REVIEW
Clarification Technologies for Monoclonal
Antibody Manufacturing Processes:
Current State and Future Perspectives
Nripen Singh, Abhiram Arunkumar, Srinivas Chollangi, Zhijun George Tan,
Michael Borys, Zheng Jian Li
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb,
35 South Street, Hopkinton, Massachusetts 01748; telephone: (978) 784-6909;
fax: (978) 784-6639; e-mail: Nripen.singh@bms.com
ABSTRACT: Considerable progress has been made increasing
productivity of cell cultures to meet the rapidly growing demand for
antibody biopharmaceuticals through increased cell densities and
longer culture times. This in turn has dramatically increased the
burden of process and product related impurities on the purification
processes. In addition, current trends in the biopharmaceutical
industry point toward both increased productivity and targeting
smaller patient populations for new indications. Taken together,
these developments are driving the industry to explore alternative
separation technologies as a future manufacturing strategy.
Clarification technologies well established in other industries,
such as flocculation and precipitation are increasingly considered as
a viable solution to address this bottleneck in antibody processes.
However, several technical issues need to be fully addressed
including suitability as a platform application, robustness, process
cost, toxicity, and clearance. This review will focus on recent efforts
to incorporate new generation clarification technologies for
mammalian cell cultures producing monoclonal antibodies as
well as challenges to their implementation supported by a case
study.
Biotechnol. Bioeng. 2016;113: 698–716.
ß 2015 Wiley Periodicals, Inc.
KEYWORDS: clarification; filtration; flocculation; monoclonal
antibody; precipitation; purification
Introduction
Clinical and commercial success of biologics has impelled the need
for a large global manufacturing capacity (Anicetti, 2009). Demand
has driven significant improvements in upstream productivity
cresting at product titers greater than 10 g/L; however, this has
increased the burden on clarification and purification steps further
downstream to remove the increased levels of biomass and
Correspondence to: N. Singh
Received 11 June 2015; Revision received 20 August 2015; Accepted 20 August 2015
Accepted manuscript online 24 August 2015;
Article first published online 29 September 2015 in Wiley Online Library
(http://onlinelibrary.wiley.com/doi/10.1002/bit.25810/abstract).
DOI 10.1002/bit.25810
698 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016
impurities. Moreover, these impurities can show significant
variations, which primarily depend on culture conditions. Concen-
tration and composition of impurities are critical for efficient process
development to meet the demands of increased product quality.
These demands may be met by either increasing capacities of filters
and chromatography resins, dimensions of filters and columns or
developing alternative means of purification that are effective,
economical and scalable (Cooper, 2005; DePalma, 2009; Gottschalk,
2008; Th€ommes and Etzel, 2007). Reducing levels of these impurities
and adventitious agents like endotoxins and viruses earlier in the
process will ease the burden on downstream purification.
Impurities generally consist of product and process related
components as well as contaminants as shown in Table I. Product
related components are molecular variants of the desired target
molecule, aggregates or product variants by different posttransla-
tional modification, and degraded products. Process related
impurities are cell components like host cell proteins (HCPs) or
DNA, chemical additives, and residual media. Among all of the
soluble impurities present in a cell culture fluid, HCPs and DNA
constitute majority of them. The vast heterogeneity in biophysical
and biochemical properties of HCPs such as Mw, isoelectric point
(pI), hydrophobicity and surface charge distribution makes them
particularly difficult to remove and demand multiple unit operations
employing distinct separation mechanisms since the presence of any
trace levels of these impurities can elicit serious immune reactions in
patients (Aboulaich et al., 2014; Jin et al., 2010; Levy et al., 2014;
Nogal et al., 2012; Shukla et al., 2007; Shukla and Thommes, 2010).
Because of this concern, identity of these HCPs has recently attracted
attentionand extensive proteomic analyses in combination with
techniques like 2D-DIGE, 2D-GE/MS and 2D-LC/MS have been
conducted (Aboulaich et al., 2014; Jin et al., 2010; Levy et al., 2014;
Pezzini et al., 2011; Zhang et al., 2014). Approximately 15% of these
proteins are found to be highly acidic (pI < 5), 60% of proteins are
mildly acidic to neutral (pI 5–7) and fewer than 25% of proteins are
basic (pI > 8) (Jin et al., 2010; Levy et al., 2014; Pezzini et al., 2011).
The profile of these HCPs that need to be removed during
downstream processing is influenced significantly by expression
conditions and primary recovery operations. Reducing the levels of
these soluble and insoluble impurities earlier in the process is
ß 2015 Wiley Periodicals, Inc.
Table I. Iso electric points, source and cause of burden for various class of molecules commonly found in CHO cell harvests and process
intermediates (Aboulaich et al., 2014; Braisted and Wells, 1996; Gagnon et al., 2014; Jin et al., 2010; Levy et al., 2014; Mengeling et al., 1988; Raetz,
1993; Zhou, 2008).
Molecule class pI Range MW Range (kDa) Hydrophobicity
HCPs 2–11 10–200 Variable
DNA 2–3 90–1000 Low
Insulin 53–5.5 5.8 Low
Viruses 4–7.5 200–7200 Variable
Protein-A 4.8–5.2 6 Low
Endotoxins 1–4 3–40 Variable
therefore a key requirement for efficient purification. Previous
attempts to implement such a strategy have been hindered by a lack
