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would cause acidic impurities to not bind to the column. The “bind and elute” modes are preferable
for a target protein with a pI ranging from acidic to below the neutral range. The target protein is
eluted rst under high salt concentration conditions, leaving most impurities bound to the column.
In CEX or AEX, optimizing the resin, loading conditions, impurity prole, amount of product
loaded, charge variant prole, and chromatography conditions is necessary to maximize efciency
and recovery. The primary variables for this step commonly include column loading, wash, and
elution buffer compositions.
The impurity prole and the target protein determine the use of hydrophobic interaction chromatography, mixed- mode chromatography, and occasionally ceramic hydroxyapatite, alongside ionexchange chromatography.
Hydrophobic interaction chromatography serves as a polishing step post- IEX or as an intermediate step after Protein- A purication. In its ow- through mode, HIC effectively eliminates
numerous aggregates, ensuring a high yield. It offers excellent resolution and separation of process
and product- related contaminants in both bind and elute modes.
Multimodal chromatography uses a resin combining different interaction types to separate the
target protein and impurities, such as ion exchange, hydrophobic, and hydrogen bonding. These
resins offer different selectivity from standard single- mode chromatography resins, making them
suitable for various conditions such as high and low conductivity or pH. Examples of commercially
available multimode resins include Capto MMC and Capto Adhere (Cytiva). The multimode chromatography method effectively removes aggregates, host cell protein, and leached Protein- A.
Ceramic hydroxyapatite (CHT) is utilized for its unique separation capabilities, unprecedented
selectivity, and resolution in efciently removing nucleic acids, viruses, macromolecules, and other
proteins. CHT serves as a polishing step in large- scale mAb purication, effectively eliminating
dimers, aggregates, and leached Protein- A using a gradient elution method.
Polishing steps are crucial to ensure the removal of adventitious and endogenous viruses. Overall,
the purication strategy should streamline process development efforts, ensuring the achievement of
product quality attributes, reducing production costs to make the process economically viable, and
minimizing downstream processing complexities.
11.11 VIRAL REMOVAL, INACTIVATION, AND FILTRATION
The end product must be completely free from any contamination, including bacteria, mycoplasma,
and viruses. Mammalian cells are known to naturally produce virus- like particles, which present a
signicant safety risk. Monoclonal antibodies (mAbs) derived from mammalian cell systems must
adhere to strict viral safety specications due to the potential for contamination from both enveloped
and non- enveloped viruses. This process includes characterizing the host cell line, scrutinizing
all raw materials for potential adventitious agents— especially those sourced from animals— and
conducting tests at various production stages as well as on the nished product. A crucial aspect
involves demonstrating through viral clearance that the manufacturing process can effectively
handle and eliminate viruses.
According to current regulations, therapeutic products derived from mammalian cells must contain fewer than one virus particle per million doses for safety. This requirement translates to approximately 12– 18 log10 clearance for endogenous retroviruses and six log10 clearance for adventitious
viruses. Consequently, strategies to remove viruses are integrated into the production process, especially during purication steps. Moreover, multiple virus clearance measures are employed in mAb
processes to ensure that viruses are eliminated by at least one mechanism if not by others.
Virus removal or inactivation is achieved by subjecting the solution containing the target protein to conditions that denature virus proteins without affecting the active ingredient. Common
methods employed include low pH inactivation, detergents, viral lters, heat treatment, irradiation,
and chromatography.
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11.11.1 Ph tReatMent
249
Treating Protein- A eluate at low pH effectively targets enveloped viruses. Typically, incubating the
protein solution at a low pH (approximately 3.0– 3.5) for at least an hour has been a common practice
in mAb processes. Additionally, high pH treatments, such as using sodium hydroxide for cleaning
chromatography columns, have proven effective against both enveloped and non- enveloped viruses.
Exposure to extreme pH conditions can signicantly reduce the virus count (approximately > 4.0
log10).
11.11.2 viRus filtRation
Virus ltration relies on a size- based approach for viral clearance, often used in conjunction with
pH treatment in mAb processes. Virus lters, available as ultralters or microlters with very small
pores, are primarily composed of polyethersulfone (PES), polyvinylidene (PVDF), and cellulose.
