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Development of a Master Plan
3.3.3 thiRd- PaRty cuRRent good ManufactuRing PRactice audit: data and
saMPle integRity
As clinical pharmacology testing for biosimilars is conducted in an at- scale current good manufacturing practice (cGMP) lot (i.e., a nal commercial lot), it is imperative that the developer qualies
its cGMP production. The audit is specic to the product and not waived based on previous audits.
The audit is conducted by third- party auditors. The auditors also conrm and assure that the samples
that will be selected for clinical pharmacology testing are valid and their integrity conrmed.
3.3.4 validated saMPles
The samples used for analytical assessment and clinical pharmacology must be validated for their
source, history, and compliance. Generally, during an audit, samples will be collected and provided
to a third- party testing facility.
3.3.5 thiRd- PaRty analytical assessMent
The nal analytical assessment must be conducted by a third party approved by a newly formed
Global Medicines Agency (GMA) as a qualied testing facility.
3.3.6 ceRtified saMPles Retained by clinical ReseaRch oRganizations foR clinical
PhaRMacology testing
Clinical research organizations should retain the samples if there is an issue regarding an outlier or
later inquiry; the time limit is through the product’s shelf- life.
3.3.7 RefeRence PRoduct
To qualify a product as a reference product, a comprehensive dossier must be used to approve a biological product, which is still being marketed in the nation of origin. Only one reference product can
be utilized. When the reference product has different strengths or presentations, the lowest- strength
product should be used. Several batches of the reference product should be used, as it will reach the
market at different time points and can be obtained directly from the market. The reference product
batches should undergo testing for the required attributes to establish the shelf- life and should be
stored as recommended. Occasionally, it may be possible to test reference product batches that
have been stored for a long period (e.g., product samples frozen at −80°C) or beyond their intended
shelf- life if reliable data demonstrate that the storage conditions have no effect on the critical quality
attributes (CQAs).
3.3.8 chaRacteRization
As dened in ICH Q6B, proper methods should be used to perform characterization of the reference product. Some of the characterization studies determine physicochemical qualities, biological activity, immunochemical properties (if any), purity, impurities, contaminants, and amount.
Developers are encouraged to adopt newer technologies as available. As the quality attribute values
of the reference product can vary from batch to batch, it is essential to establish the ranges of these
variations. The variations are either process- related (the manufacturing system) or product- related
(the expression system), the latter cannot often be resolved, requiring the developer to create a
different expression system; the same can be the case for process- related attributes, but these are
readily xed. However, any difference in both groups of attributes cannot be justied based on any
in vivo or ex vivo studies.
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Development of a Master Plan
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3.3.9 iMPuRities
When developing a biosimilar, impurity proling is required, and guidelines for product- related
variations are established with the developer. For instance, a biosimilar may show fewer impurities
in terms of type and quantity, but there must be no mismatched impurity, as it cannot be justied
through a safety study.
3.3.10 function- based tests
CQAs should be identied using analytical proling and in vitro functional levels. Functional
experiments should be pertinent to the potential mechanism of action in all therapeutic indications,
including those that examine apoptosis, complement- dependent cytotoxicity, antibody- dependent
cellular phagocytosis, and antibody- dependent cellular cytotoxicity. Functional tests (ADCC,
ADCP, and CDC) are not suitable for a reference product primarily targeting a soluble antigen.
3.3.11 test PRoceduRes
Testing of CQAs does not require validated procedures, as some test methods cannot be fully validated.
Analytical methods must be qualied, sensitive, and adequately selective to identify potential differences.
Where appropriate, the procedures described in the ICH recommendations (ICH Q2A, Q2B, Q5C,
and Q6B) for analytical assessment can also be utilized to evaluate quality attributes for batch release.
Additionally, the use of appropriate orthogonal methodologies is necessary for robust data.
3.3.12 nuMbeR of batches
Generally, eight batches will be tested, one of which should be the clinical batch. Therefore, the nal
third- party analytical assessment will include at least three PPQ lots.
