2.68
Aseptic Operations
D Pollard, Merck Research Laboratories, Rahway, NJ, USA
© 2011 Elsevier B.V. All rights reserved.
2.68.1
2.68.2
2.68.2.1
2.68.2.2
2.68.3
2.68.3.1
2.68.3.2
2.68.3.3
2.68.3.4
2.68.3.5
2.68.3.6
2.68.4
2.68.4.1
2.68.4.2
2.68.4.3
2.68.4.4
2.68.4.5
2.68.5
2.68.5.1
2.68.5.2
2.68.5.3
2.68.6
2.68.6.1
2.68.6.2
2.68.6.3
2.68.6.4
2.68.7
References
Introduction
Design and Procedural Approaches to Minimizing Contamination
Room Classification Considerations
Utility Design Considerations
Fermentation/Cell Culture Considerations
Equipment Design
Use of Disposables
Equipment Setup
Considerations for Raw Materials and Media Preparation
Seed Expansion Considerations
Vessel-to-Vessel Transfers
Considerations for Purification and Formulation/Fill
Liquid Filtration
Considerations for Applying Disposable Technology to Purification
Formulation and Fill Considerations
Maintenance Considerations
Training of Personnel
Validation and Verification
Sterilization Considerations
Sanitization Considerations
Sanitary Cleaning Issues
Sterility Analysis and Culture Purity
Procedures Used for Process Sampling
Procedures for Testing of Contaminants
Criteria for Contaminated Samples
Strategy for Contamination Investigation
Summary
Glossary
contamination Accumulation of undesired biological
particles from either a microbial, a viral, or a fungal
source, which reach levels above the acceptance criteria set
for the specific process step.
sanitization A set of conditions typically chemical, such
as disinfectant or caustic washes, for the reduction of
bioburden within equipment or upon surfaces within the
processing facility http://en.wikipedia.org/wiki/File:
Disinfection.jpg.
sterility analysis The application of test methods to
quantify the level of contamination within a process. This
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can be from online process attributes or direct
measurement from sampling.
validation Actions or activities that establish by
objective evidence that a process consistently produces
a result or product meeting predetermined
requirements.
viral clearance A measure of the capacity of a
purification process to remove virus particles from
the final drug bulk product. Typically, this includes
multiple steps for multiple log reductions such as pH
inactivation hold step and through specific filtration
steps.
2.68.1 Introduction
The aseptic operations during any bioprocess are vital for controlling the desired level of microbial load (bioburden) of a given
biological product. Manufacturers follow the standards and procedures set by the regulatory agencies [1, 2] to ensure product
quality. This minimizes the risk to patients of exposure to unacceptable levels of contamination such as failure to maintain product
sterility. Practical and experience-based practices, not captured in regulatory documents, have been defined by organizations [3–5]
and harmonization conferences [6, 7]. These regulatory compliant procedures are based upon rational, evidence-based science, and
engineering with incorporation of risk assessment analysis.
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Other Considerations
The manufacturing of sterile products is acknowledged to be the most difficult of all pharmaceutical production activities [3]. For
bioprocess production, such as aqueous protein or monoclonal antibody solutions (Figure 1), some form of aseptic operations
usually encompass every production step from fermentation, purification, formulation, and fill. It is a regulatory requirement to
assure that culture purity (single organism) is maintained from the master cell bank and throughout the upstream step (Figure 1).
During purification, maintaining the low bioburden specifications of the drug substance is completed using a combination of
chemical sanitization of equipment and filtration of all buffers, including those used for formulation. After formulation the final
drug product is filter sterilized and aseptically filled into the final container (vial, syringe, or IV bag) and, in some cases, lyophilized.
Managing contamination to minimal levels covers a wide range of activities, including facility design, equipment setup, process
operations, process validation, process monitoring, and personnel training (Figure 2). Despite all these efforts including attention
to detail for process operations, contaminations will always occur as no microbial fermentation or cell culture facility is contam­
ination free. So procedures need to be in place for vigorous investigations of contamination to build experience and a knowledge
database. Literature contamination rates of 5–30% have been described for microbial fermentation with a contamination prob­
ability of 1 out of 100 acceptable [8]. Examples of rates <1% are considered commendable and indicated as good performance
[8–10], while rates of 2% have been recorded for animal cell culture [8, 11, 12]. Facility improvements have shown to lower
contamination rates. For example, the contamination rate for monosodium glutamate production was reduced from 4.5% to 0.6%
by a combination of sparger air system upgrades, installation of a laminar flow hood in the inoculum room, and repair of holes in
the heat exchangers of the continuous sterilization system [11].
For the microbial fermentation or cell culture process, microbial contamination is the most common cause of process
failure over mechanical, electrical, or instrumentation problems that occur [10]. Microbial contamination can impact the
process by changing the chemical conditions such as the conversion of nutrients to unwanted impurities, changing the pH, and
triggering the formation of enzymes leading to product degradation [8]. Historically, there are only a few examples of facilities
that will continue to process contaminated batches beyond fermentation. These were usually for small-molecule natural
products/anti-infective products where the subsequent chemical steps achieved sufficient purity and removed contaminants.
This is certainly not an option for the injectable products for biologics and vaccines. Upon contamination detection, the entire
B
D
Raw
materials
D
Nutrient
addition
D
Media
prep
S
S
D
Buffer
D
S
D
D
S
0.22-µm
filtration
Microfiltration
Seed train
Disc stack centrifuge
B
Ultrafiltration
D
B
D
B
D
Production fermentor
(1000–5000 l)
D
D
Buffer
Buffer
D
Buffer
D
Buffer
Sterile fill for drug product
S
Sterile fill
(vials, syringe,
IV bags)l
B
D
D
Filtration (0.22 µm)
Intermediate product reservoir
(sterile disposable bag or stainless steel tank)
D
Polishing 2
chrom
B
Polishing 1
chrom
Capture
chrom
Ultrafiltration
Drug substance
Bioreduction Final formulation
Filtration 0.22 µm
D Disposable option is available
S
Sterility testing
B
Bioburden testing
B
D
Product
intermediate
filtration
B
D
B
D
Figure 1 Bioprocess map for production of a therapeutic protein or monoclonal antibody using microbial fermentation using a Pichia expression host.
Stages for sterility and bioburden testing are indicated. Steps that have disposable options are indicated.
Aseptic Operations
Raw
materials
Facility and
Equipment
design
Process
design and
operational
procedures
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Process
monitoring
Preventative
maintenance
Minimizing
contamination
Sterility
sampling and
testing
(low contamination rate and
bioburden control)
Personnel
training
Contamination
investigation
Regulatory
framework
(agency
interaction)
Microbiological
process
controls and
validation
Knowledge
database
Figure 2 Integrated approach for control of contamination in bioprocessing.
batch is discarded, equipment shutdown, and a failure investigation initiated. This causes substantial losses of time, materials,
and revenue, with disruption to the facility schedule.