They are categorized as retrovirus and parvovirus lters based on size distribution. Table 11.7
TABLE 11.7
Commercially Available Virus Filters
Log Reduction
Value Claimed by
Company Product Material Virus
Asahi- Kasei Planova 15N Cuprammonium
regenerated
cellulose
Planova 20N Cuprammonium
regenerated
cellulose
Planova 35N Cuprammonium
regenerated
cellulose
Planova BioEX Hydrophilized
PVDF
Millipore Viresolve NFR with
retropore membrane
Viresolve Pro (Viresolve
Pro device, Viresolve
Pro Shield prelters)
Pall Pegasus Prime PES Parvovirus >4 18– 26
Sartorius Virosart® CPV PES PPV, MVM >4 18– 24
Virosart® HF PES Small non- enveloped virus
PES Retrovirus >6 80– 130
PES Minute virus of mice (MVM) ≥4.0 log 18– 24
Hepatitis A virus (HAV) >6.7 27– 32
Parvovirus B19 >6.1 18– 26
Plum pox virus (PPV) >4.6 18– 24
Parvovirus B19 >4.9 18– 26
Plum pox virus (PPV) >4.0 18– 24
Xenotropic murine leukemia
virus (XMuLV)
HIV >7.3 80– 120
Plum pox virus (PPV) < 1.0 18– 24
SV40 >7.8 40– 50
Mouse minute virus (MVM) >4.8 18– 24
Amphotropic murine
leukemia virus (A- MuLV)
Plum pox virus (PPV) >5.3 18– 24
Parvovirus ≥4.0 18– 26
Xenotropic murine leukemia
virus (XMuLV)
MuLV >6 log 80– 130
(e.g., MVM, vesivirus,
parvoviruses)
Large enveloped viruses such
as MuLV
the Manufacturer
>3.1 80– 110
>5.2 80– 130
≥5.0 80– 110
>4 18– 30
>6 80– 130
Virus
Size (nm)
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summarizes some commercially available viral lters, outlining the most prevalent viruses, though
it is not exhaustive, and other potential contaminants may exist.
Filter fouling, caused by aggregates, debris, and DNA, is a common issue impacting the performance of virus lters. Prelters, such as the negatively charged Pall Mustang S used before the
Viresolve Pro virus lter, help prevent this. Controlling the membrane permeability of viral lters
within a specic range is crucial for the ltration process. However, throughput can vary between
lots due to variability in the protein solution’s viral lter burden. Thus, evaluating lters for largescale manufacturing requires testing their performance using a worst- case protein feed sample.
Ensuring the integrity of virus lters is crucial pre and post- use. These integrity tests conrm the lter’s performance, check for defects or damage, verify compliance with manufacturer
specications, validate correct installation, and importantly, conduct end- user virus retention
studies. Non- destructive tests such as bubble point, forward ow, water intrusion, and binary gas
tests are commonly used. If the post- use lter integrity test fails, the ltration process is repeated.
Filter suppliers usually collaborate with drug manufacturers to meet these requirements.
11.11.3 otheR Methods
11.11.3.1 Chromatography
Chromatography methods used in mAb purication can efciently separate enveloped and nonenveloped viruses from the target protein. For instance, afnity chromatography is an example.
However, its effectiveness as a complementary method depends on purication conditions and virus
properties. Evaluations are necessary to assess chromatography’s capability in consistently clearing
viruses.
11.11.3.2 Detergent
This method is mainly used in manufacturing blood products. Sodium cholate and Tri (n- butyl) phosphate (TNBP) effectively treat plasma- derived products to deactivate enveloped viruses. Detergent
treatment deactivates the virus by dissolving its lipid membrane, preventing it from binding to or
infecting cells. However, detergents cannot be used against non- enveloped viruses.
11.11.3.3 Heat Treatment
This method is employed for virus removal in both human plasma- derived and animal- derived
products. Wet and dry heat treatments alter the viral protein structure, rendering the virus inactive.
Heat treatment is effective against both enveloped and non- enveloped viruses. Yet, non- enveloped
viruses require extremely high temperatures for effective inactivation. This could potentially impact
the target protein’s functionality or denature it, raising concerns.
