished drug products for
the purpose of achieving sterility

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Foundations of Microbiology
Microorganisms are ubiquitous. They are in the air we breathe, food we eat, water we drink,
and surfaces we touch. They range from simple nucleic acid-free entities, termed prions (first
recognized approximately 20 years ago), to complex eukaryotic cells such as yeast that have
been known since Leeuwenhoek invented the microscope in the 15th Century.
Microbes are only a problem when their presence results in unwanted effects, such as causing
infections or contaminating drug products or intermediates. Controlling microbes when
manufacturing products that use organic materials is a demanding challenge that has existed
for centuries.
In the 19th century French scientist Louis Pasteur helped found the science of microbiology,
when he studied the causes behind the spoilage of wine. Pasteur examined properly aged wine
under a microscope and noticed the presence of yeast cells. He then discovered that soured
wine contained bacterial cells that were producing lactic acid. His findings led him to
recommend that vintners (wine producers) heat the wine, which would kill the lactic-acid-
producing bacteria. His discovery helped save the French wine industry, which was crucial to
the country's economy. This led directly to the heat-treating process named for him,
pasteurization, which is used on food products (e.g., dairy products) worldwide today.
There are many other similar instances in which food is adversely affected by inadequately
controlled microbes. Many methods traditionally used to preserve foods fundamentally rely on
creating an environment that is inhospitable to or kills microbes, such as smoking meats or
pasteurization.
As with food production, microbiological control is a key issue in pharmaceutical
manufacturing. This is particularly true in the case of biopharmaceuticals and bioprocessing,
which use various organic materials to create a range of products. Microbiological control is
vital for two main reasons:
1. The majority of biopharmaceutical medicines are designed for parenteral administration
(i.e. they are administered to the patient by injections of various types) and must be
sterile and free of significant amounts of pyrogens such as endotoxin to prevent
infections in the recipient patients
2. Biopharmaceutical drug substances are generally large, complex proteins that are
susceptible to degradation, mediated by enzymes produced by contaminating microbes.
This chapter will describe microbial contaminants, how they might enter into the production
cycle, and their impacts. It will also cover the controls that are established to prevent microbial
contamination of products.

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Bacteria, Fungi, and Mycoplasma
Bacteria and fungi are a heterogeneous group of organisms that include both simple non-
nucleated prokaryotic cell types (bacteria) and nucleated eukaryotic cells (fungi, including
yeasts and molds). All of these are ubiquitous, as they are present in air, water, soil, and even in
other living organisms, including humans. In fact our own body cells are outnumbered by the
bacteria that we harbor by a factor of a hundredfold!
Life as we know it depends on the activities of bacteria. They help certain animals digest food
and convert it to energy. They help produce oxygen by working in symbiotic relationships with
plants. They break down dead plants and animals which contributes to the cycle of life. They
even help in making foods like bread, yogurt, and cheese, and beverages like wine and beer.
The numbers of bacteria and fungi are almost incomprehensible. It has been estimated that
there are 4–6 X 1030 total prokaryotic cells on Earth and that every person has approximately 3
X108 prokaryotic cells on their skin and 7 X 1013 cells in their intestines.
This group of microbes can impact biopharmaceutical processing in various ways and at
different levels, from the cell culture to the final dosage form. With cell cultures, the process
objective is to maintain an axenic or monoseptic-type culture. This means that only the
engineered production cells of interest are present and that extraneous contaminating
microbes are excluded. This requires the stringent control of operating conditions and
equipment. Despite these controls, however, batches of product are still lost due to
contamination. This remains an ongoing problem in the industry. Typically, when a mammalian
cell culture becomes contaminated by bacteria or other cells, the contaminating microbes can
overgrow the production of the mammalian cells since the former can grow much more quickly
than the mammalian cells. This results in the microbes out-competing the mammalian cells for
nutrients. This growth is readily apparent through atypical batch parameters like visual
appearance (turbidity), pH, and DO (Dissolved Oxygen). Microbial contamination of a microbial
cell reactor is more difficult to detect, but no less of an issue.
A common type of contamination at the cell culture level is caused by Mycoplasma, the
smallest self-replicating prokaryote. These lack a cell wall, as well as the ability to synthesize
one. These organisms depend on their host cells for cholesterol and as such exist as parasites or
commensally with their hosts. They are 0.2–0.3 micron in diameter and can be observed as
filamentous or coccal forms. And though over 160 species have been identified to date,
approximately 90 percent of all cell culture contaminations are caused by only five Mycoplasma
species: M. hyorhinis, M. arginini, M. orale, M. fermentans, and Acholeplasma laidlawii.
Mycoplasma can grow to very high concentrations in mammalian cell cultures—to levels near
107–108 organisms/ml. However, it remains unobservable by regular light microscopy, and
generally requires fluorescence staining of the culture to observe. While late-stage Mycoplasma
contamination can cause cell culture media to become acidic, there are usually no overt signs
that cultures are contaminated. Mycoplasma can cause changes in growth characteristics,
membrane antigenicity, and mammalian cell metabolism. It can also produce chromosomal
aberrations, disrupt nucleic acid synthesis, alter transfection rates, and induce virus
susceptibility.

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Mycoplasma can either directly or indirectly contribute to human disease, representing
significant safety and regulatory concerns. Therefore, testing for Mycoplasma in manufacturing
cell substrates and the culture media is essential. The main sources of Mycoplasma
contamination likely arise from the production cell line, the raw materials used in the process,
the production personnel, and/or the environment. Specific methodologies are required to
examine cell cultures for Mycoplasma infection as defined in the European Pharmacopoeia
([Link].), section 2.6.7. Traditional culture methods can take up to a month or more to produce
results. Recently PCR based techniques have allowed for near real-time detection.
The cell culture process is considered a closed system – production cells are introduced into a
closed vessel containing a nutrient medium designed to support the growth of these cells. This
culture is incubated for the appropriate time, typically days (for bacteria), weeks, or even
months (for mammalian cells), before the cells or the culture medium is harvested. The process
of extraction and purification then begins. Typically purification is termed an open process and
there are opportunities for environmental microbes to enter the process. In addition, the
nature of the equipment and materials used to purify a product, such as filter membranes and
chromatography resins, may make sterilization of that equipment impossible. These
opportunities for contamination must be understood, monitored, and controlled. The typical
microbes of concern at the purification stage are bacteria and molds as the lack of cells means
there is nothing to serve as a host for viruses or Mycoplasma so these organisms are unable to
proliferate in the equipment and systems without them. Bacteria and fungi are typically carried
by people and materials and they thrive in moist environments. The concerns about bacteria
and molds in open processes are:
1. The simple control of their numbers. While it can be anticipated that they may be
present, it is important that the environmental conditions do not promote their
proliferation where they can overwhelm the capability of downstream filtration
processes designed to remove them.
2. Bacterial products or structures such as endotoxin- these can be dangerous to the
patient who ultimately receives that product
Concerning the former, certain bacteria and molds secrete proteolytic enzymes into the growth
medium, potentially degrading the therapeutic protein. Regarding the latter, Gram-negative
bacteria release a component of their cell wall termed endotoxin that can be highly toxic if
introduced into patients. It is one example of a pyrogen. Pyrogens can cause fever and other
immunologic side effects. Consequently, endotoxin levels must be carefully minimized during
production.
The majority of biopharmaceuticals are delivered as parenteral medicines. These deliberately
bypass the body’s external physical barriers against infection (e.g., skin, mucous membranes,
digestive system) and, as such, must be delivered in a sterile state (free from any viable life
forms) to the patient. Microorganisms known to cause infectious diseases in humans are hard
to distinguish from those customarily thought to be benign. Once a body's external defense
barriers have been breached, virtually any microorganism has the potential to proliferate and
cause infection or other unwanted side effects. This is especially true in immunosuppressed or
immunodeficient patients. For example, the bacterial species Citrobacter freundii, Enterobacter

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agglomerans, Enterobacter cloacae, and Klebsiella aerogenes were responsible for a spate of
fatalities in the 1970s from infusion fluids that were thought to be sterile. These four species,
found living in commensal association with healthy humans, were thought to be no more than
“opportunistic” pathogens. As recently as 2002, a 77-year old woman died from fungal
meningitis after receiving a spinal injection, which subsequently tested positive for a rare
fungus species present in the manufacturing plant.
These cases illustrate that achieving sterility in a final product is of vital importance. This effort
is complicated, however, by the fact that most biopharmaceutical molecules cannot be
subjected to the most common method of generating a sterile product—autoclaving. During
the autoclaving process, a finished product in its final container is sterilized using heat. this is
referred to as terminal sterilization. Biopharmaceuticals, however, are typically very heat-labile
molecules, meaning that heat is likely to change them and alter their function. Therefore, they
must be sterilized using a sterilizing filtration process applied to the bulk formulation, after
which they are placed into pre-sterilized individual containers under aseptic conditions and
capped with pre-sterilized closures. Though necessary for completely excluding microbes, the
process of aseptic manufacture is highly complex and demanding.
Viruses
Viruses are small, obligate, intracellular infectious agents that can cause a wide variety of
diseases in humans, animals, and plants. In their simplest form, viruses consist of genetic
information in the form of DNA or RNA packaged within a surrounding protein coat. They can
only replicate inside another organism's living cells and therefore cannot be categorized as
living.
Viruses were first identified over one hundred years ago by two scientists working
independently. Shortly after Pasteur's work with bacteria, the Russian scientist Dmitri Ivanovski
was researching the cause of tobacco mosaic disease. A few years later, the Dutch botanist
Martinus Beijerinck also studied the same plant disease. Beijerinck named the agent he
discovered, which was smaller than bacteria, a virus—based on the Latin word for poison.
Beijerinck is now considered the father of virology(a specialization of microbiology).
In the medical field there have been numerous cases over the years in which medicines have
been contaminated by viruses—unintentionally infecting patients. The best known example
was in the 1980s, when as a result of the development of the AIDS epidemic, the HIV virus that
is associated with AIDS began appearing in blood products. Viral infections were devastating to
many patients suffering from hemophilia—an inherited disease that impairs the body's ability
to control blood clotting (due to the absence of certain clotting proteins) and results in
uncontrollable bleeding when blood vessels are damaged (e.g., from a cut, wound, etc.). The
treatment of this disease was by the injection of the missing blood clotting proteins, effectively
alleviating the disease symptoms and batches of these therapeutic clotting proteins were
produced from blood donations. Before the HIV virus had been identified, however, no reliable
diagnostic tests were available to detect it. As a result, donated blood could not be screened for
the virus. Furthermore, blood donations were often obtained from individuals who were paid
to donate and these donors often led unhealthy lifestyles that made them more likely to be

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infected. If a single blood donor was infected with the HIV virus, then the entire batch of
donated blood samples could be contaminated.
As a result of the above incident, with more than ten thousand hemophiliacs contracting HIV as
a result of the tainted blood supply, more stringent controls were initiated. Screening tools for
example, are now used to accept or reject blood donors, and diagnostic tests have been
developed and are used to detect the virus in donated blood. These tools and tests are now
routinely applied to all blood donations in developed countries and the risk of HIV transmission
via blood and blood products has been largely eliminated.
In a case from 2010, an important childhood vaccine for the prevention of a deadly infection
caused by rotaviruses (which causes severe diarrhea) was demonstrated to be contaminated by
an apparently harmless, porcine-derived virus known as a circovirus. Although the circovirus
was not known to cause illness in humans, government agencies recommended that medical
professionals cease using the vaccine for a period of time until its potential impact to patients
could be assessed. The main issue with detecting and controlling viruses is that new viruses are
discovered frequently. Thus it is not practical or feasible to test for all known viruses. As a
result, the approach is to eliminate as many viruses as possible by carefully analyzing all the
biological materials used in the production of product. Furthermore, steps are included in the
production process that are designed to inactivate and/or remove viruses. Finally, specific tests
are performed to check for a range of relatively common viruses known to be of concern in the
manufacturing and administration of biologics.
Prions
Prions are a recently-discovered class of infectious agents that simply consist of protein,
meaning they do not have any associated nucleic acid but can be replicated by the body. Prions
do this because they are misfolded proteins, and once inside an organism can induce further
similarly-misfolded proteins to occur by inducing the formation of an amyloid fold. The end
result of this is a large number of misfolded proteins of the prion form, causing the disease
state. The term prion is a linguistic portmanteau of the words protein infection. Prions were
first proposed in the early 1980s by Stanley Prusiner as the causative agent of two unusual
neurological diseases, previously attributed to “slow viruses”- scrapie in sheep and Creutzfeldt-
Jakob Disease (vCJD) in humans.
At the time, Prusiner's work was a revolutionary concept that was not widely accepted;
however, his work did eventually earn him the Nobel Prize for Medicine in 1997. Since that
time, a number of other prion-associated diseases have been identified. Most neurologic prion
infections fall under the term Transmissible Spongiform Encephalopathies (TSEs). The most
well-known of theses is Bovine Spongiform Encephalopathy (BSE), more popularly known as
"mad cow disease." BSE is a disease that affects cattle, causing neurological symptoms in
infected animals. Prion diseases are generally transmitted by consumption of nerve tissue from
infected animals (i.e., brain, spinal cord). BSE is believed to have been transmitted to large
numbers of cattle that were given animal feed containing Meat/Bone Meal (MBM) from other
animals (which included ground spinal meat). While MBM has been used for many years in
animal feed, it is now thought that a change in the treatment process of this product in the