3.3.13 data evaluation
Depending on the type of data output, a visual comparison sufces for test results sent as printed
output, such as spectra. Quantiable data from multiple batches should use the 3- Sigma range,
which is derived for the reference sample as (ref − 3ref, ref + 3ref), providing the most accurate
inference. If the test sample’s MinMax range falls within the 3- Sigma range, then the 3- Sigma test
is accepted.
3.3.14 exPRession systeM
The expression system determines product- related CQAs, including primary structure, higher- order
structures, glycosylation (only in eukaryotic hosts), product- related variations, and process- related
variations. The expression system should be the same class as the one used to express the reference product, even though SRA agencies allow the use of a different expression system; this recommendation comes from the realization that switching an expression inevitably leads to variable
post- translational modications that may be difcult to evaluate in safety and efcacy studies. The
developers are also advised to select more steady expression systems; generally, high- yielding cell
lines produce more variants. Cell lines should be qualied according to the ICH Q5D.
3.3.15 analytical PRofiles
Proteins undergo addition of functional groups after translation (post- translational modications
[PTMs]), which should be comparable, not necessarily identical. In addition to PTMs, these proles
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Development of a Master Plan
include aggregates, fragments, visible or subvisible particles, acidic and basic variants, and other
product modications such as reduced, oxidized, glycated, and misfolded protein forms. These
attributes can change over the product’s shelf- life, requiring testing over the shelf- life duration.
When the environment changes during different stages of the production process, hydrophobic
regions of the protein can unfurl, causing either accumulation or fragmentation, adding to immunogenic responses. The aggregate size ranges from soluble aggregates to visible residues, depending
on the duration of exposure to various stresses such as shear, thermal, chemical, and freeze- thaw,
among others. The matrix- free size exclusion chromatography substitute analysis helps dene size
distribution, sedimentation velocity- analytical ultracentrifugation.
Charge variations are proteoforms that occur at different stages of the manufacturing process in
various colloidal matrices (such as culture medium, in- process buffers, or formulations) and have
varying charges. It is, therefore, preferable to use several types of cation exchange chromatography.
Oxidation, phosphorylation, sulfation, acetylation, methylation, and hydroxylation are examples
of nonenzymatic PTMs occurring across various manufacturing stages. Liquid chromatography is
preferable for dening PTMs and measuring associated molecular variations and contaminants.
Cell substrates are process- related variations or residuals, including host cell proteins (HCPs),
host cell DNAs (HCDs), cell culture, and downstream processing residuals. Enzyme- linked immunosorbent assay and real- time or quantitative polymerase chain reaction assay are the main HCP and
HCD detection and quantitation techniques, respectively. These variants are not tested during the
drug substance qualifying phase because they are part of the release specication.
3.3.16 Release sPecification
Release specications are based on the characterization of the reference product, except for the
legacy compendial attributes such as sterility, ll volume, and delivered volume; other characteristics
are independently established, such as sterility, invisible particles, protein content, potency, and
physical characteristics unique to the biosimilar candidate. These standards may be used to specify
the biosimilar candidate’s release specication.
3.3.17 foRMulation
A formulation different from that of the reference product is permissible for biosimilars. A formulation with the same number of inactive substances or fewer is advised, unless constrained by
patent protection. The formulation’s stability and compatibility (i.e., how it interacts with excipients,
diluents, and packaging materials) should be proved, along with the integrity, activity, and potency
of the active ingredients. If the primary packaging that is in contact with the product is different,
then further safety tests are required to verify that there is no unexpected leaching of package
components into the product. Developers are encouraged to select a primary packaging materials
as similar as possible, since it is often difcult to justify these ndings. The formulation may not
contain any unique excipients previously not used in a similar product, and all excipients must be
free of animal products.