11.11.3.4 Ultraviolet Irradiation
Gamma irradiation is utilized for animal- derived raw materials. Viruses such as reovirus and CVV
have been made non- functional using this method. Precise control of operational parameters is crucial for consistency, effectiveness, and reproducibility. Low- dose UV- C radiation (254 nm) can destroy viral nucleic acid for ultraviolet- based treatment, without affecting the target protein. Some
resistant strains of parvovirus have shown increased vulnerability to inactivation using UV- C.
11.12 VIRUS REMOVAL VALIDATION
Regardless of the virus removal method chosen, focus on robustness, reliability, and consistent
effectiveness in eliminating the virus is crucial. A risk- based approach might require multiple inactivation or removal methods in the manufacturing process to effectively eliminate both enveloped and
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non- enveloped viruses. Routine virus validation studies, utilizing model viruses reecting actual
process conditions, are necessary to meet regulatory expectations.
The FDA Guidance “Viral safety evaluation of biotechnology products derived from cell lines
of human or animal origin,” from 1997, states the following: “Condence that the infectious virus
is absent from the nal product will result in many instances are not derived solely from direct
testing for their presence, but also from the demonstration that the purication regimen is capable of
removing and inactivating the viruses.”
The virus protection evaluation procedure includes selecting virus- free cell lines, examining
unprocessed bulk, assessing the downstream process’s ability to eliminate viruses, and ultimately
testing the product to conrm the absence of contaminating viruses.
Validating virus removal throughout downstream processing involves examining cell substrates,
raw materials, virus inactivation/ removal, and nal product testing. Concerns arise with retrovirus
or adventitious viral contamination, despite no virus infectivity or reverse transcriptase activity in
the master cell bank (MCB) or working cell bank (WCB). Electron microscopy can detect virus- like
particles (VLPs). Microscopic examination does not determine the biological relevance of suspicious particles, particularly their infectivity, such as the presence of vast numbers of A- type particles
in hybridoma cells, where infectivity is extremely low or non- existent. Despite the discrepancy
between the number of virus- like particles and their infectivity, the total reduction factor is often
used to calculate the particle number.
It is challenging to dismiss the possibility of an unknown virus with unknown and potentially
hazardous physiological implications. Viral contamination complicates the development of specic assays. Without an accurate and responsive assay, monitoring the virus’s presence, removal,
or inactivation in the protein drug’s downstream phase is difcult. Preventive measures include
extensive testing of producer cells for specic viruses and testing for adventitious viruses at various
fermentation stages.
Therefore, there is a signicant emphasis on validating viral clearance steps, often facilitated by
a virus challenge or spiking study. This study involves adding viruses to the product at known titers
and monitoring them throughout each step of the process using an infectivity assay.
Viral clearance/ titer decrease (expressed as log10) is typically calculated at various stages, if
not all, of the process. The study of viral clearance or inactivation necessitates the use of identical
viruses from the same genus or family as the known or suspected virus, or non- specic viruses
closely related to them. The procedure assesses the overall level of viral reduction achieved during
operations involving known viruses. Assuming intentional virus insertion into the unit operation
application sample shows adequate clearance at specic steps, any downstream unit operation
should not be tested under such circumstances. Due to the complexities involved in viral clearance
research, focus is placed on a few efcient unit procedures for virus elimination. One- log reduction
factors generally do not signicantly impact the overall clearance factor, typically 2– 3 in a downstream process. While determining excess clearance (clearance minus risk) can be challenging, it
should be a critical aspect of any effort to mitigate viral infection risk.
Experiments involving virus spiking should be avoided in cGMP facilities, except in large- scale
production, where it’s an exception. To prevent potential virus burden at the polishing stage, virus
validation experiments should be conducted in small- scale trials using scalable equipment, especially during capture and intermediate phases. Steps likely to clear the virus should be individually considered when there is enough virus for informed conclusions. Conrmation of cleaning in
place and the efciency of frequently used chromatographic columns and lter systems is essential.
Fractional factorial designs are ideal for investigations, as downscale factors of 100– 1000 can be
achieved.
Virus clearance tests are typically conducted twice during the process and product lifecycle.
The rst is linked to the production of clinical phase I material, demonstrating the eradication of at
least two separate viruses. The second involves the eradication of four different viruses during the
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manufacturing of phase III material. Xenotropic murine leukemia virus (XMuLV), minute virus of
mice (MVM), Simian virus 40 (SV40), and pseudorabies virus (PRV) are commonly used as the four
virus models due to their representation of various traits reective of potential adventitious agents.