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1970s resulted in the prions no longer becoming properly inactivated. This change started a
cycle in which a high proportion of animals became infected with BSE.
What is the relevance of prions to the pharmaceutical manufacturing? The industry generally
uses a number of animal-derived materials, such as gelatin and amino acids, which are
traditionally harvested from cattle. For biopharmaceuticals, fetal bovine serum is used in the
process of growing mammalian cells. If this serum includes material from infected animals, it
could potentially infect the producing cell line and ultimately be transmitted to the patient via
pharmaceutical product derived from these cells. In the 1960s and 70s, the methods of
recombinant DNA and genetic engineering had not yet been developed. All human therapeutic
proteins for patients whose bodies did not adequately produce them were derived through the
isolation of that protein from human tissues. One of the best and most successful cases was the
use of Human Growth Hormone (HGH), used to treat dwarfism in children who made an
insufficient amount of this essential hormone. The HGH was extracted from human pituitary
glands obtained from cadavers. To make one batch of HGH, a large number of human
pituitaries would be gathered and the product extracted. HGH batches created in this manner
were effective in reversing the growth deficiency symptoms in the affected patients. Some
years later, however, it was discovered that some patients had developed a type of CJD and
that donated pituitaries from individuals with CJD could contaminate the manufactured HGH
and potentially transmit the infectious prion to all the children who received the product. This
event increased the awareness of the dangers posed by this type of infectious agent and
expedited the approval of the rDNA-based HGH, which eliminated the need to start with human
pituitaries and thus resolved the issue.
When the “mad cow” type of TSE was identified in the United Kingdom in the 1980s, it created
a wave of concern, as it was recognized that the prion was extremely difficult to inactivate or
remove. Thus if many parts of an infected animal were to enter the production process, the
concern was that it might contaminate other previously inert, animal-derived components of
pharmaceuticals. This led to concerted global efforts to eliminate the use of animal-derived
materials wherever possible. When this was not feasible, the effort was to mitigate the risk by
using only certain parts of the animal deemed to be of lower risk or by using only animals from
BSE-free areas. To date there have been no reported cases of CJD in humans due to
pharmaceuticals contaminated by BSE.
Prions are now well accepted as the causative agent of certain transmissible spongiform
diseases and are recognized as being potentially responsible for devastating illness that are
transmitted by contaminated pharmaceutical products. However, through careful control of the
supply chain inputs (raw materials), this issue has been more successfully controlled.
Microbiological control
The manufacture of biopharmaceutical products begins with the essentially closed, axenic
mono-culture process of fermentation, proceeds through the low bioburden process of
purification, and ends with the aseptic fill/finish process to produce a sterile dosage form.
During this cycle there are various types of microbial agents that can enter these processes in
different ways. In some circumstances, extraneous microbes may be tolerated—in other
circumstances they are unacceptable. In all instances, however, it is essential that organizations

Introduction to Biomanufacturing 289

understand, monitor, and control their products and potential impurities in those products (in
this context extraneous microbes can be considered as impurities). Often it is difficult to
measure the quantity of these impurities that are present and there are major challenges in
proving their complete absence. In order to control them, it is necessary to control the
environmental factors that impact their presence. To understand this, a closer examination of
the manufacturing process of biopharmaceutical products is necessary.
Typically the manufacturing process can be considered as two independent activities: the
manufacture of the drug substance and the manufacture of the drug product. These two
activities can occur in separate facilities (at times in separate countries) and can have their own
specific, but differing, requirements for microbiological control.
Manufacture of the drug substance
The manufacture of the drug substance consists of a relatively long, discontinuous set of
process steps in which the realities of achieving microbiological control are quite different at
the various phases. This culminates in the preparation of a single or small number of containers
of a substance, typically as a solution that is allowed to have a low bioburden. In other words, it
is not sterile and can have a small number of microbes present when tested. During the
required steps, the exclusion/control of extraneous microbes is critically important to ensure
the required quality substance is achieved. An ongoing analysis and understanding of the
microbes (in the air, water, surfaces, and the people relevant to the process) are required. A
critical piece of documentation is a bioburden control strategy. This document is used to
provide a proactive analysis of the risk that bioburden presents to product quality. It should
document how the process reduces the risks posed by microbes to acceptable levels. For
example, microbes are eliminated through sterile filtration, toxins may be eliminated through
chromatography or viruses may be reduced through an inactivation step.
Manufacture of the drug product
The manufacture of the drug product involves the preparation of individual sterile dosage form
units from a biopharmaceutical drug substance or substances. These drug substances are
usually not “sterile” but are considered low bioburden solutions. Various other substances (e.g.,
excipients, preservatives, etc.) and components (e.g., vials, syringes, stoppers, etc.) required for
the dosage form are included in this process. Compared to the manufacture of drug substances,
the manufacture of the drug product is essentially one relatively short, continuous process.
This process is similar, or in some cases identical, to that used for the aseptic manufacture of
different types of parenteral products (aseptic manufacturing is a well-established process that
has been used for decades). The process output is a sterile product produced from a series of
individual, non-sterile components. There are many controls and conditions related to the
facility, people, and process that must be in place to achieve the sterile product. It is important
to understand the terms sterile, aseptic, and axenic:
 Sterile: the absence of life. All drug products are required to be sterilized once
placed in their final container. This is performed to prevent the provision of a
product contaminated with microorganisms to patients.

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  Aseptic: acting in such a way to prevent the introduction of microorganisms.
Aseptic processing is used for those drug products that must be sterile but cannot
be subject to terminal sterilization due to their heat-labile nature; effectively all
biopharmaceutical products are in this category.
 Axenic: freedom from foreign organisms. All biopharmaceuticals are intended to
be axenic cultures; that is, they only contain the cell line desired, without other
foreign organisms
During aseptic processing, previously sterilized components, containers, and closures are
assembled within specially designed and controlled environments. These environments are
intended to minimize the potential contamination of the product by microbes or particulates. In
such processes, aseptic techniques similar to those used in routine microbiology laboratories
and in many medical procedures are widely used.

Types of Contamination
Contamination is the presence of any unwanted substance that will affect the safety, integrity,
strength, purity, or quality of a drug product. In aseptic biomanufacturing, the goal is to
maintain sterility in the final product or to prevent contamination of the sterile components
and the end product. Contaminants can be transferred to the sterile components and end
product from the environment or by direct contact. Contaminants can be grouped into two
categories, particulate (non-viable) or microbial (viable). Neither type may be visible to the
naked eye.
Particulate (non-viable) contamination
Particulates are small bits of matter, or particles, usually of microscopic dimensions.
Government agencies and industry groups have different means of categorizing particulates
based on size and the type of particle. Generally a particle is smaller than a human eye can
detect, which is an object of approximately 25 microns in size, about 39 millionths of an inch
(0.000039 inch). A micron is one millionth of a meter. Figure 8-1 illustrates the size of one
micron.

Figure 8-1. Comparison of one micron-size particulate to one human hair

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Particulates can be composed of any material, either organic or inorganic. They can be found in
gases, liquids, or solids as either suspended or settled material:
 gas: the suspended particulate is referred to as an aerosol or airborne contamination
(Figure 8-2 illustrates the relative size of common airborne contaminants—a person
cannot see up to 97 percent of particulates in the air; dust accounts for approximately
three percent of the particulates that flow through an air space).
 liquid: the suspended particulate is referred to as a suspension; when settled at the
bottom of a liquid it is referred to as silt.
 solid: the suspended particulate is referred to as included matter.

Figure 8-2. Relative size of common airborne contaminants

Particulates can be harmful to the product or process. In a cleanroom environment, common
sources of non-viable particulates include:
 cellulose fiber from paper
 glass particulate from breaking glass vials during filling
 aluminum particles from capping vials
 gown fibers
 hair
 human dead skin cells (one of the most frequently encountered particulates in the
cleanroom)
It is important to note that all particulate contaminants on this list, may also present a source of
viable (microbial) contamination as well.

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Cleanroom classifications are based on the number of particulates allowed in the air.
Particulate sizes measured are typically between 0.5 micron and 5.0 microns. These particle
sizes are considered to represent sizes of microbes. They are measured using a particle counter.

Microbial (viable) contamination

As previously discussed, microbial contamination is caused by microorganisms such as bacteria,
fungi, and viruses. All microorganisms are ultimately excluded to the fullest extent possible
from medicinal products. If not controlled, however, they can reproduce. Thus they are referred
to as "viable." Bacteria can replicate rapidly—some types as quickly as every twenty minutes.
For example, within eight hours one bacterium can multiply to over 16 million bacteria (Table 8-
1 illustrates microbial replication). This rapid increase in numbers, along with the associated
metabolic products and cell wall components such as bacterial endotoxin, is why microbial
contamination is a major concern during aseptic processing. It only takes one bacterium to
cause significant contamination of a product.
Table 8-1. Microbial contamination replication

Time of Day Number of

Bacterial Cells

9:00 a.m. 1

9:20 a.m. 2

9:40 a.m. 4

10:00 a.m. 8

10:20 a.m. 16

1:00 p.m. 4,096

1:20 p.m. 8,192

1:40 p.m. 16,384

2:00 p.m. 32,768

2:20 p.m. 65,536

4:40 p.m. 8,388,608

5:00 p.m. 16,777,216

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Bacteria are the most common microbial contaminant found in cleanrooms, followed by mold.
Bacteria can be divided into Gram-positive and Gram-negative bacteria, based on differences in
their cell walls. Table 8-2 is a summary of some of the most frequently encountered
microorganisms in a cleanroom setting. Bacteria from skin contribute the most contaminants to
cleanrooms. In a well-controlled cleanroom environment, the presence of mold and Gram
negative organisms should be minimal.

Table 8-2. Commonly encountered microorganisms in cleanrooms

Microorganism Example Source

Gram-positive cocci Staphylococcus species humans

Gram-positive cocci Micrococcus species humans

Gram-positive bacilli Bacillus species soil

Sources of contamination
Microbial and particulate contamination sources in the cleanroom include humans, air,
surfaces, water, and the components used to manufacture the product. Humans are the main
source of cleanroom contamination. The very presence of people in a manufacturing process
area poses risks to products. Contaminants from humans include shed skin particles and hair, as
well as those found under fingernails, on hands, and or on clothes (Table 8-3). People can also
expel contaminants by talking, sneezing, and coughing, even while wearing a mask (Figure 8-3).
Depending upon the type of mask and other factors, not all particulate contaminants may be
blocked.

Table 8-3. Sources of microbial contaminants from humans

Source Amount

nose secretion approximately 10 million microbes/gram

Spittle approximately 100 million microbes/gram

scalp approximately 1 million microbes/cm2

forehead 10,000–100,000 microbes/cm2

Armpit 1–10 million microbes/cm2

Hands 100–1,000 microbes/cm2

Note: During the course of a typical day, a person will shed 10 grams of skin particles. This is
equivalent to one layer of skin (40 ft) within 3–4 days.

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 Figure 8-3. Respiratory dispersal of microbes

The process of product contamination is influenced by many factors. Contamination can be

introduced by materials, water, the degree of human contact, and the manufacturing
environment. People and their specific activities may have direct contact with the product.
Contaminated work surfaces, tools, and fixtures can provide a direct transfer of contamination
to the product.