of clarification technologies with robust adsorptive capabilities. Such
a technology would need to be capable of handling large process
volumes, high impurity loads, and high stream turbidity that are
typical in cell culture supernatant.
The separation of cells and cell debris can be facilitated by
techniques like centrifugation, microfiltration, depth filtration as
well as various pre-treatment approaches based on precipitation or
flocculation techniques (Hutchinson et al., 2006; Roush, 2008).
Different flocculants and precipitants have been explored as
components to improve clarification efficiency, process yield, and
clearance of impurities during the primary recovery step of
biologics from mammalian cell culture (Brodsky, 2012; Kang et al.,
2013; Riske et al., 2007; Romero, 2010; Roush, 2008; Singh et al.,
2013a). Although precipitation relies on lowering the solubility of
target solutes in order to create solid particles, flocculating agents
trigger the destabilization of a biocolloidal suspension by causing
the adhesion of dispersed particulates into larger-size clusters,
resulting in an increase in the average particle size distribution.
Selective precipitation of either the impurities (cell debris, HCP,
DNA) or the target antibody is achieved by altering the cell culture
environment through variation of factors like pH and conductivity
of solution, or the addition of precipitants like ethanol, ammonium
sulfate, polyethylene glycol (PEG), caprylic acid, and divalent ions
(Lydersen et al., 1994; Westoby et al., 2011; Grodzki and Berenstein,
2010; Giese et al., 2013; Brodsky et al., 2012; Herzer et al., 2015).
Flocculation using polyelectrolytes can be implemented either to
selectively flocculate target proteins (McDonald et al., 2009;
Michaels and Miekka, 1961; Zhang et al., 2005) or to flocculate
impurities while leaving the protein product in the solution (Kang
et al., 2013; Ma et al., 2010; McNerney et al., 2015; Mcnerney et al.,
2013; Rathore, 2009).
Depth filters are traditionally used in the clarification of
mammalian-cell and bacterial fermentation broths to improve
capacity of sterilizing grade filters and to protect chromatography
columns and virus filters. Although optimized depth filtration plays
an important role in removing insoluble impurities and enhancing
the efficiency of the entire purification process, it has recently been
explored for clearance of soluble impurities. A typical depth
filtration involves a number of complementary mechanisms that
allow impurity removal on the basis of molecular interactions.
Known mechanisms are electrostatic, hydrophobic, and hydrogen
bonding in addition to the well known hydrodynamic interactions
(size-based sieving, interception, and cake-filtration). Recent
Source Cause of contamination
Host cells Secretion, cell rupture,cell death
Hust cells Cell rupture, cell death
Cell culture media Addition to media
Host cell, cell culture media Adventitious contamination
Protein-A resin Leaching during chromatography
Cell culture media & contamination Adventitious containation
advances in depth filtration has made it a potential powerful tool
for impurity clearance during antibody purification. For example,
positively charged adsorptive depth filters have been used pre and
post chromatographic capture step and demonstrated robust
removal of impurities (Charlton, 1999; Dorsey, 1997; Schreffler
et al., 2015; Tipton et al., 2002; Yigzaw, 2006).
This review will focus on efforts made in recent times to
incorporate the use of flocculants and precipitants along with the
new filtration media for clarification of mammalian cell cultures
used in production of antibodies supported by a case study
implementing the same. The mechanism of action of various
flocculants and precipitants are reviewed along with filtration
media for clarification and impurity clearance. Although electro-
static interactions play an important role in impurity clearance
during these clarification technologies, additional mode of
interactions like hydrophobic interactions and hydrogen bonding
do exist between the HCPs and clarification media and a better
understanding of the physicochemical properties of HCPs should
help to elucidate the exact mechanisms of removal. This will drive
successful implementation of these new techniques, while in turn
allow for process compression, cost reduction and streamlined
production of antibodies.
Precipitation Technologies
Precipitation has long been used in the plasma protein industry to
purify proteins and can play a singnificant role in separation and
enrichment of the target antibody. Precipitation not only purifies
the target product but may offer further economical benefit of
significant volume reduction in cases where the precipitated
product is readily soluble at a higher concentration in another
buffer system. However, critical issues including robustness,
scalability, precipitant clearance, and process economics are often a
concern and has resulted in slow adoption of these technologies in
antibody manufacturing processes. In addition, process economics
and scalabilty need to be evaluated before incorporating in the
platform (DePalma, 2009; Sommerfeld and Strube, 2005). In this
section, a review of the different precipitants is presented that have
been evaluated in recent times (Fig. 1 and Table II).
Acidification
Precipitation of impurities in a CHO cell culture by lowering the pH
was first demonstrated by Lydersen et al. (1994). Following this
study, several other groups also demonstrated the effectiveness of
Singh et al.: Clarification Technologies for Monoclonal Antibody 699
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 Figure 1. Chemical structures of commonly used flocculants and precipitants.