Validation model viruses must be comparable or identical to suspected viruses in the cell line or
closely related to viruses that could infect the cell, such as retroviruses for recombinant or hybridoma
cells. To achieve the ideal reduction factor for viruses, the model virus should be produced at high
titers and easily detectable with a responsive test. Caution is advised when concentrating a virus
solution to improve volumetric titer, as viral particle aggregation can lead to increased but negligible mechanical removal via ltration or decreased inactivation due to viral particle defense at the
aggregate’s center. Model viruses used as sources of infection include SV40, human poliovirus 1,
animal parvovirus, parainuenza virus or inuenza virus, Sindbis virus, RNA viruses, and murine
retroviruses.
In cases involving mammalian cell culture or biological materials, virus clearance is achieved
through downstream processing, validated similarly to sterility testing for bacterial contamination.
Spiking experiments for appropriate unit processes are used to assess the impact of viral clearance.
To ensure balanced virus distribution for individual intermediates, the virus titer of the load is
compared to the (residual) virus titer of the product- containing fraction after processing, such as the
ow- through or eluate of a chromatographic process or the permeate of a ltration process.
In most cases, virus inactivation occurs in a two- step procedure (fast phase 1 and slow phase
2). Samples taken at various time intervals, with at least one time point less than the minimum
exposure time, are used to construct an inactivation curve. Quantitative infectivity assays should be
sensitive, reproducible, and conducted with enough repetitions to ensure statistical validity. Assays
detecting viral contamination often yield highly variable ndings due to the biological nature of the
test techniques, necessitating extensive validation of assay accuracy, reproducibility, repeatability,
linearity, limit of quantitation, and detection limit. Objective statistical assessment is essential, as
emphasized in FDA’s “Points to Consider,” EMEA’s “Notes for Guidance,” ICH Guidelines, and
other sources. Plaque formation and cytopathic assays are the two most common in vitro assay
methodologies in quantitative virus clearance research, both validated and routinely used for determining viral titers. Q5A, the fourth phase of the ICH Harmonised Tripartite Guideline, provides
deeper insights into tests.
Before titrating process samples, it’s crucial to explore how buffer solutions might interact with
detector cells or diminish the infectivity of the model virus. Various detector cells for virus titration,
such as SC- 1 cells (Retrovirus), CV- 1 cells (SV 40), L 929 cells (Reovirus), and Vero cells (PI3),
are employed. The XC plaque assay is capable of detecting retroviruses over an extended period.
The procedure involves inoculating SC- 1 cells with the sample, UV- irradiating the cell layer, and
overlaying it with XC cells after a specic cultivation period. Once plaques form on the cell layer,
they are counted. Retrovirus- infected cell monolayer structure determines the titer in plaque or
concentrates forming units (pfu/ FFU). For viruses causing cytopathic effects (CPE) without plaques
or foci, the titer is expressed as TCID50 (tissue culture infectious dose for 50 percent of the total
cell number). Maintaining virus distribution across the process cycle, including washing and regeneration phases of chromatography or ltration retentate, is improbable due to virus denaturation by
commonly used caustic solutions or capture within the lter membrane matrix. Approved virus titers
typically range of 107 to 109 mL−1 but are reduced by 1 log due to a required 1:10– 20 spike for validation experiments. Titration of all process uids is technically challenging due to limitations set by
detection cell volumes, commonly using 0.1– 1.0 mL for titration.
Unit activity reduction factor computation involves process uid volumes and viral titers before
and after processing. The reduction factor, expressed in log 10 units, consists of “individual reduction factors” (Ri) for each operation. The combined specic reduction factors during purication
generate the overall virus reduction factor. Virus clearance accumulation is only valid for steps with
diverse physicochemical measures. A logarithmic reduction factor in the order of 1, indicating a
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90% titer reduction, is considered insignicant in virus clearance due to assay variability. Electron
microscopy quanties viral particles in cell culture uid, yet represents a challenge regarding sample
representativeness in various fermentation sizes.
Cells in culture, at densities of 106 to 107 cells per milliliter, reduce EM- analyzed cells to approximately 103/ mL after several logarithmic reductions. Identifying virus particles amid particulate
matter from the preparation process requires expertise. Sample preparation methods, like highspeed centrifugation, often yield complex aggregates, obscuring virus identication.