Control of Contaminants in the Cleanroom

Several measures must be taken to reduce the likelihood of transferring contaminants from
their source to sterile components and the end product. These include properly designing the
facility, controlling the air supply for the aseptic environment, sterilizing the manufacturing
components, using aseptic gowning, following aseptic techniques, and implementing a cleaning
and disinfection program.
Each individual step in the entire biomanufacturing process, from the raw materials at the start
to the final finished product, should be examined for potential risks. An approach used to
examine the steps in a process is called the Hazard Analysis and Critical Control Points (HACCP).
HAACP has been used extensively in industries such as food processing since the 1990s and has
recently become more common in biomanufacturing processes. This hazard analysis method
leads to the determination of critical control points, which helps establish a preventative
monitoring system. This system is used to observe key parameters that have a potential impact
on product quality.
Control through facility design
A cleanroom is any room or area where an attempt is made to limit, control, and eliminate the
amount of airborne contamination. Many contaminants are continuously generated within a
cleanroom by people, processes, equipment, and the facility, which are then transferred

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through the flow of air. Eliminating these contaminants is a function of appropriate facility
design (see the Facilities chapter) and in-process controls described in the following sections.
Control through air supply
It is important to note that there is no such thing as a totally clean room (a room with
absolutely no contamination)—there are only degrees of cleanliness. Therefore, it is vital for
aseptic processing that a facility's cleanroom be designed properly to maintain the necessary
level of cleanliness.
The cleanroom environment is created and maintained by controlling the air that is supplied.
Traditional air handling systems, referred to as Heating Ventilation and Air Conditioning (HVAC),
use filtration systems that remove large particles and provide air that is adequate for routine
manufacturing. Along with controlling particulate emissions, HVAC systems play an important
role in regulating a facility’s temperature and humidity. This standard HVAC filtration alone is
not adequate for aseptic processing though and is not effective in reducing sub-micron airborne
contamination.
Cleanrooms used for aseptic processing require High Efficiency Particulate Arrestance (HEPA)
filters. HEPA filters are used to supply clean air to controlled areas such as cleanrooms. HEPA
filters remove 99.97 percent of the particles suspended in air that are 0.3 microns or larger in
size. The number of air changes per hour in a cleanroom is also controlled. The air handling
system replaces the air in a cleanroom regularly, effectively purging the particulate matter
generated within the room.
Additional ways by which to maintain this environment include specific construction materials,
air classifications, pressure differentials, airlocks, restricted entry, and flow of people and
materials.
All systems should be properly validated, or demonstrated to be effective in maintaining the
proper level of cleanliness, through a rigorous protocol. This protocol will demonstrate that the
system can be relied upon with a high level of confidence to provide an environment that is
appropriate for its intended use – which is to minimize the risk of contamination.
Control through sterilization of components and equipment
The components and equipment used in aseptic biomanufacturing must be sterilized.
Sterilization is defined as the act or process, either physical or chemical, which eliminates or
inactivates all forms of life, including bacterial endospores (the most resistant of all
microorganisms). An item can only be deemed sterile when there is a complete absence of
viable microorganisms. Sterilization methods vary based on the material of the components to
be sterilized. Commonly used methods for sterilization of biopharmaceuticals include:
 dry heat
 gamma irradiation
 ethylene oxide
 Vaporized Hydrogen Peroxide (VHP)

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  filtration
Sterilizers, their use, and their load patterns need to be initially validated to ensure that each
sterilizer is functioning properly and that every item in the load is sterilized by the end of the
cycle. Ongoing validation, performed at least annually, ensures that the cycles remain within a
validated state. Preventive maintenance ensures the sterilizer is running properly.
The basic principles for validating a sterilizing process are:
 Demonstrate that the equipment used for the sterilization process (autoclave, dry heat
oven, and VHP) is capable of operating to achieve the desired end result
 Run cycles to demonstrate actual operational conditions. Ensure that the required
parameters of microbial kill (i.e., bioburden reduction) are achieved. Biological
indicators are typically used for this purpose. Biological indicators are materials that are
inoculated with a known quantity of microorganisms, typically one that is resistant to
the sterilization conditions and thus serves as indicators of the worst case scenario for
the sterilization cycle.
 Continually monitor the process parameters (e.g., temperature, pressure, etc.) during
each sterilization cycle to ensure they are operating within the validated parameters
 Perform continuing validation studies periodically to ensure that the loads are
maintained in a validated state
It is important that sterilization processes operate within validated parameters to ensure the
sterility of all equipment and components since each and every item used for aseptic
processing cannot be tested for sterility. It is also important to check the expiration date and
appearance and integrity of sterilized equipment and components before their use. Any
equipment that has been compromised or is out of date must not be used.
Steam sterilization involves exposing an item to steam under high temperature (minimum of
121 degrees Celsius) and pressure conditions (15 psi). Steam sterilization can be used for items
that are heat and moisture stable and that can be penetrated by steam. An autoclave (Figure 8-
4) is a commonly-used method for sterilization.

Figure 8-4. Steam sterilization using an autoclave

Introduction to Biomanufacturing 297

To provide a uniform temperature distribution and efficient heat transfer, air must be removed
from the sterilization chamber. This is usually accomplished using a vacuum system. Once the
correct temperature and pressure conditions are achieved in the autoclave chamber, the
sterilization time begins. A steam sterilization cycle is not valid unless the appropriate
temperature and pressure conditions are maintained for the required period of time.
Some of the items that can be steam sterilized using an autoclave include:
 culture media
 filters
 glass bottles
 miscellaneous items such as stoppers, caps, tubing and forceps
Prior to sterilization these items are prepared and wrapped using penetrable materials such as
Sterilin® bags or parchment paper. All items must be loaded according to validated load
patterns to ensure that each item in the load is sterile.
Autoclaving is a consistently effective means of sterilizing most objects, provided the process is
done correctly. The principle advantages of steam sterilization are its simplicity, relatively short
processing times, and lack of toxic residues. Its main disadvantages are the relatively high
temperature and the limited types of materials that can be sterilized in this manner (i.e. those
that are not sensitive to moisture, temperature, and/or pressure). Furthermore, there are
hazards associated with high temperature and operating pressurized vessels. To prevent burns,
operators must wear thermal gloves that allow hot materials to be handled safely.
Steam-In-Place (SIP) procedures are used for large vessels, such as tanks and other types of
equipment too large to fit in an autoclave. Clean-In-Place (CIP) procedures must be completed
first to prevent the SIP process from baking on any residuals (cross contamination). For the SIP
cycle to be effective its validated procedure must be followed exactly.
Dry heat sterilization involves exposing an item to hot air (160–170 degrees Celsius) in an oven-
like chamber. To ensure temperature uniformity within the chamber, the air is circulated using
a fan-blower system. Dry heat sterilization can be used for items which are heat stable but are
sensitive to moisture or cannot be penetrated by moist heat. Glassware can be sterilized by dry
heat.
Once the oven correct temperature is achieved in the chamber, the sterilization time begins. A
dry heat sterilization cycle is only valid if the appropriate temperature conditions are
maintained for the required period of time. As with steam sterilizers, all items must be loaded
according to validated load patterns to ensure that each item in the load is properly sterilized.
The main advantages of dry heat sterilization are its simplicity, penetrating power, and lack of
toxic residues. Components that might corrode if exposed to moisture are well suited for dry
heat sterilization. Another significant advantage is that dry heat has the ability to depyrogenate,
or inactivate, all pyrogenic substances (any substance, such as endotoxins, that produce fever
when introduced into the body). Steam does not have this ability. Dry heat sterilization’s
disadvantages are the relatively long processing time and the high temperature. This limits the

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types of products and packaging materials that are compatible with this process. Dry heat
sterilization takes longer than steam sterilization mainly due to the fact that air is a relatively
inefficient heat transfer medium. There is a time delay for the heat to penetrate the load. As
with steam sterilization equipment, operators must wear thermal gloves that allow hot
materials to be handled safely.
Gamma irradiation provides a simple sterilization alternative for moisture-sensitive and/or
heat-sensitive non-liquid items. Some types of liquids which are heat-sensitive can also be
sterilized this way. Gamma irradiation involves exposing items to gamma rays produced by
isotopes Cobalt-60 or Cesium-137 until the desired dosage is delivered. A gamma ray is an
electromagnetic radiation of extremely high energy, emitted by a radioactive atom during
decay. It is similar to an X-ray but shorter in wavelength (higher-energy). Gamma rays damage
DNA and other cellular structures in living organisms, thus making them effective in killing
bacteria and other potential contaminants. Items sterilized by this method are not exposed to
any toxic agents and no residues are left behind.
The exposure during gamma irradiation can be quantitatively monitored, making it an easily
validated method. The main disadvantage of gamma irradiation, however, is that it is a
relatively expensive process. The extremely high capital cost to install gamma irradiators makes
it prohibitive for many organizations. However, there are third party organizations that provide
gamma-irradiated items to the pharmaceutical industry. There are also safety issues associated
with the process since it involves the use of radioactive isotopes. Gamma irradiation is not
widely used for aqueous drug products and protein-based pharmaceuticals because it can
degrade such products via the electromagnetic radiation.
Vaporized Hydrogen Peroxide (VHP) is an aerosolized, low temperature chemical sterilant that
kills microorganisms, including bacterial endospores on environmental surfaces in an enclosed
area. It is used to sterilize sealed enclosures such as isolators, workstations, and pass-through
rooms. VHP's advantages include its effectiveness against a wide variety of microorganisms, the
use of low temperatures, and compatibility with a wide variety of materials and surfaces.
Furthermore, once the VHP (31% H2O2) is used, it can be sent through a catalytic converter to
produce water vapor and oxygen. These by-products are safe and environmentally friendly. The
main disadvantage of VHP sterilization is its limitation as a surface sterilant. It is only effective
on exposed surfaces. Furthermore, 31% H2O2 is toxic and must be used in enclosed or sealed
areas. And though it rapidly aerates and converts to harmless by-products, it is important to
follow all required safety precautions when working with this substance.
Filtration does not necessarily kill microorganisms but it does remove them from the filtered
material. Filtration is used to sterilize heat-sensitive materials. And the process basically
consists of passing the liquid or gas product through a porous medium such as cellulose esters
or plastic polymers that trap the microorganisms in the filtration matrix or retains them on its
surface. Filters are assigned a nominal size rating above which a certain percentage of
contaminants will be retained.

Introduction to Biomanufacturing 299

Control by aseptic gowning
The human body constantly sheds dead skin cells and hair and excretes oil and moisture—all of
which can contain microorganisms that could contaminate critical parts of the process and
products. Humans shed approximately 1000 bacteria-carrying cells per minute. Many airborne
microorganisms are dispersed in cleanrooms due to human skin cells. A person can shed one
outermost layer of epithelial cells every 24 hours which amounts to 109 cells per day. A small
but significant amount of these skin cells can be associated with microorganisms which can get
into the air flow and cause contamination.
Wearing clean, sterilized clothing is necessary to protect the product and critical surfaces from
contamination. Aseptic gowns (Figure 8-5), consisting of a gown, mask, gloves, goggles, and
boots, cover all or most of a person. Individuals working within cleanrooms must be properly
trained on aseptic gowning. The apparel provides a barrier between the individual wearing it
and the sterile components and products. However, aseptic gowning does not provide 100
percent protection. Microorganisms have the ability to penetrate woven or non-woven fabrics
to emerge into the surrounding air, thus proper gowning reduces but does not eliminate
microorganisms shed by people in cleanrooms.

Figure 8-5. Full aseptic gowning

Within a facility, different areas are assigned classifications that determine gowning
requirements (Table 8-4).

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 Table 8-4. Gowning requirements by area classification

Clean Area Classification ISO Typical Area Gowning

(0.5 micron particles/ft3) Designationa

Class 100 5 point of fill, full aseptic

sterility testing gowning, to
or other consist of sterile
aseptic gown/hood,
manipulation sterile double
gloved, mask,
goggles, and
boots

Class 10,000 7 background same as Class 100

room/area for
aseptic
manipulation

Class 100,000 8 personnel and non-shedding

equipment one- or two-piece
airlocks suit gathered at
leading into wrists and ankles;
the cleanroom hair, beard, and
facility shoe covers

Controlled NA access and exit employer-issued

Unclassified to/from uniform plus non-
classified shedding smock,
areas; hair, beard, and
packaging shoe covers
areas
a
ISO 14644-1 designations provide uniform particle concentration values for cleanrooms in multiple
industries.

Individuals working in a cleanroom must follow high standards of personal hygiene and
cleanliness to reduce the amount of microorganisms and particulates shed. Anyone with a
respiratory or gastrointestinal infection, open skin lesions, or any other condition that causes
skin to peel should not work in a cleanroom. For people working in a cleanroom environment,
the challenge is being gowned and operating in a slow, controlled manner for long periods of
time.