3.3.18 RefeRence standaRd
The in- house primary reference material is an adequately documented sample prepared by the
manufacturer from a representative lot or lots and calibrated against which the in- house working
reference material is used for biological assays and physicochemical testing of the following lots.
It is the sole source acceptable for use as a working reference. Reference standards that are openly
accessible (such as European Pharmacopoeia) cannot be used as the reference product for comparison testing.
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Development of a Master Plan
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3.3.19 stability
The stability of the biosimilar candidate must be evaluated according to the ICH Q5C including
accelerated and stress stability testing to further enable direct assessment of structural similarities
and produce degradation proles.
3.3.20 PRocess Qualification
Before any analytical assessment for similarity, the upstream and downstream processes must be
evaluated. However, on completion of the clinical pharmacology studies, no batch size adjustment
is permitted; the developer may do this only under ICHQ5E, which applies only post- approval.
Bridging studies are needed to validate whether or not the production size changes.
3.3.21 aniMal toxicology
For biosimilars, no animal toxicological testing is necessary. This conclusion is based on the recent
amendment to the BPCIA, wherein “animal toxicology” was removed and replaced with “nonclinical
testing.” As animals have no binding receptors for biological drugs, this binding results in pharmacological and toxicological reactions. This testing is currently also recommended for new biological drugs.
3.3.22 clinical PhaRMacology
An extension of analytical evaluation, PK, and PD studies reect how the body perceives the drug
molecule and vice versa. Such studies are also conducted for drugs such as aibercept or ranibizumab
that are administered locally into the eyes; these drugs do not enter the general circulation, and hence,
they are tested through parenteral administration for the same reason. For most of the chemical generic drugs, PK proling is not required when the drugs are administered intravenously, intramuscularly, or subcutaneously. However, biosimilars administered through parenteral routes require PK
proling, as PK parameters such as half- life and distribution volume can also correlate with the kinetics of receptor binding, an essential assessment because all biological drugs act by receptor binding.
One misunderstanding in the design of PK/ PD lies in the traditional goal of characterizing the
proles in a wide range of subject qualications such as age, sex, body mass index, body weight,
and race. All these variables add much inter- and intra- subject variability that requires a larger population. None of it is necessary for comparative PK/ PD proling, as these studies aim not to characterize but to compare the prole attributes. A robust design should accommodate crossover or
parallel designs. A crossover approach is better at identifying differences but might not be appropriate for reference products with robust immune responses or a long half- life. The equivalence
margins must be prespecied, and the appropriate range is often 80.00%– 125.00%. The key PK
parameters, typically AUC0–
, should be equivalent in the PK experiment.
Cmax
3.3.23 iMMunogenicity
Immunogenicity is an inherent property of proteins, and it is best tested in healthy subjects during
clinical pharmacology proling. However, the immunogenicity of a specic protein can be assessed
through preclinical and clinical studies during drug development. These studies evaluate the protein’s
potential to elicit an immune response, including production of antibodies against the protein.
3.3.24 clinical efficacy
No clinical efcacy and safety testing is required for molecules with a PD response; this will exclude
mAbs until similar waivers allow them. Comparative clinical efcacy testing requires hundreds of
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Development of a Master Plan
thousands of patients to obtain statistically meaningful results; thus, such studies have never failed.
This requirement will vary in different parts of the world, but over time, a concurrence will be
reached that such testing is not necessary.
3.3.25 naMing
Biosimilars should have a brand name and share the same international nonproprietary name as
that of the reference product and any additional designations required in the local jurisdiction.
Biosimilars should also have a different brand name.
3.3.26 label
The label must, without exception, include all risks related to the reference product and have the
same indications. The developer is not permitted to ask for fewer or more indicators.
3.3.27 substitution
The European Medicines Agency (EMA) very recently approved that the reference product and
other biosimilars authorized using the same reference product can be replaced or interchanged with
biosimilars.
3.3.28 PediatRics
For biosimilars, no pediatric compliance studies are necessary.