Risk assessment for validating a purication process involves evaluating multiple factors on a
case- by- case basis, including cell substrate, virus nature, culture methods, target protein, process
design, intended product use, patient population, and administration dosage/ frequency.
Although outsourcing viral clearance validation to specialized labs is common, replicating certain viral setups in a manufacturing facility might pose challenges due to time, cost, or resource
constraints.
11.13 PRODUCT CONCENTRATION
Tangential ow ltration (TFF) is a standard procedure utilized for products derived from E. coli
and CHO systems. Its primary functions involve clarifying, concentrating, and purifying proteins.
In a TFF system, uid is injected tangentially along the membrane’s surface, where some feed
components are pressured across the membrane onto the ltrate side. Larger particles and molecules
unable to pass through the membrane pores are retained and swept away by a crossow, preventing
membrane clogging and improving throughput (Figure 11.10).
TFF is categorized further based on the size of isolated molecules in the feed. Ultraltration
(UF) is the most prevalent form, separating proteins from buffer components (e.g., buffer exchange,
desalting, and concentration). Membrane pore sizes, ranging from 1 to 20 nm, effectively separate
proteins within the 1kD to 1000 kD range (known as the nominal molecular weight limit, NMWL).
The pressure- driven ultraltration process retains larger molecules (protein products), enabling
smaller molecules to pass through. This separation occurs primarily due to variations in ltration
rates of components through the membrane under applied pressure.
Ultraltration membranes often employ polymers like polysulfone, polyethersulfone,
polyvinylidene uoride, and regenerated cellulose. Their microporous structure controls selectivity,
molecule retention, and ltration ux (e.g., polyethylene). These membranes can be reused without
signicant performance decline or contamination risks, thanks to their resistance to robust cleaning
agents, acids, and high temperatures. Cellulose membranes, superior in permeability and retention,
are widely used for protein applications, prioritizing high retention crucial for successful processes.
When selecting the right membrane, considerations include NMWL rating, chemical compatibility,
and fouling limit (Table 11.8).
FIGURE 11.10 Tangential ow ltration
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TABLE 11.8
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Commercial Membranes and Properties
Commercially Available Membranes Properties
®
Ultracel
Ultracel PL Regenerated cellulose, NMWL– 1 to 300 kD
Ultracel PLC Composite regenerated cellulose, NMWL: 5– 1000 kD
®
Biomax
Regenerated cellulose
Polyethersulfone- based, NMWL– 5 to 1000 kD
In manufacturing processes, two common UF systems include virus ltration and high-
performance tangential ow ltration (HPTFF). HPTFF achieves separation by leveraging
differences in size and charges. Efciency is maximized by altering buffer pH and ionic
strength to increase disparities between the product and impurities. Particularly in mAb purication techniques, HPTFF effectively removes host cell proteins (HCP) and host cell DNA
contaminants.
Dialtration (DF) is a TFF technique that converts a protein buffer into a preservable and
stabilizable form. It collaborates with ultraltration or other separation methods to enhance product
yield or purity. During dialtration, the buffer enters the recycle tank while the ltrate exits the
device service. Typically, the target protein product resides in the retentate, and dialtration ushes
components out of the retentate into the ltrate, combining buffer exchange and undesired impurity
removal/ reduction into one technique. Dialtration can be performed in batch or continuous volume
modes. Batch mode involves adding a large buffer volume to the recycling tank, concentrating the
retentate, and repeating the process until achieving the desired number of diavolumes. Continuous
volume mode maintains the retentate volume by adding buffer at the same rate as ltrate removal,
offering more regulated product concentration than batch mode. The primary aim of the DF step
is to remove undesirable buffer or contaminant species from the retentate product, affecting yield
based on the number of dia- volumes utilized.
The UF/ DF formulation step stands as an indispensable and widely adopted stage that handles
the highest product concentration, primarily in its nal formulation— an integral part of downstream
processing. Evolution persists within UF/ DF operations, emphasizing robustness, high yield, and
cost- effectiveness. Moreover, UF/ DF holds a standard position for buffer exchange and product
concentration processes before or between chromatography steps.