Contaminants vary by product type but the most frequent microbial contaminants are from two forms: (1) fast-growing sporeforming Gram-positive bacteria such as Bacillus subtilis, associated with incomplete sterilization such as from large-medium particles
or residual dried batch in vessel crevices [10, 13]; and (2) Gram-negative rods, which are indicative of cooling water leak [14], water
in the inlet air, or incomplete filter sterilization. Gram-positive bacteria often enter from non-sterile air [8], owing to improper air
filter installation, sterilization, or integrity [15]. Multiple contaminants are usually indicative of general sterilization failure [16].
Mycoplasma is an important contamination to monitor for cell culture processes [17–19]. Mycoplasmas lack a cell wall, have
filterability at 0.22 μm, and are easily killed at 60 °C. Cell culture media components are heat-labile sensitive, so sterilization by
filtration is the only option. Mycoplasma infections can overwhelm production cell cultures achieving high densities (106–107
colony-forming unit (CFU) ml–1) but visually no turbidity is observed [19]. Twenty species of mycoplasmas are known to cause cell
culture issues and five have shown to give >95% of contaminations (Mycoplasma arginini, M. fermentas, M. hyorhinis, M. orale, and
Acholeplasma laidlawii) [19]. Contamination sources are commonly from human operators or from the cell lines. Viral contamina­
tion, via endogenous viruses or adventitious viral agents, is an important concern for cell culture [17, 20]. Cell lines contain
retrovirus-coding sequences in their genomes and therefore inherently express retrovirus particles during production. Adventitious
viral agents may be introduced through the use of cell lines derived from infected animals or virus-contaminated reagents or serum
components. Safety assurance is accomplished by the combination of raw material control/testing, master cell and working bank
testing, in-process control testing, and virus clearance studies. Typical viral testing includes a panel of viruses, ranging in size from 17
to 400 nm, such as bovine viruses (viral diarrhea virus, adenovirus, polyoma virus), reovirus, cache virus, and murine minute virus.
Reported viral infections of recombinant CHO cell lines include murine minute virus, a parvovirus [21], and epizootic hemorrhagic
disease virus [22].
The risk of contamination has to be evaluated for each particular bioprocess. Subtle changes in operating conditions between
processes can have a large impact on the susceptibility to contamination. Certain factors have been identified that lower the risk of
contamination such as pH range (<5 and >8), low initial bioburden of the media before sterilization, high osmotic pressure, high or
low carbon concentrations, switching from complex media with insoluble solids content to soluble defined media [16, 23], or
applying temperatures above 60 °C.
Minimizing the risk of microbial contamination is a combination of prevention activities and contamination monitoring
(Figure 2). Contamination risks occur in aging facilities that are susceptible to mechanical failures, and also new facilities that
have operational unknowns [16]. A balance needs to be addressed between increasing time for preventative maintenance (PM) to
reduce failures versus fast turnaround times to maximize the productivity of the facility [11]. Process design, testing, and training are
all important. Protocols must be in place for each new bioprocess to minimize and investigate microbial contaminations [10, 16].
This is influenced by the nature of the fermentation/contaminant, equipment design, process operation procedures, and the
microbiological process controls implemented (Figure 2).
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Other Considerations
2.68.2 Design and Procedural Approaches to Minimizing Contamination
2.68.2.1
Room Classification Considerations
For biologics and vaccine production, the use of validated classified areas with room engineering controls is a regulatory require­
ment to minimize the potential for contamination of the process. Details of classified area design have been previously described
[3, 13, 24]. The flow of airborne particles is controlled by the facility design using high containment areas with HEPA. Prefiltered air
with climate control (18–20 °C, <50% humidity), personnel gowning areas, and equipment air locks. The HEPA filters have a
retention of >99.97% of particles larger than 0.3 µm [3]. Floors are typically monolithic with integral drains to prevent standing
water, while the ceilings are impervious, windows are flush mounted, and all surfaces can withstand chemical sanitization. A grade
C or class 100 000 room is controlled environment for processing with unidirectional air and people flow segregation with airflow
locks. A grade B or class 10 000 area has segregation and airlocks used to control flow with inlet only and outlet flows for personnel
[3]. Typically, this uses 100–600 air changes per hour airflow with strict standards of free from microbial contamination and particle
specifications such as <3500 particles at greater than 0.5 µm m–3 [3]. For sterile operations such as making of master cell banks or
formulation and filling of final product typically requires a grade A, class 100 environment. Environmental monitoring plans are
used to quantify the room classifications, which will be discussed in the monitoring and sampling sections. Access to utilities for
repair or maintenance must also be considered. For example, separating the utility header in a separate service corridor adjacent to
the classified area, process area allows greater flexibility for ease of repair and maintenance.
2.68.2.2
Utility Design Considerations
The design and quality of the utilities that are in the product (steam and sparger air) and non-product contact (instrument air and
chilled water) can affect contamination. For example, it is recommended for fermentor sparger air to locate the air compressor
intakes at elevation above and upwind from fermentor exhausts and cooling tower mist [11, 16]. There is estimated to be a 1-log
reduction in live organism concentration with every 3 m of increased elevation [11]. As air flows through the compressor, it is heated
to >100 °C and can be cooled directly downstream of the compressor outlet. It is recommended to maintain the air at elevated
temperature for as long as possible by relocating the coolers between the retention chamber and the drier [16]. This utilizes the heat
of compression to sterilize the air. Retention chambers (typically 5400 l for pilot plant facility) have been designed to include
exterior insulation and interior baffles to extend the travel path of hot air in plug flow. This minimizes the inlet/exit temperature
drop to ≤2 °C [16]. In a facility at 11 000 l min–1 and 2.8 kg cm−2, the compressor discharge and retention chamber temperatures
typically are around 93 °C with residence times in the retention chamber of 20 s (>16 000 l min–1) to 1 min at 5000 l min–1 [16].
Condensate in the sparger air is removed using heat exchangers with driers and then filtered to remove desiccant before reaching
fermentors. Moisture in fermentor inlet filters can be caused by poor system design or maintenance, or excessive pressure/
temperature drop in the air supply header. Typically, the dew point is monitored when sparger air leaves the utility building and
as it enters the process building [16]. Target dew points after the dryer are below –20 °C and can change up to ±15 °C after flowing
150 ft depending on ambient temperature and demand [16]. High-quality dried and filtered instrument air ensures automatic valve
reliability. Replacing plastic instrument airlines with copper minimizes leaks (particularly at fittings) and enables reliable instru­
ment air pressure to control valves. Installing backup compressors and receiver tanks can prevent fermentor backpressure loss caused
by automatic valves reverting to their failure states during instrument air supply interruption. Installation of filter housings with
lines that slope away from the filter enables moisture drainage, preventing condensate collection and blinding of the filters.