3.3.29 huMan factoR studies
These investigations are necessary to ensure that the appropriate dose is administered when a patient
receives a product. However, these studies are not required if the device utilized is very similar to
that of the reference product. Furthermore, no such studies are necessary when a healthcare expert
uses the product.
3.3.30 RisK ManageMent
A biosimilar product uses the same risk management plan as that of the reference product.
It is anticipated that a developer is planning to distribute the product globally; while most developers
avoid securing the assistance of a rapporteur, the use of a rapporteur is highly recommended to save
time and cost. Holding meetings with regulatory agencies with an available rapporteur report will
always be helpful and earn greater condence of the regulatory agencies.
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Trends in the Manufacturing
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of Recombinant Proteins
4.1 BACKGROUND
Manufacturing of therapeutic proteins utilizes either unmodied or genetically modied living
entities, bacteria, mammalian cells, viruses, and yeast to produce large protein molecules. Some
of the manufacturing systems are straightforward, for instance, the manufacturing of naturally
produced products such as penicillin, and are therefore not included in this chapter.
To gain a clear understanding of the manufacturing processes for therapeutic proteins, it is
important to understand the roles of DNA and RNA in our body. The manufacturing processes are
tightly connected at each unit of operation during upstream and downstream processing. Variation
in yield, diverse impurities, and protein product potency achieved are the factors that signicantly
affect the steps involved in the manufacturing process. Therefore, the manufacturing process should
be carefully laid out in a lengthy denition and development process in the form of a ow chart that
identies sizing issues.
The manufacturing process starts with establishing a genetically modied cell line through culturing to express the desired therapeutic protein in a bioreactor. The term “bioreactor” refers to a
device or vessel that supports the growth of a living entity, and the term “fermenter” also refers to
a bioreaction vessel that involves the production of gases and heat; in many cases, these terms are
used interchangeably. The correct term that should be used for the production of therapeutic proteins
is bioreactors.
In the rst step, the living entities are allowed to express the desired therapeutic protein (product),
and in the next step, the expressed protein products are removed from the culture media either by
ltration (if the therapeutic protein is in solution) or by rst disrupting the cells (in case of bacteria)
and then removing the protein through a multistep process.
The subsequent steps involve purication of the therapeutic proteins and, in some cases, induction of proper protein folding. If mammalian cells susceptible to virus infection are used as the
expression entities, then any viral contamination should be removed.
Finally, the puried therapeutic protein should be labeled as a drug substance when it is diluted
with a buffer and stored at −20°C.
After production, the drug substance should be formulated into a drug product in the form of a
solution in a vial or a prelled syringe or as a lyophilized powder. Thus far, therapeutic proteins are
being administered parenterally, but efforts are being taken such that, in the future, these proteins
may be administered orally, transdermally, through inhalation, or other routes.
The therapeutic protein manufacturing process is expensive and subject to strict regulatory control
because even subtle changes in the molecular structure can alter their immunogenicity and efcacy.
More recent technologies of single- use, continuous manufacturing (CM), and online monitoring
DOI: 10.1201/9781003392026-4
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Trends in Recombinant Proteins Manufacturing
are rapidly changing the manufacturing risk prole. They offer many long- term advantages in the
planning of manufacturing facilities, which are described in detail in this chapter.
4.2 PROCESS OPTIMIZATION
4.2.1 cell line develoPMent
The development of a traditional cell line is time- consuming. A critical step in early- stage manufacturing can take nearly 40+ weeks starting from concept to creation in establishing a high- yielding
and high- quality clone. This includes choosing a suitable cell line, construction of an expression
vector, transfection of cells, cell sorting, clone selection, and evaluation based on cell growth and
productivity. Single- cell isolation and screening are critical in the workow of cell line development
from a regulatory perspective. The conventional screening approach involves seeding a single cell
per well in a 96- well plate and then screening multiple plates to choose a high producer.