Introduction to Biomanufacturing 301

Proper practices for aseptic gowning include:
 wash hands with soap and water prior to entering the gowning room
 keep fingernails trimmed to preserve the integrity of the gloves
 remove watches and jewelry prior to gowning, with the exception of smooth wedding
band and medical jewelry; special measures such as additional gloves may be necessary
to lower the risk for these exceptions
 follow the organization's established SOP for the gowning process, maintaining the
order in which gowning attire is put on to assure that gown cleanliness is maintained as
much as possible
 wear face mask for facial hair
 gown, making sure the gowning apparel does not contact walls, floor, bare skin, or
equipment; gown again if the apparel contacts a potentially contaminated surface
 wear gloves when entering the cleanroom area; disinfect gloves with sterile 70%
isopropyl alcohol; inspect gloves and gowning apparel initially upon entering the
cleanroom and periodically to ensure that they have not been compromised; and
replace damaged items as necessary
 use gowning materials that fit properly as inadequately-sized gowns or materials may
cause problems in performing aseptic work; loose-fitting gowns create the potential for
contact between the gown and a critical surface area.
 keep talk to a minimum; avoid sneezing and coughing as particulate matter can be
dispersed through such actions
 if necessary, adjust gloves and the sleeves of the gown during aseptic manipulations, as
it is possible for the gown and the gloves to separate at the wrist, leaving the wrist
exposed
Control through aseptic techniques
Every activity that is done for aseptic processing should be performed with the mindset of
reducing the probability of risk to the lowest-acceptable level. Because of its inherent
probabilistic nature, risk can never be completely eliminated, but one should understand that
his or her behaviors directly and immediately impact the chance for success during aseptic
processing. Even though most biosafety cabinets have a rating of Class 100, this rating still
allows for the presence, however limited, of particulate matter to circulate.
The most important factor in reducing contamination during aseptic processing is operator
technique. This is due to the fact that the most frequent physical interventions in close
proximity to the open cell culture are the operator’s movements. Given proper equipment and
material asepticity, as well as process design (e.g., appropriate filtration steps, SIP following
CIPs, etc.), the operators are the major potential sources of contamination for the cell culture.
Extrinsic to aseptic technique, the three major components most important for reducing
probability of contamination are: environment, equipment and materials.

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Environment
• the environment must be clean with HEPA-filtered air circulating
 the aseptic area should be cleaned before and after use. Cleaning after use reduces the
risk of cross-contamination in a multi-product suite and reduces the microbial load on
working surfaces
Equipment and materials
 Ensure that equipment and materials are cleanable and fit for purpose, i.e., use
equipment that is designed for cleanrooms. Equipment would not be considered
cleanable if the presence of air exhaust on the equipment leads to non-aseptic turbulent
airflows, surfaces are difficult-to-access or reach for cleaning, or non-suitable materials
of construction (e.g., non-slip felt/fabric pads on underside of equipment) are present.

Aseptic technique guidelines when working under a biological safety cabinet

When working with a biological safety cabinet (BSC) as part of the aseptic process, it is
important to understand the associated guidelines and limitations of their use.
BSCs and cleanrooms are categorized as Class 100 environments. This means that one cubic
foot of air may have no more than 100 particles of 0.5 microns or larger. The critical surfaces in
the aseptic processing area in the biological safety cabinet must see the first air from the HEPA
filter. No body part, equipment, objects, or contaminated air should come between the HEPA
filter and the critical surface. An example is passing a gloved hand over an open bottle of
product. Anything on the hand or sleeve could potentially be shed into the product by the flow
of air under the hood and subsequently contaminate the product. Techniques for activities as
basic as removing or replacing a bottle cap should be well thought out and practiced in order to
minimize the chances of contamination. This is called the First Air rule (Figure 8-6).

Figure 8-6. Violation of the First Air rule

Introduction to Biomanufacturing 303

List of behaviors that should be adhered to in order prevent contamination
The following list of behaviors should be adhered to when working in a biosafety cabinet or
cleanroom to prevent contamination.
 verify the proper operation of the BSC - that it has been certified prior to the start of the
procedure and that it’s indicator lights, alarms, and/or gauges are working properly.
The BSC should be on a periodic certification schedule
 plan all activities - arrange materials and plan operations so that there is a minimum of
movement and physical contact with materials once aseptic operations begin. Place
non-critical materials (e.g., tube racks) in a location where they do not need to be
moved or manipulated during the aseptic activity
 movement of personnel into and out of the cleanroom area should be kept to a
minimum as fast and unnecessary movements cause turbulence which can disrupt the
air flow and contaminate critical surfaces, including the product. A vacuum can be
created near the sides of a BSC work area which could pull contaminated air from
outside into the BSC and thus it is important that no work be performed near the sides
of the BSC work area
 perform any aseptic manipulation above waist level for BSCs and cleanrooms. Based on
the concept that the rebound effect of the air may extend approximately one foot
above the floor surface, all aseptic manipulations and connections should be performed
at least two feet or higher from the floor
 bring only the minimum equipment and materials necessary to perform the activity into
the area
 clean all equipment and materials brought into the BSC area with 70% isopropanol
 use materials that are suitably constructed to withstand cleaning without being
compromised
 place all materials and equipment in such a fashion as to allow for the activities being
performed to be accomplished with a minimum of movements and in a manner so as
not to break the first air rule
 Segregate clean and dirty (waste) materials within the BSC
 Prohibit movement of materials and personnel into or out of BSC or cleanroom until all
activities being performed in the cabinet are brought to a stage where no product is
open to the environment
 Perform all aseptic manipulations at least six inches from the front edge and sides of a
biosafety cabinet or as per the manufacturer’s recommendations
 Avoid touching critical surfaces. Gloved hands are not considered sterile even after
disinfection with alcohol as disinfection reduces the number of microbes but cannot
sterilize a surface
 Avoid excess turbulence and particulate shedding by:

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 – minimizing the number of people and equipment under the hood; only those
essential to processing should be present
– limit talking
– working in an unhurried, deliberate manner. Motions should be controlled and
methodical (even while sitting or standing still, a person can emit 100,000 particles
per minute—the more a person moves the more particulate load is emitted in the
area). Perform only those actions that are necessary to accomplish the task being
performed. Do not perform unnecessary movements or touch objects when doing
so is not needed. Table 8-5 lists the amount of particles that a person can
generate.
 Avoid placing equipment on the air intake grate of the BSC as this disrupts air flow.
 Avoid using equipment if its sterility has been compromised—critical surfaces remain
sterile if they are handled correctly and the package is intact. Replace any component
with a fresh sterile one if contamination is suspected
 refrain from storing materials that generate fibers or particulates (e.g., cardboard,
paper, wood, etc.) where aseptic work is performed
 when entering a cleanroom via an airlock adhere to the following behaviors:
– All equipment must be disinfected in the airlock prior to entering an aseptic area
and before an aseptically-gowned person contacts it
– Opening and closing of airlock doors should be kept to a minimum. They should
be opened only to perform necessary activities
– both doors of an airlock should never be opened simultaneously

Table 8-5. Estimated particles (0.3 micron and larger)

generated by various activities

Particles Emitted Activity

100,000 motionless—sitting/standing

500,000 upper body motion

1 million upper body and minor leg motion

2.5 million sitting to standing or vice versa

5–10 million walking >2.0mph

Introduction to Biomanufacturing 305

If specifications require measurement of viable and non-viable particulates during your
operations the following experiments can be performed:
 Viable particulates: surface monitoring plates may be used to measure viable
contaminants on personnel and equipment surfaces. Air viable devices may be used to
measure viable air contaminants.
 Non-viable particulates: a particulate measuring device can also be used to measure the
total of both viable and non-viable particles.

Control by cleaning and disinfection

Cleanrooms do not have self-cleanup capabilities to offset any contamination brought into the
room or generated by people and/or equipment. Most contaminants introduced in this fashion
settle to the floor or other horizontal surfaces and could be introduced into the air by changes
in air currents or activity in the room. Thus contamination needs to be removed from these
surfaces by frequent cleaning and disinfection. A one-micron anthrax spore can take
approximately twenty minutes to float a one meter distance to the floor or horizontal surface.
Cleaning is often confused with disinfection; however, they are not the same. Cleaning is
applying a detergent (along with the physical removal of particles and microorganisms from
surfaces) by mopping, wiping, or brushing. Disinfection is the elimination of most recognized
disease-causing or harmful microorganisms but not necessarily all microbial forms and is a less
lethal process than sterilization. Although disinfection might make use of ultraviolet radiation,
boiling water, or steam, the term is typically associated with the use of chemicals.
Cleaning
Cleaning is used to remove contaminants and residues that can interfere with the effectiveness
of disinfectants. Proper cleaning is important for a successful disinfection. Cleaning does not
have to be performed at the same frequency of disinfection. An example of a four-step cleaning
process of a surface involves:
1. scrubbing the surface with a mop or wipe and using a detergent solution
2. rinsing the surface before the surface dries
3. collecting any remaining liquid on the surface by wiping or vacuuming
4. allowing the surface to dry then disinfecting
Though vital to the cleaning process, disinfectants can be burdensome. One issue with
disinfectants is that they can leave residues. Air pockets can form in the residue when a
disinfectant dries and these air pockets can harbor bacterial endospores. If the air pockets
break, they can release the endospores. The residues can also be transported on personnel to
critical surfaces as well as the product and cause contamination. They can also be extremely
corrosive and damage the surfaces on which the disinfectants are applied. Routine removal of
these residues through careful cleaning is necessary.

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Disinfection
A disinfectant is defined as a chemical agent used on surfaces that destroys disease-causing
microorganisms. No single disinfecting agent or procedure is adequate for all disinfection
purposes. For example, most disinfectants do not ordinarily destroy bacterial endospores. Thus
a variety of disinfectants and disinfectant procedures are used to maintain aseptic processing
equipment and facilities.
The effectiveness of a disinfectant depends upon:
 the disinfectant concentration
 the length of exposure to the disinfectant
 the amount of organic matter (e.g. soil, blood) present
 the nature and amount of microorganisms on the surface
 the material to be disinfected
When selecting a disinfectant for an aseptic processing area, these criteria must be considered.
All disinfectants must be used at sufficient concentrations and given sufficient time to work.
The disinfectant vendor’s recommendation for contact time must be followed.
Disinfectants include antimicrobial components. Many different types of antimicrobial
components can be used in disinfectants, each with varying degrees of success depending on
the type of microorganism. Most disinfectant antimicrobial components require a minimum
contact time of five minutes. Disinfectants can also have a detergent component which is used
to help remove soil from the area being treated. A disinfectant is more effective once soil has
been removed and the surface is clean.
A disinfectant can have an acid or alkaline pH level. The pH of an environment can limit
microbial growth. Very few microorganisms can survive in an environment with a pH of less
than 4 (acidic) or more than 10 (alkaline). Most microorganisms grow well in an environment
close to pH 7. The proper disinfectant can create an environment that is not conducive to the
survival of microorganisms.
Chemical agents used as disinfectants in biopharmaceutical manufacturing fall into the
following categories based on their antimicrobial component (Table 8-6):
 alcohols
 aldehydes
 halogens
 peroxygens
 phenolics
 surface active agents
Each type of disinfectant is also categorized by its use and the microorganisms against which it
is effective- bactericidal agents, fungicidal agents (fungi), virucidal agents (destroy/inactivate

Introduction to Biomanufacturing 307

viruses), sporicidal agents (bacterial and fungal spores), and bacteriostatic agents (prevent the
growth of bacteria but do not necessarily kill them). Solutions of these disinfectants must be
sterile when used in Class 100 and Class 10,000 area classifications. Examples of disinfectants
are sodium hypochlorite (halogen), 70% isopropyl alcohol (IPA), and peracetic acid (peroxygen).
Table 8-6. Summary of disinfectant categories and their efficacy
4 = most efficacious in killing
1 = least efficacious in killing
0 = not effective in killing

Gram- Gram- Endospores Fungi

Positive Negative
Bacteria Bacteria
Disinfectant
Category

Alcohols 3 3 0 2

Aldehydes 4 4 4 4

Halogens 3 3 1 2

Peroxygens 4 4 4 4

Phenolics 3 2 1 3

Surface Active 3 1 0 2
Agents

Many facilities typically rotate the types of disinfectants used in cleanrooms by alternating the
use of two different disinfectants. This is usually performed on a monthly basis. The rationale is
that one disinfectant would not be effective in killing a particular population of microorganisms,
whereas second disinfectant could possibly prove efficacious against a different microorganism
or microorganism population.
All equipment and containers should be disinfected in the equipment airlock prior to being
transferred into an aseptic processing area. Equipment and containers should be wiped from
either top to the bottom or from the cleanest to most contaminated area. Sterile 70% IPA is
commonly used to disinfect equipment and components immediately prior to transferring them
under a laminar flow hood, as well as immediately prior to use within the laminar flow hood.
While working under the laminar flow hood, workers should frequently apply 70% IPA to their
gloved hands.