The appropriate design and operation of the water systems that contact the process are critical to minimizing contamination.
For biologics and vaccines manufacture, water for injection (WFI) made from thermal distillation is commonly used for buffers
and final product formulation. It is common for supply loops to recirculate water at 70 °C to prevent microbial growth [11]. Then
cold water is established at point of use drops using an individual heat exchanger.
Proper utility system operation requires monitoring using a combination of automated data acquisition system and manual
review via utilities checklists. Periodic reviews are necessary to confirm consistent execution of the operation. Measures undertaken
due to utility failures should be documented to build a knowledge database. Experience of these procedures has shown to reduce the
impact of product and non-product contact utility outages and promote uniformity of response [16]. After a utility failure, affected
fermentors should be evaluated to determine whether to abort the batches. Evaluations are conducted using online trends and
visual monitoring of field gauges during utility loss and subsequent restoration.
2.68.3 Fermentation/Cell Culture Considerations
2.68.3.1
Equipment Design
To achieve desired sterility and culture purity goals, bioreactors are to be of high-quality design and well maintained [10, 16]. Many
design features that are typically incorporated for sterility are also useful for containment (i.e., minimization of microorganism
release) [25]. The steam seal heat barrier plays an important part against the entry of contaminants. Diaphragm valves are
commonly used for sterility although constant steam service has shown to deform Teflon-backed ethylene polypropylene diene
monomer (EPDM) diaphragms [10]. Alternatives include the use of three-piece ball valves that allow for easy ball replacement
(75% less time) without the cutting and welding necessary for existing two-piece valves [16]. The most efficient facility allows for
Aseptic Operations
937
piping arrangements that allow localized shutdown of equipment without impacting the steam seals of adjacent equipment.
Establishment of piping installation preferences contributes to maintaining sterility control such as the minimization of dead legs,
installation of steam entries on the top of piping, installation of condensate removal legs on the bottom of piping, smooth aligned
manual welds, line sloping for drainage, and use of 316-l material of construction [16].
The agitator seal on a fermentor is a main area of contamination concern. For steam-lubricated, top-driven seals, steam should
remain applied to the seal, even when not in use. For condensate-lubricated, bottom-driven seals, residual condensate should
remain between batches. After long periods (2–3 months) of inactivity, fermentors with bottom seals should undergo sterility
testing [16]. The use of high-temperature fluorinated elastomers (Kalrez, DuPont, Wilmington, DE, USA) on both double
mechanical seal faces is recommended. Seal failure, due to external or internal leakage, can be determined visually and by measuring
air pressure decay rates. A 2- to 3-year typical seal service life has been established based on tracking seal failures [16]. Ideally,
stainless steel grade of 316 l is recommended for bioprocessing along with smooth welds and joints to give high cleanability. Often
facilities use electropolished systems with an ultrasmooth finish, such as a 0.4 ra where no point is higher than 0.4 μm above the
surface of the metal [26].
2.68.3.2
Use of Disposables
For cell culture, the industry is moving away from stainless steel bioreactors for inoculum expansion and production to single-use
technologies such as Wave rocking bioreactor (500 l) or disposable stirred tank reactors (up to 2000 l) [20, 27]. This has mostly been
driven by the industry’s desire to speed up timelines and reduce costs by the removal of clean-in-place (CIP) and steam-in-place
(SIP) infrastructure and the associated validation/monitoring of such systems. The disposable reactors are based upon gas
impermeable multilayered bags of polyethylene, polystyrene, or polypropylene (polyethersulfone, polyvinylidene fluoride) of
300 µm thickness. Sterilization is by steam or gamma irradiation using cobalt 60 isotope (25–40 kGy) to provide the electro­
magnetic energy that penetrates materials of construction, destroying any microorganisms and viruses. Reactor bags are held in
support vessels that enable temperature control and process monitoring such as pH or dissolved oxygen (DO) through fluorescence
patch technology [27].
The disposable bag technology is also used for preparation and storage of cell culture media, purification buffers, and product
intermediates at 200-, 500-, and 1000-l scale [20, 28]. This removes the need for additional stainless steel vessels and the associated
CIP infrastructure and validation. Analysis shows that total production costs of a 2000-l process are 30% cheaper using disposables
rather than using stainless steel vessels [20]. However, at 8000-l scale bioreactor, there is little difference due to lower facility
dependence and high material costs from the larger number of disposable bags required [20]. Issues still remain to be resolved such
as the control of leachables, limitations of scalability, and process monitoring with the lack of standardization for materials of
construction [27]. Despite these issues, an often overlooked advantage of single-use technology is the reduction in cross contam­
ination and improvement in sterility assurance. Single integrated disposable systems can now be purchased that combine the
disposable bag and capsule filters using the highest purity silicone tubing (platinum cured) [29] (Figure 3). The single system
includes the necessary clamps, adaptors, and connection devices into one single system as specified by the customer. This has greatly
reduced the number of sterile manipulations that would have been required for sterile connectivity of each piece and to the process.
The need for open connections has been eliminated by the ability to connect tubing-by-tubing weld connections. Tubing welder
systems (Terumo, Terumo Medical Corp., MD, USA) provide sterile welds to dry or liquid-containing tubing but can be limited to
the size of the tubing. Another approach is the use of disposable aseptic connectors (such as ReadyMate™, GE Healthcare) that
provide high flexibility by the genderless design (Figure 3). This allows cross-size connectivity between a different size tubing and a
tamper proof connection that can withstand process pressures up to 15 psi.
Figure 3 Integrated design of sterile bag integrated with filter assembly and genderless connector.
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Other Considerations
2.68.3.3
Equipment Setup
The accurate setup of the equipment will assist in the minimization of contamination (Table 1). For contained systems, pre-batch
integrity testing of hydrostatic and air pressurization hold testing is a routine operation for fermentors. A pressurization hold test of
<0.07 kg cm−2 loss over 12 h is routinely deemed acceptable. This allows leaks to be identified and corrected that otherwise may
cause grow back, especially if a channel (fluid layer) is involved [16]. This is usually followed by a 7–10-day sterility test. Before the
production batch, it is usual to perform routine gasket and O-ring replacements, such as for probes and blind plugs, and clean up of
all ports and threads. The next step is steam sterilization of the empty vessel at 1.3 kg cm−2 and 124.5 ± 1.0 °C for 1 h without
installation of the sparger air filter or probes and is usually completed within 48 h before media batching [16]. The fermentor then
remains under air pressure until ready for media batching. Personnel visually examine inside the vessel during setup and before
batching. Often an agitator seal or head plate O-ring leak can be detectable simply by checking for unusual amounts of accumulated
condensate. During and after sterilization, the temperature and pressure profiles should be evaluated to identify any unusual
occurrences that should be documented to aid contamination investigation. Examples include difficulty in obtaining airflow
through the sparger filter after sterilization, foaming during heat up and/or sterilization hold periods, and Templstiking failures
[16]. Steam traps should be regularly checked, at least daily, during the fermentor operation as well as directly before sterilization.