Several tools available today can accelerate some of the steps involved in cell line development,
and they can help reduce the overall timeline. Cell line developers can use an effective targeted transfection process that allows gene insertion into “hot spots” when compared with random transfection
events used previously. This specic manipulation allows the creation of a high- yielding cell line
and signicantly reduces the follow- on screening process. Additionally, instead of screening multiple plates (50– 100 plates in traditional processes), wherein each well contains one clone, seeding
cells into mini- pools and then selecting a high- performing pool of cells yields a smaller cell population that enables choosing the nal single clone. Fluorescence- activated cell sorting together with
glutamine synthetase is a rapid clone- screening approach. Today, microuidics- based technology is
being widely used by cell line developers. These systems can sort and deposit single cells and can
also provide evidence of clonality, proliferation rates, and specic productivities within a short span
(as little as 5 days). When used as a combined technique, they can signicantly reduce the overall
timeline for cell line development to nearly 8– 10 weeks, thus helping the process development progress faster toward early- stage deliverables.
Several manufacturers continue to focus on traditional techniques such as plating into semisolid media and determining an individual clone’s protein titer at the static phase that does not correlate well with fed- batch. The most common enrichment method for potentially higher producers
but equally for pre- selection includes, uorescence- activated cell sorting or vector optimization for
sorting.
One of the myths related to bioprocess technology is that the cell line must be forced to produce
higher yields. However, the extent to which the cell line is being pushed is limited before it becomes
unstable; the cells will produce a higher titer that may not be desirable for monoclonal antibody,
but for a monoclonal antibody, variations in glycosylation other DNA- based changes will become
inevitable. Developers must therefore calculate the overall cost based on the cost of media serving
as the carbon source needed for pilot production before switching over to higher- yielding cell lines.
In many cases, a higher- yielding cell line helps reduce the bioreactor size but not necessarily the
cost of goods.
4.2.2 cell cultuRe Media
Cell culture medium is the primary raw material for cell culture and exists in more than 100 different
formulations comprising critical components, including amino acids, vitamins, fatty acids, and
lipids. The chemical constituents of a medium, formulation, and concentration are crucial factors
that determine the suitability of a medium to support cell growth, cellular metabolism, yield, and
protein quality. Developments in cell culture media signicantly affect cell density, increase specic
productivity, and improve product quality (less variability). The following section presents some of
the culture media optimization strategies and challenges that can enhance process intensication.
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Media optimization approaches cannot be universally applied for all cell lines or clones; a medium
that may enhance productivity in one clone may not necessarily enhance the same for another clone.
Likewise, product quality can be affected. A platform approach, particularly one that uses a single
basal medium, is always unlikely to yield satisfactory results. While it is advantageous to use an
available medium (ease of sourcing media components and having a xed and validated approach)
for the production of all monoclonal antibody products using the same host cell line, variations in
product quality (glycosylation, charge variants, etc.) may be necessary for the functionality of the
nal product. As such, having unique solutions, even with a nite number of compounds, is highly
recommended.
A traditional approach to the development of a culture medium and evaluation of its suitability
for a particular cell line involves changing one component in the media at a time. A design of
experiments approach enables the study of the relationship between the components to optimize
the composition. Depending on the number of components being used in a medium, multiple
iterations are possible, which makes the assessment relatively time- consuming. Media blending,
an alternative to the traditional design of experiments approach, allows simultaneous optimization of the several components within a medium, and it has become a commonly used method.
Additionally, successful medium formulation requires reliable analytical methods such as capillary electrophoresis, high- performance liquid chromatography (HPLC) coupled with mass spectrometry, and gas chromatography to quantify metabolites present in the culture medium during
the entire duration.