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Examples of surface disinfectants for different area classifications are shown in Table
8-7. Table 8-8 shows the frequency of application of various disinfectants.
Table 8-7. Surface disinfectants usage
on different surfaces in different area classifications

Area Ceilings Walls Floors Plastic Critical Work

Classification Curtains Surfaces

Class 100 phenolic phenolic phenolic PA or PA or

and and and hypochlorite hypochlorite
monthly monthly monthly and monthly and monthly
sporicide sporicide sporicide sporicide sporicide
all followed all followed
by IPA wipe by IPA wipe

Class 10,000 as above as above as above as above NA

Class 100,000 phenolic phenolic phenolic NA NA

PA: Peracetic Acid (peroxygen)
Hypochlorite: Halogen
IPA: Isopropyl Alcohol, NA: Not Applicable
Table 8-8. Frequency of application for disinfectants a

Area Classification Ceilings Walls Floors Plastic Critical

Curtains Work
Surfaces

Class 100 daily to daily to daily daily daily wiping

weekly weekly mopping mopping, or spraying
mopping mopping wiping, or
or spraying
spraying

Class 10,000 weekly weekly daily NA NA

mopping mopping mopping
or
spraying

Class 100,000 monthly monthly daily NA NA

mopping mopping mopping
or
spraying
a
The frequency of disinfectant use should be determined by environmental monitoring results.

Introduction to Biomanufacturing 309

Environmental Monitoring
Aseptic biomanufacturing environments require strict controls to minimize the potential for
microbiological and particulate contamination of the product. A comprehensive environmental
monitoring program is required for facilities, personnel, and process utilities to check that the
state of control is maintained. Such control is mandated by cGMPs for the consistent
production of products that are safe, pure, and effective.
Monitoring alone, however, is not enough. A key disadvantage of environmental monitoring,
particularly with microbial monitoring, is that often a potential problem is not identified until
several days after the occurrence. Prevention is the preferred means of control over
monitoring.
Prerequisites for an environmental monitoring program
To assure a high likelihood of achieving acceptable testing results from the environmental
monitoring program, the following vital elements must be in place prior to implementing the
program:
HEPA filter certification program: For HEPA filtered equipment supporting classified areas,
such as laminar flow hoods and HVAC systems, an ongoing HEPA filter certification program
is necessary. Minimal certification includes routine biannual HEPA filter leak testing and air
flow testing in Class 100 areas. HEPA filters serving Class 10,000 and Class 100,000
environments should be certified at least annually. Certification must also include the
verification of the appropriate pressure differentials between rooms and the room air
exchange rate.
Cleanroom cleaning and disinfection program: Procedures must be in place for the cleaning
and disinfection of all classified areas, and all individuals performing these tasks must be
properly trained.
Cleanroom qualification/requalification program: Prior to use, the cleanroom and its
process utility systems must be formally qualified. Performance Qualification, also known as
PQ, consists of a series of extensive tests to ensure that the area or process utility is
operating as intended and that the environmental requirements are achieved. Cleanrooms
and process utilities also need to be re-qualified in response to changes in area integrity
(such as power failures affecting operation of laminar flow hoods and HVAC systems).
Airflow pattern visualization program: Smoke studies must be performed to demonstrate
the unidirectional flow of HEPA filtered air across product contact surfaces in Class 100
areas and to confirm the appropriate flow of air from the areas of highest criticality outward
to less critical areas. These studies should be performed both in static and dynamic modes
of operation and recorded on video. Smoke studies are performed prior to PQ testing. They
should also be performed on an annual basis to reconfirm the unidirectional nature of the
airflow to determine that no routine drift of airflow parameters has occurred.

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 SOPs on the performance of environmental testing, investigations, documentation of
results: SOPs must be in place to provide a written description of the program specifics for
the monitoring and testing of classified areas, personnel, and controlled utility systems,
along with the alert and action levels. Also, an SOP must document the process for
recording, accessing, tracking, and storing of test results.
Personnel training program: Relevant personnel must receive documented training in
environmental monitoring and testing procedures. This training must be conducted prior to
the performance of any testing. The performance of those conducting the test(s) should be
evaluated on an ongoing basis in the field by a trainer or supervisor.
Components of an effective environmental monitoring program
An environmental monitoring program must be established at each biomanufacturing facility to
continuously monitor the environmental conditions to which products are exposed during
manufacturing. An effective environmental monitoring program should generally include
scheduled monitoring of:
 airborne viable microbial and non-viable particulate levels
 microbial contamination on personnel, work surfaces, floors, walls, and equipment
 microbial contamination of clean utilities
 pressure differentials
 direction of air flow
 temperature
 humidity
Awareness of the microorganisms present, as well as the particulate levels, can assist in
designing an effective control system. However, no environmental monitoring program,
regardless of its design, can provide the level of confidence desired without overall cleanroom
systems management. This management is accomplished by adherence to cGMPs, facility
design control, effective supervision, sound corrective action steps, and proper employee
training.
A critical element to designing an effective environmental monitoring program is developing a
well thought-out sampling plan. A sampling plan is a documented plan that:
 describes the procedures and methods for sampling in a cleanroom
 identifies the sampling sites, the sampling frequency, and the number of samples
 describes the method of analysis and how to interpret the results
The specific requirements for environmental monitoring programs are often interpreted
differently by different international regulatory agencies. Although there have been efforts
among industry groups and regulatory agencies to harmonize guidelines and requirements,
significant variations still exist. Therefore, it is important that the quality group within the

Introduction to Biomanufacturing 311

organization keep abreast of environmental monitoring guidelines and requirements for the
different countries in which the product is to be sold.
Sample site selection
Environmental monitoring programs are designed to provide periodic evaluations of
environmental quality and system performance within classified environments where
biopharmaceutical manufacturing occurs.
This is accomplished in part by conducting air, surface, personnel, and process utility
monitoring at sample sites that are representative of those locations that:
• come in contact with exposed product and/or components
• are in close proximity to exposed product
• are areas of high personnel and equipment flow
• contribute to the particulate and microbial levels within an area (e.g. personnel,
equipment, etc.)
• represent the most difficult or inaccessible areas to clean or disinfect
Collectively the resulting test data represent the systems’ performance over time and provide
the evidence to assess whether the systems are performing as intended for the production of
quality products. Each manufacturing process should be carefully evaluated when selecting
sample sites for a sampling plan. Representative locations should be identified through the use
of a formal mapping process, based on analysis of risk, through smoke studies; and/or through
data obtained from the Performance Qualification.
An important consideration is the extent of exposure or contact that each point in the
biomanufacturing environment has with the product. Sites determined to have a greater
opportunity for contaminating the product should be sampled and monitored. Critical test sites
or potential product contact sites can include critical surfaces, manufacturing equipment,
gloved hands of personnel, tools, and room air.
It may not always be practical, however, to select a sampling site at the most critical sampling
locations. When selecting sample sites, consideration should be given as to whether the
sampling location would actually increase the probability of product contamination (e.g. a
sampling location that could violate the first air rule). It would also be impractical to have
numerous critical site locations as extensive monitoring during aseptic processing alone would
add unnecessary bioburden.
Routine test sites are non-product contact sampling locations that include locations such as
floors, walls, doors, and ceilings. Sampling locations may not be monitored if there is a low
probability of contamination during processing of those sites. Table 8-9 provides an example of
recommended critical and routine sampling locations. It is important when developing a
sampling plan to have a rationale for selecting each sampling location.

312 Chapter 8 - Microbiological Control

 Table 8-9. Example of critical test site and routine
test site locations and the rationale for selecting each
Tests Test Location Rationale
Critical Sites (Laminar Flow)
Microbial Air Sample within approximately 1 ft. of Sample is to monitor the air where
the critical process point or product there is increased activity
Particulate container opening. (mechanical or otherwise) at a
Air location that is close to and
representative of the air in contact
with the open product.

Surface Sample within approximately 1 ft. of Samples are to monitor the critical
Contact Plate (or at the nearest obtainable surface surfaces where there is increased
to) the product container opening or activity (mechanical or otherwise) at
critical aseptic process step. locations that represent surfaces
close to or in contact with exposed
product or product components.

Personnel Sample gown in 2 locations (chest, Samples are to reflect the parts of
Contact Plate forearm) and gloved fingertips of the gowned and gloved person that
each hand. are in the closest proximity to
exposed product.

Introduction to Biomanufacturing 313

 Table 8-9. Continued
Tests Test Location Rationale
Routine Sites (Non-Laminar Flow)
Microbial Air Sample in a central location in the Sample is to reflect general
room or otherwise representative area/room conditions which are
Particulate location. representative of room conditions.
Air

Surface Sample on a representative wall, Sample is to reflect the general

Contact Plate curtain (outside), door push condition of the area surfaces.
plate/handle/push button, floor
surface, or other frequently-utilized
surfaces as determined by the
sampling plan.
Personnel Sample gown in 2 locations (chest, Routine personnel testing is to
Contact Plate forearm) and gloved fingertips of ensure that gowning is performed
each hand. properly and that glove disinfection
is effective.

Sampling frequency
Environmental testing frequencies in the cleanroom vary depending on the room classification
and the nature of operations. The testing frequency typically can be divided into two
categories- per process testing and routine testing. For biopharmaceutical manufacturing,
microbiological and particulate testing must accompany manufacturing. This testing is referred
to as per process or critical site testing. A process is generally defined as a set of
interdependent steps or manipulations that are conducted within the same set-up or process. It
begins with the area set-up for aseptic processing and concludes at the completion of
processing. Recommended monitoring frequencies for critical site testing are shown in Table 8-
10. Routine monitoring, or monitoring not performed during processing, is generally performed
on a weekly basis (at a minimum). Both per process and routine monitoring provide assurance
that operations, cleaning, and HVAC systems are continuing to operate and perform
satisfactorily and consistently.

314 Chapter 8 - Microbiological Control

 Table 8-10. Example of critical site test requirements
and frequency of testing for Class 100 areas

Environmental Test Class 100 – Critical Site Testing Frequency

Microbial Air (active) once per process (minimum of 1m3 of air)

Microbial Air (passive) one settling plate per designated location

Surface Contact Plate minimum of one site per process

Product Contact Surface Contact minimum of four product contact surface sites to be
Plate (Sterile Filling Only) performed during filling prior to the breakdown of a
line at the end of a fill; to include all component bowls
and representative fill needles

Personnel Contact Plate the fingertips of both gloved hands and the chest and
forearm of personnel who perform aseptic operations
or testing during processing

Particulate Air minimum of once per hour per process (minimum of

1m3 of air)

Interpreting environmental test results and establishing action and alert levels
Environmental testing results fall into one of three categories - passing, alert level, or action
level (Figure 8-7).
• Passing result indicates that the test result for the sampling site was within acceptable
established levels. Tests that do not pass are defined as environmental test excursions,
which include alert and action levels. Alert and action levels are assigned for each room
classification and each type of test performed.
• Alert level indicates a test excursion above the environmental norm for the site and is
usually set at 10–50 percent of the action level. It is based upon historical statistical data
for the sampling site and is typically re-evaluated on a yearly basis. An alert level
highlights a potential problem.
• Action level is a test result that reaches or exceeds the area classification as established
from industry/regulatory guidelines and/or a statistical analysis of historical data. An
action level indicates a possible problem and requires that corrective action be taken.
An investigation is conducted to determine the impact on the product and the root
cause. Examples of environmental action levels for commonly performed tests are
indicated in Table 8-11.