Inexpensive bleed valves can be installed for easy draining/clearing of plugged traps [16].
2.68.3.4
Considerations for Raw Materials and Media Preparation
Understanding the bioburden of raw materials and how it changes during raw material preparation can be important for
minimizing contamination. For example, the media components for fermentation use complex components such as yeast extract
and soy peptone [30] that supply an initial bioburden to the media, which can increase during media batching preparation.
Minimizing the time between media batching and sterilization to <6 h is routinely employed. Other possible methods include
lowering the media to a low pH (<4.0) and cooling the media (6–8 °C) before sterilization. For media with insoluble components
or clumps, an in-line mixer (homogenizer) can be employed to ensure that solids are sufficiently wetted. Media are recirculated
through the mixer for up to 1 h (3–10 turnovers), then passed once through the mixer before transfer to fermentors. For vessel steam
sterilization, it is important to maintain a positive pressure thoughout the sterilization and cool down cycle. After sterilization,
vessel pressurization with sterile sparger air before cooling is recommended to avoid vacuum upon cooling. Raising the backpressure to 1.5 kg cm−2 before introducing sparger air and start of cooling avoids foam, and large pressure drops [16]. Other facilities
shut off steam and start cooling while permitting backpressure to decrease as low as 0.2 kg cm−2 before applying sterile air. This
approach potentially increases the risk of pulling in nonsterile air [16].
2.68.3.5
Seed Expansion Considerations
Inoculum expansion using disposable shake flasks with vented caps is used routinely and replaces the use of glass Erlenmeyer flasks
with cotton bungs that are prone to wetting and provide a potential contamination risk. Similarly, the use of tubing welders and
sterile disposable connectors allows closed connections of inoculum to the process vessel. Presterilized, single-system disposable
Table 1
Fermentor pre-batch setup procedure for lab, pilot, or manufacturing scale facility using steam in place for sterilization
Procedure steps
Operating conditions
Acceptance criteria
1
Pre-batch integrity testing
<0.7 kg cm–2 loss over 12 h
2
3
Sterility test
Gasket and O-ring replacement
4
Empty vessel steam sterilization
5
Visual fermentor inspection prior
to media batching
Media batching: minimize hold
time before sterilization
Fermentor + media sterilization
Reactor leak testing by hydrostatic and air
pressurization hold testing
Sterilization of media for 7–10 days
Gaskets/O-rings change out for fermentor,
probes, blind plugs
steam sterilization at 1.3 kg cm–2, 124.5 °C for
1 h without sparger air filter or DO, pH
probes. Reactor remains under pressure
until media batching.
Agitator seal or head plate O-ring leak can be
detectable by condensate accumulation
Media makeup and transfer to fermentor
6
7
Maintain temperature and pressure conditions
during entire sterilization. Frequently
templstick key areas – filter housing, harvest
point, side ports, steam traps checked
Free from microbial contamination
All new gaskets/O-rings installed
Acceptable sterilization maintaining desired
temperature and pressure for duration
No visible condensate accumulation in the fermentor
Sterilize media < 6 h after media make up
Sterilization maintained at temperature and pressure
with no templstik failures and no steam trap
failures.
Adapted from Junker B (2006) Sustainable reduction of bioreactor contamination in an industrial fermentation pilot plant. Journal of Bioscience and Bioengineering 102(4): 251–268.
Aseptic Operations
939
bags with capsule filters are used as nutrient feeding containers to avoid repeatedly assembling and autoclaving plastic containers
with numerous fittings.
2.68.3.6
Vessel-to-Vessel Transfers
Transfers between reactors such as inocula or media can be made by hard pipe or presterilized tubing connections using pressurized
sterile air via dip tubes [16]. Transfers between harvest valve of the seed vessel side port of the receiving vessel maybe preferred as dip
tubes can be difficult to clean additional validation difficulty. Hard transfer piping can be steamed constantly or steamed for 30 min
at 130 °C (in excess of the required Fo). During tank-to-tank pressure transfers, backflow of broth is prevented by using a higher
pressure in the source tank. Pressurized transfers are conducted with appropriate transfer line cooling such as emptying the initial
hot broth to waste or using sterile airflow to vent.
2.68.4 Considerations for Purification and Formulation/Fill
For purification, the first step is typically to separate out the cells via disc stack centrifugation (Figure 1) or high-pressure
homogenization for cell breakage if the product is intracellular. Adding sterilization capability to these intricate machines adds
significant complexity and cost. An additional 20–30% is required for a manufacturing capacity centrifuge, which is already a high
capital expense item [31]. So the majority of the industry uses CIP and sanitization procedures to minimize bioburden for most
purification unit operations. This includes chromatography and especially filtration (ultrafiltration (UF) and microfilteration (MF))
where the membranes are not capable of withstanding SIP [28, 31]. Chemical sanitization is usually completed with 0.5–1 N NaOH
for a hold time of >1 h that provides adequate bioburden control. Regulatory authorities will expect maximum hold times to
be used and justification for specifications needs to be defined. Water rinses are needed to remove the chemical sanitizer
residues. Sufficient rinses to return the conductivity to <5 mS cm–1 can be used or specific unit operation criteria such as
150-l rinse of WFI m−2 membrane surface area for membrane filtration.
2.68.4.1
Liquid Filtration
All buffers used for purification are filtered using presterilized 0.22-μm filter capsules and then typically stored in disposable bags.
The disposable filter capsules are extensively used throughout bioprocessing for sterile filtration of gases (sparger air) or liquids such
as nutrient feeding, bioburden reduction of purification buffers, product intermediates, and final product sterile filtration [31–33].
A sterilizing grade filter rated as 0.22 μm has been validated for bacterial retention to defined specifications. Bacterial retention
occurs by the sieving action on the membrane surface but also by size exclusion entrapment in the membrane structure. For
example, 10 layers of 0.8-µm cellulose is required for a reduction of 2.4 × 108 of a challenge organism such as Brevundimonas
diminuta [31]. Membrane filters are porous materials so retain a certain amount of open area. This allows for fluid flow through the
membrane as defined by the porosity of the membrane. The membranes are usually pleated and formed into a cylinder, then cast
into a capsule of polypropylene. Layers of membrane are corrugated along with upstream and downstream layers of coarser
material, allowing support and drainage for the membrane. Hydrophobic membrane filters (materials such as polytetrafluoroethy­
lene (PTFE) can be in situ steam sterilized such as 30 min exposure at 125 °C for repetitive cycles and are often used for gases, such as
sparger air, as the hydrophobic nature allows them to dry quickly. Sterile liquids are often filtered using polymeric microporous
hydrophilic materials such as polyvinylidene fluoride [31].