However, today’s newer technology, for example, high- throughput microarray analysis, involves
an integrated approach toward whole- media analysis rather than measuring these metabolites and
ions individually. Microarray analysis (EMD MilliporeSigma) can be used to identify medium
components to which cells respond, and this approach reduces the need for random testing. Several
manufacturers of cell culture media are backed by biotechnological companies and therefore offer
high- throughput technologies to drug manufacturers for medium optimization (e.g., MilliporeSigma,
Sartorius). Given that modeling has the ability to reduce the need for additional research or experimental analysis, it presents many opportunities to the eld of culture medium development that can
signicantly reduce time and cost. With such stoichiometric models and kinetic models, online or
inline analysis during cell culture can provide valuable inputs to these models to optimize media
or even feed solutions to establish a more robust process. In view of this approach, companies can
create their platform cell culture media specic to their cell lines.
Cell culture medium optimization is further catered to fed- batch processes that utilize intermittent feeding (mostly concentrates of specic media components). However, given the high volumes
of media requirements, having a cost- effective solution for medium preparation is of paramount
importance for a perfusion culture. A concentrate in a cell culture medium is one option to reduce
operational footprint; for instance, a medium whose components are concentrated three to four
times their original amount will be equivalent to approximately 60,000– 70,000 L, which can easily
support a perfusion process, provide signicant space, and reduce resource utilization. Alternatively,
a medium explicitly designed for the perfusion process is also another option to meet the perfusion
process media demands. Such medium will be more suitable to support high- cell densities, improve
volumetric productivity, and reduce costs (lower perfusion rates) rather than adapt media used for a
fed- batch process (Table 4.1).
4.2.3 high– cell density cRyoPReseRvation
Cell bank manufacturing is an optimal opportunity for process intensication. Briey, in a typical
mammalian cell banking process, a vial of cryopreserved cells sourced from a high– cell density cell
bank is thawed, and the cells are inoculated into at least a 25- mL culture medium. The seed culture is
subsequently expanded to generate adequate cells that can be banked. In industrial practice,
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TABLE 4.1
Trends in Recombinant Proteins Manufacturing
Decision Matrix to Address Bottlenecks and Develop Next- Generation Process
Technology Solution Pros Cons Comment
Bottleneck: Limited facility footprint
Perfusion High volumetric productivity Operational complexity Needs more process development
than fed- batch
Bottleneck: Limited CAPEX
Perfusion Smaller bioreactor Operational complexity Needs more process development
than fed- batch
Bottleneck: Limited capacity in a production bioreactor
N- 1 perfusion Cell expansion time shifts to
N- 1; shorter N time with
the same titer
Perfusion High volumetric productivity Operational complexity Identify a separation technique that
Bottleneck: QC/ QA release
Online sensors and PAT
implementations
Multi- attribute release
methods
Bottleneck: Low- yield process
Perfusion High volumetric productivity Operational complexity Identify a separation technique that
Concentrated fed- batch High volumetric productivity Operational complexity
Bottleneck: Several products and processes
Perfusion Higher level of exibility Operational complexity Scale- out depending on batch
Reduce number of release
methods through tighter
process control
Drastic reduction in release
methods
Operational complexity,
possible effect on process
performance
Resource demanding to
develop
Resource demanding to
develop
Ensures capacity for shorter
turnaround times
will suit your process
Spectral methods combined with
multivariant analysis can offer
several process parameters
Still in development
will suit your process
volume needed
Source: www.gen engn ews.com/ magaz ine/ 324/ sup plem ent- next- gen erat ion- bio proc ess- tec hniq ues/
approximately 1 million cells per vial are generally used. Compared with the scale- up of a production
bioreactor for commercial purposes, the scale- up factor or the number of seed trains required
to generate a sufcient quantity of inoculum from the vial to the production bioreactor is high. This
process is time- consuming and may require additional resources and measures to ensure no failure
during the seed generation stage. The seed expansion stage can take approximately 20– 30 days from
a low- density cell bank to a production bioreactor. To negate the additional time and processing, a
high- density cell bank is being recently used to accelerate the process by providing a larger working
volume at thaw. The use of a high- density cell bank to inoculate the rst seed train bioreactor (N- 3
bioreactor) can signicantly reduce the time. For example, use of a 100- to 150- mL high- density
cell bag (cryopreserved) at 50– 100 × 106 cells/ mL can reduce the seed expansion process by 10 days
to 2 weeks. This innovative process eliminates the need for handling multiple vials, minimizes
the variability in the cell density, reduces contamination risks, and, most importantly, signicantly
decreases the time to start a production bioreactor. In high– cell density cryopreservation, it is preferable to use a specic medium capable of supporting high cell density while ensuring no cell damage
due to freeze- thaw. Additionally, modern technologies required to design single- use bag assemblies
that can handle large volumes for cell freezing and banking (uoropolymer 2D bags) are currently
under development.