Introduction to Biomanufacturing 315

 Figure 8-7. The three categories of environmental test results

Table 8-11: Example of industry/regulatory guidelines

for action levels for commonly performed environmental testing

Environmental Test Class 100 Class 10,000 Class 100,000

Particulate Air > 3,520 > 352,000 > 3,520,000

(≥ 0.5 microns/m3)

Particulate Air > 29 > 2000 > 20,000

(≥ 5 microns/m3)

Active Microbial Air ≥1 > 10 > 100

(Colony-Forming Unit or
CFU/m3)

Passive Microbial Air ≥1 N/A N/A

(CFU)

Surface Contact Plate ≥1 >5 > 25

(CFU)

Floor Contact Plate >3 > 10 N/A

(CFU)

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 Table 8-11. Continued

Compressed Gas > 3,520 > 352,000 > 3,520,000

Particulate
(≥ 0.5 microns/m3)

Compressed Gas ≥1 > 10 > 100

Microbial
(CFU/m3)

Microbial identification must be performed for all alert and action level results. The identity of
the microorganisms found is useful in understanding the potential impact to product safety and
the possible source of the contamination. For some categories of non-sterile pharmaceutical
products, certain microorganisms are deemed as “objectionable” by the FDA and other
agencies. These microorganisms include:
 Burkholderia cepacia
 Escherichia coli
 Pseudomonas aeruginosa
 Salmonella species
 Shigella species
 Staphylococcus aureus
The organisms specified were chosen because they are either pathogenic (Salmonella),
indicator microorganisms for fecal contamination (Escherichia coli), or index organisms for poor
production hygiene (Staphylococcus aureus). Biopharmaceutical parenteral products must all be
sterile and thus any microorganism in the product must be considered as objectionable. In the
low bioburden processes associated with the manufacture of biopharmaceutical drug
substances, however, each identified microorganism must be assessed on its merits in the
context of its potential downstream impact on the product and the possible routes by which it
entered the production stream rather than by its presence on a list of objectionable microbes.
Environmental excursion investigations
Regulatory agencies require that an investigation be performed whenever an action level or a
recurring alert level is identified from a sampling location. Investigations are performed to
assess the state of environmental control during manufacturing operations in support of the
product release process and the ongoing environmental control requirements for the classified
areas. Investigations must identify the time, location, and conditions related to the sampling
performed. Additionally, the result in terms of particulate count or microbial count versus the
alert and action level criteria for the sampling location must be included in the investigation,
along with the representative microorganism identification. This is so that a possible
contamination source can be determined. The investigation should also include information

Introduction to Biomanufacturing 317

regarding the activities in progress during the environmental sampling, the previous
cleaning/disinfection performed, and any potential causal events. When performing an
investigation, transient events that occurred during sampling and sampling and testing
problems are important points to consider. The investigation should determine a probable
cause, unless it is determined that the excursion is a spurious result, where data do not identify
a likely cause.
Other elements that should be included in an environmental excursion investigation are:
 review of maintenance or other activity in the area that may have contributed to the
excursion
 review of environmental trending results of the sampling site for at least a two-month
time period, looking for other environmental excursions or an adverse trend with
environmental testing results
 review of environmental data from other sites and personnel tests in the room or
system for the week in question. Alert and action levels from these sites should be
included in the investigation report.
 identification of a likely source of the microbial isolate(s) found. A review of other
sampling locations from which the same microorganism was isolated (to determine if
there was any association among the sampling locations) should be performed. This
may help to determine how the microorganism got into the cleanroom.
 performance of a physical inspection of the test site to reveal possible causes of the
action level. This would include checking other environmental parameters as applicable
(e.g., pressure differentials, air flow) to determine what effect any deviations could have
had with the excursion.
 pertinent input and findings in the investigation from personnel involved with the
process
 possible cause and investigation recommendations to the individual(s) responsible for
the affected area
 corrective actions that are being performed at the time the investigation is still being
conducted. Interaction with affected area personnel as needed to facilitate
improvement of the site/area and to obtain follow-up on recommendations
Examples of some corrective actions that may be included as part of an investigation are:
 restricting activities that may increase the bioburden or the total number of
microorganisms detected in or on an article
 increasing the area ventilation, room pressure, or quality of air delivered (e.g. HEPA
filtration)
 testing of process HEPA filters and correcting leaks
 increasing or altering cleaning and disinfecting procedures and using a sporicidal agent

318 Chapter 8 - Microbiological Control

  conducting additional personnel training to reduce practices that may add bioburden
 increasing gowning requirements for the area
 restricting improper personnel flow from less clean areas
 improving facility surfaces (e.g., painting, eliminating rough and irregular surfaces,
eliminating or covering hard to clean surfaces)
 containing process particulates (via hoods or design modifications)
 improving air lock access (e.g., both air lock doors not opened simultaneously)
 removing extraneous items from the area
Excursions that occur in locations that are critical to the biomanufacturing process could
require more rigorous investigation and corrective actions than those occurring in locations
that are less critical.
Environmental test results must be reviewed by a quality group to determine the suitability of a
particular product lot for release to patients. Alert and action levels results are used to monitor
and control aseptic process and should not be treated as product specifications for each
manufactured product lot. The focus, therefore, should primarily be on trending environmental
data and a thorough investigation rather than individual testing results.
Trending environmental monitoring test results
Although non-viable environmental test results are available in real-time, the results of
microbiological environmental monitoring are generally not available until five or more days
after sampling. Product is often filled and even packaged by the time microbiological
environmental results are available. However, near real-time analytical instrumentation is
gaining in use and reducing the time delay in identifying bacteria.
One way in which to combat such delays is to rely on other means for identifying potential
problems or issues. Other systems, such as shift and trend analysis of environmental data must
be established to enable efficient assessment of control and to allow statements to be made
regarding the likely state of cleanliness of locations within the cleanroom. The particulate air,
microbial air, and microbial surface data for each monitored cleanroom site should be
evaluated regularly for the detection of shifts and trends. This evaluation, which involves
plotting the data as graphs, provides for an early warning of a location’s potential inability to
adhere to previously established environmental conditions.
A shift is defined as the tendency for the most recent six-month period to have either higher or
lower results than the previous six-month period. A statistically-significant shift means that
there is strong evidence from the data that the shift is real and not due solely to chance
variation in the results. A trend analysis, on the other hand, is concerned only with the data
from the most recent six-month period. A trend is defined as a tendency for the data to exhibit
a continuous movement, either upward or downward, over a six-month period. A statistically-
significant trend means there is strong evidence from the data that the trend is real and not
due solely to chance variation in the results. Provided there are sufficient data available, trends
toward deteriorating conditions can be detected reliably. Operating parameters within the

Introduction to Biomanufacturing 319

cleanroom, such as temperature, humidity, air flow, and pressure differential between rooms
are typically trended hourly. The data are reviewed on a periodic basis to provide additional
assurance that cleanroom operations and maintenance are performing consistently and to
assure that the appropriate data are available for investigational purposes.
Test methodology
Environmental monitoring is accomplished using a variety of equipment, such as airborne
particle counters for non-viable particles and active air samplers for microbial detection. Any
movement of the environmental monitoring equipment between production areas must be
controlled to prevent cross contamination. Sample takers must disinfect all equipment prior to
use. Ideally environmental monitoring equipment should be dedicated to a specific production
area in a cleanroom and not moved to other production areas. Simpler methods of
environmental monitoring, which require no sampling equipment, include the use of direct
contact plates and settle plates for microbiological sampling.
Environmental testing is normally performed under dynamic conditions. Dynamic, or “in
operation,” testing confirms that the environment remains under control during processing
with personnel and activities in a normal operational mode. Static, or “at-rest,” testing is
typically only performed as part of the initial Performance Qualification or requalification of a
cleanroom. Static testing ensures that the facility environment continues to perform as
designed, with no personnel present and no activities in progress.
Microbiological testing requires the use of growth media to demonstrate the recovery of
microorganisms from sampling, while non-viable testing does not require the use of growth
media. A general growth medium such as soybean casein digest agar (i.e., tryptic soy agar, TSA)
is used in Petri plates for microbial air and microbial surface monitoring. Where the media is
used for surface contact, the plates should contain a disinfectant neutralizer, such as
polysorbate 80 for phenolics or lecithin for quaternary ammonium compounds. The growth
promoting quality of the culture media must be demonstrated as part of the normal laboratory
quality control. Various guidance documents suggest incubation times and temperatures.
The following example is an acceptable method used in the United States. After sampling, the
microbial plates are incubated at 20–25◦C for 72 hours, followed by incubating the plates at 30–
35◦C for 48 hours. At the end of this five-day time period, the plates are examined for microbial
growth and compared to established values for sampling locations. Those sampling sites
exceeding action and alert levels require identification of each microorganism recovered from
testing. Any method used for sampling and testing must be validated. The sample must be
reflective of the sampled location. Aseptic techniques must be used in the sample collection.
The samples must then be promptly transported to the lab for microbiological testing.
Furthermore, sampling must be performed in a way that has no adverse effect on the aseptic
process being monitored. It is important to note that the sample taker and the equipment are
always potential sources for product contamination.

320 Chapter 8 - Microbiological Control

Air monitoring
Research has shown that airborne contamination (versus surface contamination) has the
highest potential for adversely affecting the product quality. It is for this reason that air
monitoring is routinely conducted for both microbial and non-viable contamination in
cleanrooms.
Microbial air monitoring
There are two general methods by which microbial air testing for viables is performed in the
cleanroom environment by active microbial sampling and passive microbial air sampling. Active
microbial air sampling requires the use of equipment, typically requiring a vacuum source to
draw air over the surface of an agar plate. Passive microbial air sampling does not require any
specialized sampling equipment and viables in the air fall on an agar plate through
sedimentation. Neither method provides real-time results, however, as incubation of a growth
medium is necessary.
The standard unit of measurement for microbiological monitoring is the Colony-Forming Unit
(CFU). Active air microbial measurements are stated as CFUs per cubic meter. Passive microbial
air sampling measurements are stated as CFUs/4 hours or per unit of time period as dictated by
process duration and/or operational factors. Test results for each of these methods of microbial
monitoring are available after a minimum five-day incubation of test plates.
Active microbial air monitoring
Active microbial air monitoring is the preferred approach for classified areas due to the greater
likelihood of obtaining actual microbial counts. Active sampling devices draw both air and any
microorganisms contained in the air into the instrument. The air is diverted over a culture
medium and any microorganisms are deposited onto it. Due to this active mechanism, the
sampling duration is less than that of a passive air sampling. The volume of air sampled should
be adequate enough to provide counts in most samples. This is normally one cubic meter in
Class 100 and 10,000 areas and one cubic foot in Class 100,000 areas. Active airborne microbial
air samplers must be calibrated to manufacturer’s specifications on an annual basis.
There are a variety of samplers/methodologies used in the biopharmaceutical industry for
active microbial air sampling. All the samplers measure known volumes of air, allowing
quantification of microbial contaminants by unit volume of air. The selection of a sampler or
methodology should address the volume of air that can be sampled, the availability of culture
media plates, and the level of technical and vendor support. Following is a brief description of
some commercially available active microbial air samplers/methodologies. This list is for
informational purposes only and not intended to be all inclusive. It does not constitute
endorsement or support of any vendor/product.
 Sterilizable Microbiological Atrium (SMA): The SMA uses a simple stainless steel
chamber (Figure 8-8). The unit’s cover, or atrium, contains uniformly-spaced orifices.
The base accommodates one standard 100mm agar plate. Air is drawn through the unit

Introduction to Biomanufacturing 321

by vacuum and allowed to impact the agar surface. Units are available that use a house- vacuum
source, while other models use a self-contained vacuum source.

Figure 8-8. SMA is one method used

for active microbial air sampling

 Slit-to-Agar (STA): with the STA sampler, a known volume of air is drawn in by a self-
contained vacuum pump through a standardized slit. Below the slit is a slowly revolving
agar plate. Particles in the air are drawn through the slit and impact the agar surface.
The STA machine requires a large 150 mm agar plate.
 Centrifugal Air Sampler: the centrifugal air sampler, such as the Reuter Centrifugal
Sampler (RCS), consists of a propeller or turbine that pulls in a known volume of air then
propels the air outward to impact on a tangentially-placed nutrient agar strip.
Each of the above active microbial testing methods requires an agar incubation period. The
agar plate or nutrient agar strip is incubated for a minimum of five days. Colonies of microbial
growth, bacteria, and fungi are then counted and expressed as CFUs.
Passive microbial air monitoring
Passive microbial air monitoring uses settling plates, or exposure plates. The process involves
exposing agar-filled Petri dishes to the environment. To perform the test, a 100 – 145 mm agar
plate is placed in the sampling location. The lid of the agar plate is removed and placed in a
location to ensure the lid itself does not become a contamination vector (edges down and on a
freshly disinfected flat surface). Airborne microorganisms randomly settle by sedimentation on
the agar surface during a specified time period. Exposure times of settle plates greater than
four hours should be avoided so that the media does not dry out.
Although using settling plates best reflects the number of CFUs that naturally settle on surfaces
by gravity, it is qualitative rather than quantitative in nature. This may mean lower count results
in classified areas. Therefore, settle plates are used to supplement active air sampling. This

322 Chapter 8 - Microbiological Control

method provides more data to support the demonstration of control within an aseptic
processing area and can also be used in areas where active air sampling is not feasible. Settle
plates can also be useful if continuous monitoring is required (e.g., close to the product's
exposure position).