2.68.4.2
Considerations for Applying Disposable Technology to Purification
Presterilized disposable technology has also been applied to the filtration unit operations of depth filtration, UF, and MF (Figure 1)
[20, 31, 34]. Depth filters are primarily cellulose fiber based combined with filters aids such as activated carbon or diatomaceous
earth to give two modes of clarification: size exclusion of large particles entrapped on the surface and retained within the interior
walls of the membrane, while amine chemistry enables particle adsorption [32]. Depth filters are used to remove cellular debris after
centrifugation, but the charge surfaces also reduce contaminants such as residual DNA and host cell proteins. Modular flat sheet
disposable stacks are available presterilized by gamma irradiation. A system with 1 m2 capacity enables the processing of between
250 and 500 l and disposable systems are available up to 12 000-l scale (43 m2). UF is used for product concentration and buffer
exchanges that occur at multiple times during bioprocessing (Figure 1). Single-use tangential flow filtration (TFF) cartridge systems
are available for UF. For example, single-use and single pass flat sheet membrane systems can be manifolded together to create a
serpentine flow path of progressively smaller membrane areas. This configuration allows the cross flow across the membranes to be
maintained, while the feed stream volume and volumetric flow rate is reduced [31]. For a cell culture process, the demonstration of
viral clearance is required. The reduction of viral load (4–6 log) is typically achieved from a combination of a chromatography step,
pH inactivation, and addition of a viral filtration step such as single-use nanofilter system, run in tangential flow UF mode [28]. Prebatch setup tests for purification equipment can include pressurization tests for centrifugation and filtration skids. The proper
installation of filters assemblies can be confirmed from pressurization of the wetted and rinsed filters and the generation of stable
volumetric flow rates.
940
2.68.4.3
Other Considerations
Formulation and Fill Considerations
For final drug formulation, UF (TFF) is used for concentration and buffer exchange into the final formulation. The presterilized UF
disposable capsules can be used such as the Pellicon filters (Millipore) with disposable capsule availability of up to 2.5 m2 per
capsule. Final drug product sterilization after formulation is completed by filter sterilization (0.22 μm) typically into a disposable
presterilized bottle. This improves sterility assurance by removing the need to CIP or SIP of a suitable container and also reduces the
validation issues. The sterile material is then filled into the desired container often using in-line sterilization. The vials, prefilled
syringes or IV bags, and associated parts, such as vial caps, vial crimps, and syringe barrels, are washed before depyrogenation and
fill. The depyrogenation of glass surfaces is typically at 250–260 °C. Any sterile gas requirement (air or nitrogen) such as for the
automated filling stations or for lyophilization for freeze drying applications is filtered using disposable capsule filtration.
2.68.4.4
Maintenance Considerations
Implementing a PM program is vital to maintaining control of contamination and minimizing downtime [16]. Examples based
upon facility-specific experience include (1) quarterly infrared steam condensate trap surveys to identify traps that were plugged,
blowing through, or leaking; (2) annual testing of transfer line valves for internal and external leaks; (3) annual replacement of
diaphragms, especially those exposed to constant steam service; (4) annual testing of diaphragm valves, both before and after repair,
using pressurized air to detect leaks from valves submerged in water; (5) annual internal vessel inspections to ensure bolts are
present and tightened; and (6) annual inspection of vessel ports to remove burrs to prevent sticking [16]. Routine maintenance of
key instrumentation is important such as backpressure and temperature control for fermentation sterilization reliability. Pressure
gauge accuracy should be verified against pressure transmitters at 1.1–1.3 kg cm−2 and resistance temperature detectors (RTDs)
characterized at three temperatures (0, 65, and 130 °C) using an oil bath. For fermentation the most common reasons for probe
failures are compromised DO membranes and broken pH glass sensors [16]. These failures can be significant sterility risks owing to
potential release of non-sterile electrolyte. DO probe failures are about threefold higher than pH probe failures. A pre-batch pH/DO
probe response checkout can be conducted to ensure reliability. pH probe sensors should be replaced every six batches while DO
probe membranes replaced every other batch, and DO probe sensors replaced annually. Both pH and DO can be tested using offsets
between (1) pre- and poststerilization probe readings and (2) poststerilization probe readings and laboratory-analyzed grab
samples. When a poststerilization failure of a DO or pH probe is detected, the vessel is usually discarded. The replacement of
gaskets and external O-rings after each batch, such as on probes or vessel plugs, is common practice. This avoids the inconsistencies
associated with visual inspection and reuse. It can be made cost effective by finding a suitable disposable, single-use gasket that can
withstand the process conditions such as contact with steam. Examples include high-temperature peroxide-cured EPDM that can
withstand >275 °F for >400 h and up to 600–800 h without splitting or substantially sticking to the vessel manway [16]. Examples
of detailed maintenance to process filters include the uniform torquing of larger scale sparger filter housings to assure a uniform
seal, implementing a air pre-filter and steam filter before the main air sparger filter [8] and testing the steam supply valve to the
sparger air filter for leaks.
This maintenance work must be documented using a suitable database that allows the tracking of work orders for initiation,
completion, and repair testing. The confirmation of repair testing ensures that the repair meet expectations before returning
equipment to service [16]. Regularly weekly review of the database identifies if additional investigation or more extensive repair
might be required for repeat issues. Change control procedures for equipment and computer systems ensure that the changes are
documented, communicated, and appropriately evaluated for potential effects on contamination as well as validation [16].
2.68.4.5
Training of Personnel
Consistent bioprocess operations are needed to minimize contamination and aseptic processing is highly dependent upon the
proficiency of the personnel assigned. This proficiency must be firmly established before allowing personnel to conduct critical
aseptic operations. Training and documented communication of procedures and best practices are vital to maintaining the
operational effectiveness of a facility and minimize contamination [16]. This training should include both classroom and
‘hands-on’ dummy runs on the process floor to gain familiarity. Building the skill set of new staff should include monitoring
their ability to execute procedures. Operational procedures should be explained with the reasons behind operational procedures to
generate a logical framework to assist personnel to recall the order of execution. Benefits were acknowledged by Junker [16] from
effective training of the maintenance personnel. This included training to identify problems, understanding of project status, and
regular review of maintenance job prioritization for timely completion of the most critical repairs.