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4.2.4 cell cultuRe oPeRations
47
The last few decades have predominantly focused on fed- batch cultures. While perfusion systems
are available, the industry has not widely adopted them, mostly because of the need for a high
volume of medium and vessel capacity to support high cell densities and increased productivity.
Although cell densities achievable with fed- batch are relatively lower than those in perfusion culture, the advancements made toward increasing productivities have kept the fed- batch process in
continuous use for a long time. With the need to reduce manufacturing costs and facilities to gain
more exibility, steady- state perfusion and perfusion- based processes, including concentrated fedbatch, are now being fostered by the increasing adoption of single- use technologies. Perfusion
processes in seed train and production phases can result in a threefold increase in volumetric productivity. Upstream process intensication can be achieved through perfusion- based operations such
as by compressing the seed train duration by reducing the size and number of bioreactors required,
maximizing bioreactor utilization, increasing volumetric productivity, and reducing overall footprint, thereby maximizing facility utilization and efciency.
N- 1 perfusion, a form of seed train intensication, refers to the intensication of cell growth
during the step before the production bioreactor (N). In N- 1 perfusion, process intensication is
carried out by attaching a cell retention device to the N- 1 bioreactor to achieve high cell density
and viability, thus seeding the production bioreactor at a higher starting cell density and shortening
the production bioreactor run time. This modication can dramatically increase the facility output
without direct change to the core production process. A robust cell retention device is required to
attain a high- density cell inoculum for the production bioreactor.
Benets of N- 1 perfusion include
• Increased fed- batch process efciency.
• Time- saving and cost- effective.
• Reduced operational risk.
• Smaller bioreactor footprint.
N- 1 perfusion can help optimize fed- batch production in two ways:
• High– cell density seeding of the production bioreactor (N), achieved by attaching the cell
retention device to the N- 1 bioreactor.
• Removal of the N- 1 bioreactor, achieved by maintaining continuous attachment of the cell
retention device to the N- 2 bioreactor, provided the N- 2 bioreactor can provide sufcient cells
for the production bioreactor (N).
The N- 1 bioreactor is the most widely used application for perfusion. Increasing the cell density
of the N- 1 bioreactor allows for starting the production bioreactor with a high seeding density and
possibly reducing the bioreactor’s overall cycle time to achieve the desired titers. Higher productivity and the possibility of inoculating multiple production bioreactors from a single N- 1 bioreactor
can increase overall upstream capacity and production volumes. Such increase in production can
also be achieved with a reduced footprint; for example, a traditional process requiring a 20,000- L
bioreactor can now be tted with a 2,000- L perfusion bioreactor. Furthermore, the use of singleuse components eliminates the need for cleaning validation, i.e., clean- in- place (CIP)/ steam
sterilization- in- place (SIP). Additionally, the perfusion- based process can be extended further down
to the N- 3 seed train and high– cell density cryopreservation, as discussed previously. A single- use
bag with approximately 150- to 500- mL high– cell density culture can be frozen during storage and
can then be thawed to inoculate a seed bioreactor, thereby eliminating shake asks and a lengthy
seed expansion step.
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