Non-viable particulate monitoring

Area classifications within cleanrooms are generally based upon the level of non-viable
particulates in the air. Unlike microbial air monitoring, non-viable or particulate air monitoring
provides real-time data on the environment and is a useful tool to demonstrate that the
environment remains in a state of control with respect to particulate contamination.
It is difficult to draw a direct correlation between particulate monitoring data and microbial
contamination—a high particulate count for a specific testing location does not mean that
there will be a concurrent high microbial count at the same location. Airborne Particle Counters
measure particles in a number of size ranges then display the number of particles in each range
as a cumulative count or differential count. Although monitoring non-viable particles in
different size ranges may seem practical, particles of 0.5 microns and larger are generally
recognized as indicators of environmental contamination. Particulate monitoring for products
used outside the United States may also include the need to monitor 5-micron particles.
There are numerous manufacturers for airborne particle counters, however, the
sampler/methodology is very similar. A commonly used non-viable particle monitoring method
is optical particle counting in which the vacuum pump of the particle counter pulls sample air
into the sensor through the inlet nozzle. Particles in the sample pass through the optical
detection view where a laser light source is concentrated. Particles scatter the laser light, which
is then focused onto a photodiode. The photodiode detects and converts the light signal to
electrical pulses. The height of the electrical pulses is directly proportional to the particle size.
The electrical pulses are counted and measured by electronics on a circuit board containing
counter/threshold circuitry, a microprocessor (CPU), and communications circuitry. The
microprocessor displays the count on the front panel as the total particle count in specified size
ranges (Figure 8-9). Non-viable particles test results can be expressed as the number of
particles ≥ 0.5 microns and ≥ 5.0 microns per cubic meter or cubic foot.

Introduction to Biomanufacturing 323

 Figure 8-9. Non-viable portable particle
counter displaying real-time test results
Particle counters come in two general varieties, portable and permanent. Portable particle
counters are easily transportable, allowing an individual to test one location at a time.
Permanently-installed particle counters use dedicated sample probes that allow for remote
sampling capabilities in biomanufacturing areas, thus minimizing equipment and sampling
personnel in these areas. They also allow for automatic continuous particulate monitoring of
the biomanufacturing process in multiple locations, provide for centralized data storage, and
have alarm capabilities once particulate action or alert levels for a monitoring site are
exceeded.
The minimum particle count sample volume is one cubic meter of air for Class 100 and Class
10,000 areas. For Class 100,000 areas, a lesser sample volume may be used, but should not be
less than 1 cubic foot. Results are expressed as either particles per cubic meter or particles per
cubic foot.
Surface and equipment microbial monitoring
Microbial monitoring of surfaces is performed either per process testing, routinely, or as an
investigative tool. All techniques must be qualified using standard laboratory procedures to
demonstrate the recovery capability of the method. Contact impression plate and swabbing
techniques provide a means for direct microbial monitoring of surfaces.
Contact plates are Petri plates containing solid nutrient agar where the culture medium’s
convex surface extends above the walls of the base plate. A commonly used contact plate
method is called Replicate Organism Detection and Count (RODAC). RODAC impression plates
are specifically constructed so that an agar medium can be over-filled, producing a meniscus or
dome-shaped surface that can be pressed onto a surface for sampling its microbial burden

324 Chapter 8 - Microbiological Control

(Figure 8-10). RODAC plates are 24–30 cm2 in area. During sampling the raised surface of the
agar plate is gently rolled against the test surface. Once sampling is completed, the lid is placed
back on the agar plate and the plate is incubated to determine the number of CFUs recovered
(Figure 8-11). Contact plate measurements are stated as CFUs/plate.

Figure 8-10. RODAC plate is a contact impression plate used for

microbial monitoring of surfaces

Figure 8-11. RODAC plate showing colonies of bacterial growth

Swabbing is used for irregularly-shaped cleanroom surfaces, such as equipment surfaces, that
are generally difficult to reach with contact plates. The swab should be pre-moistened with
sterile culture media prior to sampling. Approximately four square inches of the test surface is
swabbed by rolling it until all surfaces are in contact with the swab. Swabs are then submitted
to the microbiology laboratory for processing. After collecting the surface sample by either
contact plate or swab, the surface sampled must be immediately disinfected by the sample
taker.

Introduction to Biomanufacturing 325

Microbial monitoring of gowned personnel
Since people are the greatest source of contamination in a cleanroom, proper aseptic gowning
and aseptic technique is vital to ensure product quality. Personnel microbial monitoring serves
as a tool to demonstrate that workers are adequately trained and following proper gowning
procedures and aseptic technique. All individuals working in a cleanroom must undergo initial
qualification testing to demonstrate proper gowning techniques prior to being permitted into a
cleanroom. Routine periodic gown and fingertip microbial testing is performed on all personnel
entering the cleanroom. This testing is performed at a greater frequency (usually based on the
process) for those individuals involved with aseptic processing and for those who perform
environmental monitoring. At a minimum, microbial sampling using contact plates should be
done on the chest, forearm, and the fingertips of both gloved hands (Figure 8-12 and Figure 8-
13).

Figure 8-12 Microbial personnel testing: gloved fingertip RODAC testing

Figure 8-13. Microbial personnel testing: chest RODAC testing

326 Chapter 8 - Microbiological Control

Utility monitoring
During the Initial Qualification (IQ) of a process utility system, it is assured that the actual
performance is consistent with the required performance. A system is put in place to assure
that these performance criteria are met when used during biomanufacturing processes. Thus,
all process utilities that have contact with the product must be monitored on an ongoing basis.
Examples of the utilities used in cleanrooms include compressed gases, clean steam, and Water
for Injection (WFI). Test data from each process utility system should be evaluated on an annual
basis to ensure that required quality is consistently met, to characterize seasonal variations,
and to reveal adverse trends. This section of the text will only focus on the methods used to
monitor one of these process utilities -high quality water systems. The methods used for
monitoring compressed gases for viables and non-viables are the same as those described in
the previous sections for air monitoring. Clean steam is tested to the same criteria as high
quality water.
High purity water system monitoring
Even though tap water is safe to drink, it should never be used to make sterile medicinal
products or to clean equipment used in their manufacture. Although tap water (potable water)
must meet the regulations for drinking water of the country in which it is used, it still contains
numerous microorganisms and it is not considered high quality water. In the United States up
to 500 CFUs of microorganisms per ml of water (with the exception of coliforms) are allowable
in tap water.
Since a greater level of control is needed for water used in biomanufacturing, high purity water is
used. One type of high quality water is Water for Injection, which is made by filtering and
distilling potable water (Figure 8-14). For more information of WFI, refer to the Facilities
chapter.

Figure 8-14. WFI system

The United States Pharmacopeia (USP) specifies that water used as an ingredient in the

Introduction to Biomanufacturing 327

manufacture of sterile pharmaceuticals and for cleaning equipment must meet their WFI
standards. Once a WFI system is validated for use in a facility and the WFI system is shown to
be in-control, appropriate water samples should be taken periodically from the water system
holding tank and distribution system. For water that is used as an ingredient, samples should be
taken daily at the points of use. Water used for other purposes can be tested weekly. The tests
must assess the microbiological quality (colony count, indicator microorganism, and endotoxin
testing).
Regardless of the type of sampling or the frequency, water samples need to be collected in a
manner consistent with appropriate biomanufacturing practices. For example, if points of WFI
use are routinely flushed prior to use, it is appropriate for water samples to be collected with
the same flush cycle. If points of use are not normally flushed prior to use, there should be no
flush prior to water sample collection. If biomanufacturing practices require the use of hoses,
the sample should be taken from hoses and not directly from the point of use. If the WFI
sample cannot be processed immediately for microbial testing, the sample needs to be
refrigerated at 2–8◦C .
The minimum quantity to be aseptically collected in a sterile container for WFI microbial testing
is 200 mL. Multiple containers are required for collecting WFI samples, as samples for other
testing, such as endotoxin and chemical testing, are collected concurrently. Water from some
sampling sites may be extremely hot, so to prevent the sample taker from getting burned, the
appropriate safety equipment (protective gloves, apron, etc.) must be worn.

To perform microbial colony counts on WFI, a plate count is performed by membrane filtration
of the water sample with subsequent incubation of the 0.45 micron membrane filter on a low-
nutrient agar. Incubation time of the nutrient agar is a minimum of five days at 30–35 degrees
Celsius. Representative colonies from all action and alert level growth need to have microbial
identification tests performed. Membrane filtration with selective culture media is used to
screen WFI for objectionable bacteria such as Pseudomonas aeruginosa, Burkholderia cepacia,
and coliforms. When a WFI sampling site test fails, it must be investigated to determine if
product quality is in jeopardy. A failing WFI site test result can result in product quarantine, at
the very least, and could result in product discard.
Media fills
In addition to monitoring the cleanroom environment through an appropriate environmental
monitoring program, media fills of each process must be performed. The purpose for
performing media fills is to demonstrate the capability of a specific aseptic process to produce
sterile drug products, to qualify or certify aseptic processing personnel, and to comply with
regulatory requirements. A media fill is a simulation of an aseptic process that uses microbial
growth-promoting culture media in the place of actual product. In new cleanroom facilities, or
when an aseptic process is developed, it is generally necessary to qualify the microbiological
status of the process by running at least three consecutive media fills. For existing facilities or
processes, a routine media fill should be conducted on a biannual basis for each type of aseptic
process performed. It is important that the conditions that would normally occur during the
aseptic process (e.g., full complement of personnel present, all processing steps performed) be
included as part of the media fills. The resulting containers of media are incubated for a

328 Chapter 8 - Microbiological Control

 minimum of 14 days to ensure no microbial growth. Any growth in the containers, along with
any microorganisms recovered during the environmental monitoring performed during the
media fill, is identified in order to aid in the investigation of the sources of contamination.
Failure to pass a media fill requires an investigation and additional satisfactory media fills
before processing of product can either begin or continue.
Microbial identification
Identification of microorganisms recovered during microbial monitoring is an important
component of the environmental monitoring program. Identification of microorganisms
isolated from environmental monitoring should start with a pure culture. Normally, distinct
colonies are sub-cultured from the original sampling culture medium to a nutrient agar using
the streak plate method. Once a pure culture of a microorganism has been isolated, the
microorganism is examined microscopically to determine its morphology and response to the
Gram stain.

Following microscopic examination, colonies from the pure culture of the microorganism are
identified using either phenotypic methods (i.e., observable physical or metabolic properties) or
genotypic methods (i.e., characterization of some portion of a bacterium’s genome using
molecular techniques for analysis of DNA or RNA). All microorganisms isolated from Class 100
and Class 10,000 areas must be identified at the genus and species level. Microorganisms from
Class 100,000 areas should be characterized at least to Gram stain reaction and microscopic
morphology. Identification of mold from all sampling sites at the genus and species level is
typically performed by both macroscopic (i.e., visual description of colony) and microscopic
methods. Periodically all microbial growth (not only excursions but passing level sampling as
well) should be identified to the genus and species level to ascertain the microbial flora of the
cleanroom. A change in the microbial flora or the introduction of a previously undetected species
should be investigated, as this might signal a breakdown in established controls for the
cleanroom.
Utilization of information derived from environmental monitoring
Environmental monitoring programs typically generate ample amounts of data on an ongoing
basis. It is not uncommon to have hundreds or thousands of environmental monitoring results
in one week. The amount of data will typically be a function of the size of the facility and the
level of production activity. These data are gathered in a highly programmed manner, based on
environmental monitoring SOP requirements.
Once the data is gathered (generally the data is gathered and interpreted by the specialists in
the facility's QC Microbiology lab), the information must be converted into knowledge, which
then needs to be actively managed and communicated. It is vital to understand what is
happening to the microbiological profile of the facility and this knowledge needs to be
communicated to people at various levels in the organization, especially if any subsequent
action is required. The data must be monitored to ensure that the area either remains in
control or regains control if previously lost.
The pattern of data is an outcome of the actions and activities of all those who work in the facility,
whether they are production personnel, warehouse personnel, or various support personnel (e.g.,

Introduction to Biomanufacturing 329

engineers). Part of the Knowledge Management (KM) task is to ensure that the right personnel
receive the right information/knowledge at the right level of detail so their actions can be
productive. The practice of Knowledge Management was formally introduced into pharmaceutical
manufacturing in ICH Q10 and involves acquiring, analyzing, storing, and disseminating
information (Figure 8-15). According to ICH Q10, KM and Quality Risk Management are concepts
that “will enable a company to implement ICH Q10 effectively and successfully” and “will facilitate
achievement of the objectives described in Section 1.5 above by providing the means for science
and risk-based decisions related to product quality.”