2.68.5 Validation and Verification
2.68.5.1
Sterilization Considerations
To define the sterilization conditions for a particular piece of equipment and media, it is highly recommended to complete actual
SIP studies, rather than relying on theoretical analysis of kill calculations [35]. For example, at the 120 000-l scale, a cycle of 122 °C
for 12 min was reported based upon on computer kill calculations [11], whereas SIP studies with spore strips confirmed that batch
Aseptic Operations
941
sterilization times for growth media were 45 min at ≥122 °C for 800–19 000 l were required [16]. Thermocouples with spore strips
using Geobacillus stearothermophilus can be placed inside the equipment to be heat sterilized such as the vessel headspace. Spore
solutions can be placed in the liquid-containing part of the equipment. Relative D-values of spores in the solution to be sterilized
can be compared to those in water to adjust sterilization conditions (pressure and temperature for a particular piece of equipment)
[16, 35]. Similarly the validation of autoclaves and load patterns can be conducted using thermocouples and spore strips/solutions.
The SIP effectiveness can be confirmed by conducting three successful, successive, sterility tests, or inoculated batches. A typical
sterility medium consists of 6 g l–1 yeast extract (autolyzed code 106; BioSpringer USA, Minneapolis, MN, USA), 6 g l–1 cerelose, and
1 ml l–1 polypropylene 2000 (P2000; Dow, Freeport, TX, USA), adjusted to a presterilization pH of 7.0 [16]. A low-end sterilization
temperature range of 122–123 °C for 40 or 45 min depending on tank size is used, followed by a 7–10-day hold period at
35–37 °C. At least one sterility batch is conducted after each contaminated batch.
2.68.5.2
Sanitization Considerations
To define the sanitization conditions for purification equipment requires experimentally confirming the hold times and chemical
sanitizer concentration to give sufficient bioburden reduction to meet specifications for the particular bioprocess. The validation
of filtration processes can be completed using bacterial challenge testing to demonstrate consistent removal of the standard bacteria.
A sterilizing grade filter rated as 0.22 μm has been validated for bacterial retention which is defined as one capable of retaining 107
B. diminuta cells per cm2 of membrane surface under specific conditions as accepted by regulatory agencies [2]. Filter validation also
includes integrity testing using a combination of bubble point, forward flow, and pressure hold tests. The bubble test point is to
determine the pressure at which a continuous stream of gas bubbles (air or nitrogen) is initially seen downstream of a wetted filter
under gas pressure. This can be accomplished using an automated filter integrity testing device (Integritest4, Millipore). The testing
is applied to any sterilizing filter and associated vent filter of a process. The membranes are tested wet by water for hydrophilic
membranes and using 60% IPA for hydrophobic filters.
The demonstration of sufficient viral clearance (4–6 log reduction) can be validated by viral challenge studies of the chromato­
graphy step, pH inactivation, and viral filtration. The industry typically uses two species including enveloped and nonenveloped as a
demonstration of two different viral particles with different sizes, examples of which are shown in Table 2.
2.68.5.3
Sanitary Cleaning Issues
Controlling microbial contamination of bioprocesses requires effective sanitary cleaning procedures for all areas of operations. This
even includes janitorial training for maintaining the sanitary conditions of the raw material warehouse and routine inspection of
sanitation and pest control programs. The accumulation of solids within equipment can compromise the sterilization effectiveness, so
good cleaning procedures are important to minimize contamination. Automated CIP systems with spray balls can be implemented.
Alternatively, a manual approach can be used with high-pressure nozzles of high-velocity water streams to remove adhering residues
located on areas such as the upper sidewalls or agitator mounting flange. This is typically followed by filling the tank with cleaning
agent up to contact with the tank dome, which ensures coverage of all internal crevices. The cleaning agent such as CIP 200 or sulfamic
acid (1.2 wt.%) is maintained at its optimal temperature of 80 °C (±10 °C), under agitation at 75% of maximum speed, for a 1–3-day
soaking duration. Cleaning and rinse solutions should also be passed through the fermentor internals by air-pressurization or
recirculation using a pump and include the flushing the vent line if a contamination occurred during the batch [16]. For fermentors
with repeated contaminations, boiling of the vessel with Na2CO3 or Na3PO4 (high pH, metal-chelating agents) and a germicide can
then be applied. For each new process, a cleanalibity assessment is completed using media and samples of the process to verify the
cleaning process. Clean ability is assessed by a combination of methods: visual inspection for surface cleanliness, direct swab testing of
surface, and direct testing of rinsate. Typically, rinsates are tested for pH, conductivity, total organic carbon (TOC), bioburden,
and endotoxin, and acceptance criteria are defined by the agencies [1, 2]. Cleaning validation criteria will be process specific as
cleaning residues may be removed by subsequent purification steps so the earlier cleaning processes may not require cleaning
validation. TOC analysis is considered to be a good measure of overall cleanliness as it will incorporate residues from
all possible sources, such as the product, cell culture or fermentation media, and buffers [4]. The removal of cleaning agents
Table 2
Typical viruses used for viral clearance challenges studies for validation
Virus cell
Murine leukemia
Human herpes simplex virus
Parainfluenza virus SF4
Murine virus of mouse
Reovirus type 3
Simian vacuolating virus 40
(SV40)
Virus
classification
Envelope containing
Genome
Retroviridae
Herpesviridae
Paramyxoviridae
Parvoviridae
Reoviridae
Polyomaviridae
✓
✓
✓
✗
✗
✗
RNA
DNA
RNA
DNA
RNA
DNA
Size
(nm)
80–130
150–200
150–300
18–26
60–80
45–55
Shape
Indicator cells
Spherical
Spherical
Spherical
Icosahedral
Icosahedral
Icosahedral
PG4
Vero
BT cells
324 K
LLC-MK cells
Monkey kidney cells
942
Other Considerations
such as CIP 200 is indicated by phosphorus or rinsate conductivity. Examples have been shown that rinsate analysis can be
more effective as swab sampling routinely exhibits lower recovery [4]. The cleaning process should have documentation that
the procedure does not allow microbial proliferation. The bioburden is monitored during cleaning validation and clean hold
time studies. Bioburden acceptance criteria are based upon the equipment process step, the final rinse water quality, and the
capability of the cleaning and sampling processes.
2.68.6 Sterility Analysis and Culture Purity
Sampling for contamination is required from multiple steps during bioprocess manufacturing (Table 3), can prevent the use of
contaminated inoculum, and assist the investigation of the source of contamination. Testing includes confirmation of culture purity
throughout the fermentation or cell culture process, from working cell bank vial to production vessel as well as sterility of
uninoculated media and nutrients. Additional areas include monitoring of sterile raw materials, possible product intermediate
steps during purification, and the final product. A cell culture process will also need to confirm virus reduction after the first
chromatography step. The excursions during fermentation online profiles such as pH or oxygen uptake rate from vent gas analysis or
excessive foaming are indicators that can quickly identify to contamination issues. Occasionally, low-level contaminations cannot
be detected by online parameters resorting in reliance on off-line culture purity testing.