Within this process, a retentate tank contains the target protein product, while a membrane
assembly retains the feed. A feed pump propels the feed through the membrane via tangential ow and
pressure. The traditional batch mode operation involves recirculating the feed and retentate through
the lter assembly multiple times until achieving the desired protein concentration. Additionally,
process monitoring tools, equipment for testing product recovery, and cleaning methods ensure
operational consistency and desired yields. TFF systems encompass both traditional stainless steel
and more recent single- use methods, constructed using hollow bers or cassettes. The advent of
automated single- use systems with disposable ow routes and lower volume hold- up enhances exibility, reduces cross- contamination risks, and accelerates turnaround due to minimized cleaning
tasks (Figure 11.11).
In recent years, single- pass TFF (SPTFF) has gained prominence— an advanced version of batch
mode operation. Here, after a single pass through the lter assembly, the retentate achieves sufcient
concentration, eliminating the need for recirculation. This high- throughput and recovery owe credit
to increased residence time in the feed channel, brought about by reduced ow rates or longer path
lengths. SPTFF proves particularly benecial for volume reduction steps between chromatography
or other intermediate steps with large processing volumes.
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FIGURE 11.11 Schematic of a UF/ DF system
255
FIGURE 11.12 Schematic for DP preparation activities
Regardless of the operational mode, delineating the process objectives— nal product concentration, impurity removal, volume reduction, and buffer exchange— is crucial. Such clarity maximizes
process efciency, enabling precise planning for robust and consistent scale- up operations at the
commercial level. Crucially, optimizing process parameters for TFF (UF/ DF) minimizes product
loss. Several critical process parameters merit consideration for the TFF (UF/ DF) process:
• Transmembrane pressure (TMP) refers to the pressure difference across a membrane. It
dictates the force needed to drive the feed through the membrane. Filtrate ow increases until
it reaches a plateau at a specic TMP level. When protein concentration rises or feed ow rate
decreases, TMP decreases until ux hits a plateau (Figure 11.12). Optimal activity occurs at
a TMP that sustains high ux without excessive pressure or high protein concentration at the
membrane.
• Crossow rate is determined by the feed channel type and is when the solution moves across
the membrane in the feed channel. Higher crossow rates at the same TMP result in increased
ux, potentially causing more product to pass through, leading to product degradation. To
counter this, balancing increased ux with more pump passes and holdup volume is crucial
for the appropriate crossow rate.
• Membrane area is determined based on total processing volume and process ux. While opting
for a longer process time might reduce membrane area and holdup volume requirements,
excessively low values might compromise product quality (causing degradation or contamination). It’s advisable to include a safety margin to account for fouling and variations in the
feed stream.
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• Filtrate control, used in most applications where the target product remains in the retentate,
does not have a set point for control and might operate without restrictions.
For dialtration operations combined with ultraltration, determining the starting point for the
dialtration process is essential.
High concentration formulations are generally preferred, especially when the injection volume is
low (common in subcutaneous administration of most monoclonal antibodies). However, different
drug modalities like antibody- drug conjugates, bispecic antibodies, cell, and gene therapies
have prompted adaptations to existing TFF systems (e.g., single- use systems, SPTFF, enhanced
monitoring, and pre- sterilized plug- and- play options).
11.14 ANALYTICAL METHODS
Downstream processing primarily aims to reduce or eliminate process and product- related
contaminants to yield a pure product. In- process testing during manufacturing adjusts process
parameters, ensuring overall process control. These analytical procedures are the foundation of phase
analytical technology (PAT). Monitoring product- related impurities such as high- molecular- weight
and low- molecular- weight species, glycan variations, and charge variants is critical for overall protection of the products (for mAb products). Currently available methodologies have evolved greatly,
making in- process testing a critical tool. Improvements include high- throughput technologies that
enable rapid sample processing, a faster turnaround that can support downstream operations effectively and efciently, particularly for operations that are dependent on a specic analytical testing,
example, loading and optimal operation of the downstream unit operation requires knowledge
of the product concentration (i.e., titer) in the cell culture media, and subsequent product pools,
similarly for product concentration steps. While determining the suitability of testing for largescale operations, the nature of the product at that stage, e.g., susceptibility to degradation, must be
considered for evaluating process hold conditions and minimizing the testing time in such instances.