Figure 8-15. Knowledge Management methodology

ICH Q10 describes the expectations regarding Knowledge Management as follows:

“Product and process knowledge should be managed from development through the
commercial life of the product up to and including product discontinuation. For
example, development activities using scientific approaches provide knowledge for
product and process understanding.”
It defines KM as:
“...a systematic approach to acquiring, analyzing, storing, and disseminating
information related to products, manufacturing processes, and components.
Sources of knowledge include, but are not limited to, prior knowledge (public
domain or internally documented); pharmaceutical development studies;
technology transfer activities; process validation studies over the product lifecycle;
manufacturing experience; innovation; continual improvement; and change
management activities.”
In environmental monitoring, Knowledge Management plays a vital role in the collection and
dissemination of information, and in order for the process to be successful, the following
questions should be considered:
 What is the data?

330 Chapter 8 - Microbiological Control

  What does the data mean (i.e., what knowledge can we obtain from the data)?
 How is this knowledge owned by the area management?
 How are those who work in the area kept informed of the requisite knowledge?
 How does one monitor the effectiveness of the actions undertaken and have effective
investigations in this area?

Quality Control Practices in the Microbiology Laboratory

A quality control program is essential in ensuring reliable, reproducible, and accurate test
results in the microbiology laboratory. Quality control practices help to both assure effective
laboratory operation and avoid delays in issuing test results. Both governmental agencies and
industry professional organizations provide guidelines for developing and maintaining QC
programs. And though many expendable supplies procured from commercial sources have
been tested for quality control by the manufacturer, in many cases a repeat assessment must
be performed in the microbiology laboratory prior to using the supplies. The amount of time
allotted to QC will vary with the size of the laboratory and the variety of procedures employed.
While there are a variety of facets to a QC program, many of which are discussed in the Quality
Assurance chapter, the following section will focus on two key aspects that are unique to
microbiology laboratories - the quality control of microbiological culture media and the
maintenance of microbiological stock cultures.
Quality Control of microbiological culture media
The quality of the work performed in a microbiological laboratory depends in part on the
quality of the culture media. The importance of maintaining the quality of the culture media
cannot be overstated, as there are few things in the microbiology laboratory that will lead to
problems with every aspect of laboratory operations. Culture media are either at or near the
top of that short list.
The three primary aspects of the quality control of culture media used for microbiological
product sampling and environmental monitoring are:
 the control of the culture media preparation and storage conditions
 the physical and chemical characterization of culture media
 growth promotion testing
Each laboratory must have procedures in place to ensure that culture media that have not
undergone quality control testing are not used for microbiological testing procedures. Ideally
this would best be accomplished by having a separate storage room for tested and non-tested
types. This can also be accomplished through tagging quarantined materials and placing them
in a clearly identified area within the same room. All quality control checks on the quarantined
culture media should be completed before its documented release for general use. Storage
conditions of the quarantined culture media should match those of the released media.
Control of culture media preparation and its storage conditions
The culture media that microbiology laboratories use are obtained in one of two ways - either

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through producing the culture media in-house or by purchasing the culture media pre-made
from a vendor. These preparation methods must be considered separately.

Culture media prepared in-house offer several opportunities for quality control. The raw
materials (either the dehydrated complete media or the components) must be stored under
appropriate and controlled conditions and used within established expiration dates. The
compounding of the media must be controlled to ensure the media is prepared correctly to
prevent such errors as incorrect weighing, incorrect water measurement, improper mixing of
ingredients, and use of deteriorated stock powders. Agar media, for example, must be pre-
warmed to dissolve the agar prior to sterilization but not heated so extensively as to damage
any heat-labile components. Also, the sterilization procedure must be under control. This
means using a validated autoclave cycle shown to hold the media at 121◦C for at least 15
minutes. Each batch of culture media made should be clearly labeled to allow for unambiguous
audit of each stage of preparation.
For culture media that is purchased from a vendor there is little opportunity to control the
preparation. However all media used should be checked for physical and chemical parameters
and growth promotion. As with in-house culture media, commercially-prepared media must be
stored under controlled conditions to ensure its quality through its expiration date. Key
conditions to be controlled during culture media storage include temperature, humidity, and
exposure to light. Although these factors may not be a concern for all culture media, they can
be a concern for certain types. For example excesses of heat and cold, in particular, must be
avoided for all types of media, while exposure to light will only affect certain ones.
There is variance in the expiration dates of culture media as well. The expiration date for in-
house culture media must be established by the laboratory and the expiration date for
commercially produced culture media is set by the manufacturer. Furthermore, there are
specific regulatory requirements regarding the expiration of the culture media used for certain
laboratory tests.
One of the most common problems with culture media storage is the failure to take adequate
steps against dehydration of the media. Dehydration should not be an issue with liquid or solid
media kept in well-sealed containers but can potentially occur when agar plates prepared in-
house are stored, as they are typically not placed in sealed containers after preparation. Media
that show obvious signs of dehydration (e.g., separation from edges or cracked surfaces) must
be discarded. Dehydration can be significantly reduced by sealing agar plates in plastic bags
immediately after preparation. The packaged culture media plates should then be stored in a
refrigerator.
Control of physical and chemical characteristics
The initial goal of evaluating the physical and chemical characteristics of culture media is to
rapidly screen the culture media for acceptability before investing time in labor intensive tests
such as growth promotion testing. If the physical and chemical characteristics do not meet the
specified requirements of the culture media, the culture media should be discarded. When a
new batch of media is prepared in-house or received from a commercial vendor, it should first
be examined visually for clarity and color. Unless the media contain a normally insoluble
component, the presence of a precipitate or turbidity indicates that a component of the media
332 Chapter 8 - Microbiological Control
has come out of solution. If the precipitate goes back into solution when the culture media is
brought to temperature by incubation, the media is satisfactory to use. Otherwise it should not
be used. The color of the media should then be examined and a decision should be made as to
its correctness. It should also be checked for any crystal formations or variations in color. The
pH of the finished culture media is another critical attribute to confirm. The containers of media
should also be thoroughly examined for cracks or defects and all defective units discarded.

Growth promotion testing

It is required that each new batch of culture media either prepared in-house or received from a
commercial manufacturer be selectively tested with compendial microorganisms to ensure that
the culture media supports their growth. In addition to compendial microorganisms which are
required for the growth promotion testing by regulatory agencies, other stock microorganisms,
such as those frequently recovered by a specific microbiology laboratory in its testing of a
facility, should be included as growth promotion testing as well.
One commonly used method for growth promotion testing is the Miles and Misra method, also
known as the surface variable count method. In this method, culture media plates are divided
into sectors, and drops of serial dilutions of bacterial suspensions are deposited into the
separate sections. The culture media plates are incubated for a minimum of 18 hours at 37◦C
and observed for bacterial growth expressed as CFUs. If there is either no growth or insufficient
growth, the batch of culture media must not be used.
Maintenance of microbiological stock cultures
Along with culture media quality, preserving the stock culture microorganisms is also vital to
the success of a microbiology laboratory. Since lab procedures require a pure culture, stock
culture microorganisms must be handled carefully at all times to avoid contamination.
The care of the stock culture microorganisms begins upon receipt. A careful stock culture
keeper in the laboratory will confirm the identity of the received cultures even those received
from a respected source (e.g., a national culture collection), as mistakes can occur with even
the most reputable of providers. The use of an incorrect strain in a compendial test has the
potential to bring the results of weeks or months of work into question.
Standard strains of microorganisms that conform to the typical morphological, physiological and
biochemical characteristics of species represented are needed not only for quality control
testing of culture media but also for reagents and various other microbiological procedures. The
strains used for stock cultures possess sufficient stability to display these characteristics
reproducibly if they are maintained under proper conditions.
There are several methods commonly used for maintaining stock cultures (including simple ones
like storing the culture media at the right temperature—either room temperature or in a
refrigerator as necessary) and a sufficient stock culture collection can be maintained at little
expense and requires relatively little lab personnel time. The culture media used to maintain the
stock cultures must be chosen carefully for their ability to maintain the stability and viability of
microorganisms over long periods of time without permitting excessive growth or metabolic
activity. There are also more complex methods for maintaining stock cultures that require
special equipment, such as lyophilization or freeze drying. Lyophilization is particularly effective

Introduction to Biomanufacturing 333

for long-term preservation of most microorganisms and most can be preserved indefinitely with
this technique.

Barrier Isolation Technology

There is an intrinsic incompatibility between human beings and aseptic processing. This means
that there will always be the risk of product contamination from people involved with
performing the processing unless the two are completely separated. Barrier isolation
technology can accomplish this.
Barrier isolators are airtight enclosures constructed of an impervious material, such as
transparent PVC, that protects an internal Class 100 environment from the outside uncontrolled
environment. Processing operations within this barrier are conducted either through remotely
controlled machinery or from the outside, with individuals wearing half body suits or rubber
arm length gloves. Although many organizations in the pharmaceutical industry still rely on
aseptic processing in cleanrooms, some are moving toward increased use of barrier isolation
technology. It is outside the scope of this chapter, however, to address this technology in more
detail.

334 Chapter 8 - Microbiological Control

Check Your Knowledge
1. Contaminants can be introduced into the product by:
a. equipment
b. people
c. work surface
d. all of the above
2. Microbial contamination is caused by:
a. gown fibers
b. dead skin cells
c. bacteria
d. dust
e. all of the above
3. The term aseptic means:
a. sterile
b. the absence of microorganisms capable of causing disease or contamination
c. gamma irradiation
d. all of the above
4. is an example of a non-viable particulate.
a. yeast
b. cellulose fiber
c. bacteria
d. mold
5. All of the following are effective means of sterilization except:
a. steam
b. gamma irradiation
c. alcohol
d. dry heat
6. Which of the following is a means of controlling contaminants in the cleanroom?
a. aseptic gowning and utilization of aseptic technique
b. sterilization of manufacturing components
c. controlling the supplied air
d. all of the above
7. RODAC testing is performed on:
a. product
b. fingertips
c. gowned personnel and critical sites within the cleanroom
d. b and c

Introduction to Biomanufacturing 335

 8. Proper cleanroom conduct includes the following:
a. disinfecting gloves upon using a BSC
b. never touching a critical surface
c. limiting talking
d. all of the above
9. One of the basic principles of aseptic technique is that product and critical surfaces
must see "first air" from HEPA filtered air. This means:
a. only product and components should be under the BSC
b. nothing must pass between the HEPA filter and the product
c. all aseptic manipulations should be the first things performed for the day
d. a new HEPA filter should be installed prior to performing aseptic manipulations
10. Which of the following is not a practice to keep biopharmaceutical products
contaminant-free:
a. careful visual inspection of all final product containers
b. BSCs
c. controlling the environment, employees, and equipment
d. filtering prior to filling the final product containers

Activities
1. Working in groups of three, each person is to prepare three different samples of the
environment. Using a sterile swab for each, sample two inanimate objects (e.g., the
floor, a table top, a doorknob, a cell phone, etc.). Using a different nutrient agar plate
for each sample, roll the swab over the surface of the nutrient agar plate. Next, expose a
third nutrient agar plate to the room air by removing the lid from the plate for 30
minutes then replacing the lid. Ensure that you label each agar plate with the sampling
location, your initials, and the date sampled. If you have a 37 ◦C incubator available,
incubate the agar plates for 48 hours—otherwise leave the agar plates at room
temperature. After 48 hours observe each of the three nutrient agar plates and record
the number of microbial colonies, along with their color and size. What surfaces and
what area of the room had the most microbial colonies? Write a paragraph explaining
the variation in the number of colonies found at the different locations. Compare your
group’s findings with the rest of the class.
2. Perform microbial sampling of your fingertips by gently pressing four fingertips from
your right hand on the surface of a nutrient agar plate. Repeat the sampling of your
fingertips using another nutrient agar plate after washing your hands. Ensure that you
label each agar plate with the sampling location, your initials, and the date sampled. If
you have a 37 ◦C incubator available, incubate the plates for 48 hours. Otherwise leave
the agar plates at room temperature. After 48 hours, observe both nutrient agar plates
and compare your results with those of your classmates. What is the primary role of
hand washing and or frequent disinfection of gloved hands? Were you able to
demonstrate this with the activity?

336 Chapter 8 - Microbiological Control

 3. Using the Internet, research the FDA website ([Link]) and find two different
regulatory inspections involving a deficiency (FDA 483) in the environmental monitoring
program. Write a one-page paper summarizing these deficiencies as well as any actions
that could have been taken to prevent them.

Introduction to Biomanufacturing 337