2.68.6.1
Procedures Used for Process Sampling
For each process, the inoculum seed train should be sampled from the vial, first-stage flask, and pooled inoculum. Sterility and culture
purity analysis for fermentors should be conducted daily, as well as after sterilization, after any additions such as inoculum or nutrients
and at the final harvest point. Fermentor samples can be taken from the vessel’s sample nipple into presterilized test tubes (open
transfer) with push on caps. A constant steam bleed is used for steam- sterilization of this nipple (maintaining the surrounding area
hot between samples) and is cleaned regularly. Alternatively, a contained (closed transfer) sample system can be used, as for biologics
processing, with a 0.2-µm filter-vented glass sample bottle and an integrity-tested valve array to provide a steamable connection.
For purification, samples for bioburden may be routinely analyzed from buffer makeup, after sanitization rinses, process
intermediates, drug substance, and from the final formulation. During sterile fill samples, the routine procedure is to take samples
for sterility at the start, mid, and end of the fill.
For the classified grade areas, environmental monitoring sample collection is a regulatory requirement. Sampling includes
surface wipe tests where swabs are moistened with water, wiped on the surface, and then placed into a tube with sterile water,
sonicated, and mixed [13]. The absorbency of the swab can have significant impact on the quantification of the organisms. For
example, cotton swabs can produce lower cell counts than calcium alginate swabs, as cotton contained fatty acids that may inhibit
microbe growth [37]. Calcium alginate swabs were deemed superior for quantitative analysis as they can dissolve in 1% sodium
hexametaphosphate [37]. An alternative to swabbing surfaces is direct contact of the test surface with an agar plate to determine the
number of viable CFUs of bacteria and mold on a test surface. For this RODAC (replicate organism direct agar contact) procedure,
the tryptic soy agar (TSA) surface is ‘rolled’ on the test surface such as table, wall, or personnel gown or finger tip and then incubated
for set times and temperature.
Other techniques for surface sampling include the vacuum suction sampler that has a filter sample wand that contacts the surface
and the vacuum suction sends exposed water through a filter that is then incubated on agar plates [38]. The contamination of the air
can be sampled by passive air monitoring using settling agar plates placed throughout the facility. Air filtration systems can be
implemented that can collect more than 98% of particles >0.3 µm [24, 39]. The slit to agar sampler (New Brunswick Scientific or
Table 3
A sterility and bioburden testing matrix for bioprocesses
Microbial and cell culture processes
Cell culture processes
only
Step
Type
Bacteria
Mold
Endotoxin
Virus
Mycoplasma
Raw materials
Master and working cell banks
Growth medium (sterile)
Fermentation/cell culture
End of fermentation (pre-harvest)
Product intermediates during purification
Drug substance
Drug product
Bioburden
Culture purity
Sterility
Sterility/culture purity
Sterility/culture purity
Bioburden
Bioburden
Sterility
✓
✓
✓
✓
✓
✗
✗
✓
✓
✓
✓
✓
✓
✗
✗
✓
✓
✓
✗
✗
✓
✓
✓
✓
✓
✓
✗
✗
✓
✗
✗
✓
✓
✓
✗
✗
✓
✗
✗
✓
Adapted from Adamson SR (2000) Process validation and characterization: Animal cell culture process. In: Sofer G and Zabriskie D (eds.) Biopharmaceutical Process Validation, 1st
edn., pp. 101–128. New York, NY: Marcel Dekker, Inc.
Aseptic Operations
943
Barramundi Corp.) directs air onto an agar plate, which is subsequently incubated. Caution is required such that sufficient
disinfection is completed to minimize contamination of the sample air and the generation of false positives [40]. Agars such as
TSA are used for non-fastidious microbes and Sabouraud dextrose agar (SDA) for the cultivation of fungi. After incubation, the
plates can be read on a colony counter for total viable cell count. An alternative system is a compact centrifugal air sampler (RCS
plus, Biotest AG, Dreieich, Germany), which can allow 1 m3 of air to be sampled onto soybean casein digest medium strips that are
then removed and incubated.
2.68.6.2
Procedures for Testing of Contaminants
A common monitoring approach of samples is direct microscopic examination of liquid broth samples using Gram staining. The
preparation of multiple slides can be aided by a commercially available automated Gram stainer (Midas III; EMD Chemicals,
Gibbstown, NJ, USA/Merck KGaA, Darmstadt, Germany) [16]. A second technique is direct streak of the process sample onto agar
media plates such as SDA, blood agar, or potato dextrose agar and incubated at 25–37 °C for 7–10 days. Plates are examined for
unusual colony formation on an interim and final basis, respectively. A third technique is the subculturing enrichment technique
that selectively enriches for contaminants. Tryptic soy broth (TSB) is commonly used as it detects most bacteria. Test procedures for
fermentation broth samples usually involve inoculating 1 ml of sample into 4 ml TSB in inoculation tubes shaken at 220 rpm for
15–30 h at 34–38 °C. After shaking, tubes are used to streak onto TSA plates, incubated statically for an additional 6 days, then
examined visually. Viability has been defined as the ability to form colonies, thus turbidity in a TSB tube without subsequent colony
formation was not considered contamination. An alternative is the phenol red (PR) dextrose broth that detects bacteria by turning
yellow with acidic contaminants as well as acidic production cultures.
The bioburden analysis for raw materials, process purification, and final product sterility samples is usually tested by concen­
trating cells by membrane filtration using a 50-ml sample filtered through a membrane filtration disc (47 mm diameter), then
incubating the filters on two types of agar at two different temperatures between 3 and 7 days. Typical limits of detection are down
to <5 CFU per 50 ml. Alternatively, serial dilutions can be completed and spread plate technique used on agar to quantify a yield
count between 30 and 300 CFU. Although these standard microbiological methods for contaminant detection are reliable, they are
time consuming as the typical duration of sterility testing for injectables is 14 days.
Alternate rapid methods include total and viable microbial population measured using total ATP and intracellular ATP [38].
Commercial luminescence systems are available (Celsis RapiScreen from Celsis, Milliflex from Millipore, and PallChek from Pall)
for ATP assays that give similar total viable count to conventional aerobic plate counts. The ATP assay methods can be sensitive to
the sample matrix particularly from cleaning solution residues, which can denature the luciferase enzyme [41]. Alternative methods
such as quantitative polymerase chain reaction (Q-PCR) methods are becoming mainstream tools for rapid microbial detection.