For recombinant proteins produced by E. coli, scanning electron microscopy (SEM) assesses
inclusion body morphology and size using Cell Disruption and Recovery Analysis (CDRA). SEM
helps evaluate the impact of cell disruption methods and culture conditions on inclusion body
development. Traditional plating methods involve plating lysate samples before homogenization
on selective and non- selective media, followed by incubation for 12– 24 hours. Counting the cells
on agar plates determines homogenization efciency post each pass. Turbidity measurements, as in
harvest and clarication procedures, monitor centrifugation processes for inclusion body recovery.
SDS- PAGE serves as a valuable tool in assessing the purity of inclusion bodies during recovery
operations. It proves especially useful in devising wash solution strategies and evaluating the reduction of host cell- related protein contaminants. Additionally, this method aids in examining protein
solubilization and refolding. Detection of intermolecular (and occasionally intramolecular) disulde
bonds can be achieved analytically through SDS- PAGE under nonreducing conditions. This involves
sequentially treating proteins with iodoacetamide (to prevent false disulde exchange) followed by
SDS in the absence of reductant. Techniques such as RP- HPLC, SDS- PAGE, and analytical SEC
are clear options for promptly assessing denaturation or refolding degrees. However, even Surface
Plasmon Resonance (SPR) and light scattering can be utilized. Both qualitative and quantitative
methods prove useful in identifying conditions conducive to solubilization and refolding.
Various standard analytical tests aim to determine protein titer while monitoring processrelated and product- related removal across unit operations. Methods for estimating protein titer
include UV280nm, RP- HPLC, Bradford Assay, Lowry Assay, Octet, and ProA HPLC. For aggregate analysis, SDS- PAGE, CE- SDS, and analytical size- exclusion methods are commonly used for
estimating low and high molecular weight species. Charge variant analysis typically involves CEXHPLC and iCE.
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Several commercial HCP kits are available to monitor process- related impurities, offering options
to customize anti- HPC- antibodies. These kits are primarily ELISA- based, and automated immunoassay systems also provide their HCP kits.
11.15 DOWNSTREAM PROCESSING EQUIPMENT AND SYSTEM
COMPONENTS
Downstream processing encompasses diverse equipment and supporting components, often referred
to as skids, varying in complexity. Equipment specic to downstream operations typically include
• Buffer Preparation and Storage Systems.
• Cell Disruption and Centrifugation (Microbial Processes).
• Purication Systems/ Chromatography Separation.
• Filtration Systems (membrane ltration, depth ltration, etc.).
The equipment used in these processes must offer exibility across various scales (lab, pilot,
and commercial- scale production) while ensuring no degradation or damage to the product due
to equipment selection. Collaboration between drug manufacturers and equipment manufacturers
is crucial to meet user requirement specications (URS) tailored for the specic process needs.
Signicant advancements in bioprocessing have led to the development of fully automated skids
for purication and membrane ltration operations. Disposable chromatography skids are gaining
popularity due to their rapid turnaround and elimination of cleaning and cleaning validation
requirements. Skids enable efcient setup and streamlined processing, each unit or system component designed considering the process requirements and functional operation.
Key design features include
• Process control unit/ functional system in which the product is processed (e.g., chromatography columns, membrane lter, etc.).
• The automation system enables the sensors’ connection/ communication with the detection
units in the process control system to allow operation and monitoring. Various hardware/ software platforms with optional customizable congurations are provided.
• Inlet and outlet systems (e.g., a buffer tank, holding tank, etc.) are connected to the process
control unit and other secondary accessories, including piping, valves, pumps, etc.
• Measurement devices/ probes/ sensors are used to control and monitor the process (e.g., pH,
conductivity, UV, pressure sensors).
Manufacturers typically conduct factory tests before delivering manufacturing skids to user facilities. Subsequently, on- site installation and qualication exercises (IQ, OQ) are performed to verify
specications before using the equipment in manufacturing operations.
11.16 SENSORS, PROBES, AND METERS
For upstream bioreactor operations, online or inline monitoring and controlling of process parameters
are common. Downstream processes monitor parameters
• ow rate, pH, conductivity, and UV for chromatography unit operations,
• pressure, ow rate/ speed for clarication, ltration, and
• ow rate, concentration factor, pressure, conductivity, protein concentration, temperature.
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