Methods have been demonstrated for the estimation of total bacterial population by determination of 16S RNA copy number using
a fluorescent label probe. The process takes <1 day as an autolyzer is used to automate the nucleic acid extraction and has been
shown to perform equally to manual phenol/chloroform extraction [38]. Favorable detection limits (1 CFU ml–1) of such PCR
methods have shown to be demonstrated.
The detection of a contaminant, based upon isolation of the contaminant cultivation on nutrient media, is followed by
morphological and biochemical tests. This commonly includes transmission electron microscopy to assist virus typing. The most
likely match for a genus and species can come from database profiling from fatty acid methyl ester (FAME) analysis and DNA
sequencing. A number of commercial identification systems are available (Microcompass (Lonza) and MicroSeq system
(AB biosystems)) which use PCR techniques by automating the sequencing of the universal 16S ribosomal RNA gene and then
comparing it to known libraries to find the closest match.
Q-PCR methodology has been developed for endogenous retrovirus load qualification and adventitious viral agent testing in
production cell culture [42, 43]. Cell lines contain retrovirus-coding sequences in their genomes and therefore inherently express
retrovirus particles during production. The viral load in the final product needs to be quantified and has traditionally used in vitro cellbased infectivity plaque assays [43], taking 28 days for cell culture passaging as opposed to 1–2 days for PCR assays. The Q-PCR
methods allow virus identification by comparison of rRNA sequences to known libraries [42]. New technology such as Virochip virus
detection has been demonstrated to identify new virus families via hybridization to similar sequences of established virus strains [44].
The chip system has an automated workflow of random PCR, hybridization, and fluorescent detection. A typical current chip array has
>30 000 oligos from >3000 viruses [44, 45]. Virus detection by rapid sequencing from mRNA nucleic acid pools is also feasible using
massively parallel sequencing that allows entire genomes (25 million bases) within 4 h then screened against the viral database.
The mycoplasma testing of cell culture is usually completed using standard agar plating (7–14-day incubation) and culturebased testing by exposing Vero indicator cells (28-day incubation). Specific PCR methods have also been implemented [19, 38]
which can be completed in <1 day with limits of detection to 10 CFU ml–1. The Q-PCR method amplifies a 280-bp DNA fragment of
the gene encoding for the 16S rDNA and has been validated using reference strains M. orale and M. pneumoniae with sufficient
detection ranges for all mycoplasma species found in cell culture.
2.68.6.3
Criteria for Contaminated Samples
Early contamination detection avoids wasting resources for continued processing of contaminated batches. Wherever possible, it is
routine operation to confirm seed inoculum sterility before transfer to the next growth stage. This includes microscopic evaluation
944
Other Considerations
of the broth samples and enrichment culture tubes. The detection of two consecutive samples (taken >2 h apart) is usually used as a
confirmation of contamination. The frequency of false positives, when a contaminated sample is followed by a sample that is
contamination free, is around 0.18–0.42% per year (from 5000 samples) [16] and is more common with direct broth samples and
TSB tubes than in plated agar medium.
2.68.6.4
Strategy for Contamination Investigation
Thorough investigation of contaminations can significantly help to reduce the contamination rate of a process or facility.
Performing the investigation by promoting information gathering in a non-judgmental manner promotes openness [10, 16] and
can lead to a more useful and faster narrowing down of the key issues. It is often experienced that these investigations find a matrix
of interrelated issues carrying through from different operational steps. So it is recommended to investigate a number of key points
of the process rather than a narrow focus on a single issue. Visual examination of the equipment setup and operation is vital,
including evaluation of alarms action items of planned and unplanned should be reviewed. Where ever possible the equipment
should be disassembled and evaluated including any probes and valves assessed for leakage. Integrity testing of all associated filters
is recommended. Steam traps should be checked for function, condensate traps for blockage, and internal vessel surfaces for
cleanliness. An agitator seal pressure test is conducted for internal and external leaks. For a repeat contamination, particularly of
Gram-negative rods, the jacket should be leak tested using 90 psig air and a vessel full of water.
Common issues for fermentation contamination include raw material issues, inadequate sterilization of equipment/air/media,
instrumentation errors, inadequate procedures, operator errors, insufficient training, procedures not followed, and lack of routine
PM-contaminated transfers [10, 16]. These issues need to be tracked to improve awareness of key issues. For example, Junker [16]
showed that the highest cause of contamination in a microbial pilot plant facility during the period 1Q1990–3Q1997 was to
unknown causes (55.3%), followed by contaminated tank-to-tank transfers (25.1%). Substantial efforts were then implemented to
improve thoroughness of postcontamination mechanical checkouts (to reduce unknowns) and pretransfer clearing of seed tanks (to
improve detection methods). These actions not only reduced the overall number of contaminated batches during the following
period of 4Q1997–3Q2004, but shifted the highest percentage to equipment (54.6%) followed by unknown causes (25.5%).
Further reduction in the percentage of equipment-classified contaminations might be realized by enhanced setup or PM procedures.
Tracking the timing of the contamination detection and the cause of the failure is also worthwhile. For example, Junker [16] showed
that 65–70% of fermentation contaminations arose <48 h into the batch, and 55% were equipment related. This suggests that
focusing on equipment problems resulting in early contaminant introduction might have the greatest impact on further contam­
ination rate reduction. These facility metrics should be highlighted to all, especially operators and mechanics, on a regular basis.
2.68.7 Summary
Schedule delay and loss of production capacity from contamination have been observed across the fermentation/cell culture
facilities of the industry. Literature examples of unresolved contaminations include facilities switching to different products and
specific vessels only being used for a particular product [14, 46]. This can be particularly challenging in today’s need for multiuse
facilities using different expression systems and platform technologies. The control of low contamination rates from a proactive
rather than reactive approach results in less time and expense spent on investigations and fewer interruptions to the facilityprocessing schedule. The continued control of low contamination rates requires the sustained effort from a multidisciplinary team.
Diligent attention to detail is required along with effective training and constant communication of contamination awareness. As
we move forward, there will be continued momentum to reduce contamination rates with the increasing pressure on the industry to
control costs and improve efficiency. The expansion of disposable single-use equipment will increase as scalability and standardiza­
tion of materials of construction improve. The use of Q-PCR and DNA detection methods such as DNA chip arrays will likely
become common industrial practice. This will lead to rapid identification of contamination (hours instead of days) and should
ultimately lead to improvement of identifying the sources of unknown contaminations especially in regard to raw material quality.
The use of risk assessment tools will likely increase as we move toward a risk-based regulatory scrutiny. This will challenge the level
of understanding of scientific knowledge/understanding of how the environment and the process impact product quality.
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