lowering the pH below the isoelectric point (pI) of the target Acidification of cell culture results in the transition across the
antibody for selective precipitation of process related impurities isolectric points of these impurities making them insoluble. Since
such as HCPs and DNA in aqueous solutions leaving the target HCP predominantly span the pI range of 4.0–6.5, a pH range of
protein in solution (Liddell, 2011; Takeda and Ochi, 2011). 4.5–5.0 is often found to be the most effective in isoelectrically

700 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016

 Table II. Comparison of various precipitants and flocculants used in clarification of biologics processes.

Pre-treatment Precipitant/F Operation Yield

method locculants Mode of action pH Dosage window (%) Impurity reduction Technical challenges References
Precipitation low pH- metals Charge neutralization 4.5–5.5 N/A >85 HCP:<1LRV DMA: 1–3 LRV Yield loss scalability product (Liddell, 2011; Lydersen et al., 1994;
HMW: none viruses: NT stability Singh et al., 2013; Takeda and Ochi,
2011; Westoby et al., 2011)
Organic solvents Solubility reduction 6.5–7.5 10–40% (w/v) >85 HCP:<1 LRV DNA:<1 LRV Prolonged stirring product (Hammerschmidt et al., 2015;
HMW: none viruses: NT stability hazardous waste Tscheliessnig et al., 2014)
PEG-metals Steric exclusion hydrophobic 5.5–8.5 5–15% (w/v) >80 HCP: <1 LRV DNA:<3 LRV Selectivity residual clearance (Giese et al., 2013; Page and Thorpe,
interaction HMW: yes viruses: NT 2002; Ramanan and Stenson, 2008;
Sommer et al., 2014)
Capryylic acid Coagulation and charge 4.5–5.5 0.1–10% (w/v) >90 HCP: 1–3 LRV DMA: 3–5 Yield loss solid liquid (Arunakumari et al., 2012; Brodsky,
neutralization LRV HMW: none viruses: separation scalability 2012; Herzer et al. 2015)
>3LRV
Affinity (eg. ELPs) Affinity stimulus response 6.5–7.5 ELP to Product ratio of >90 HCP: 1–3 LRV DMA: 3–5 High cost toxicology (Meyer and Chilkoti, 1999; Sheth et al.,
4:1 LRV HMW: none viruses: NT concerns 2013, 2014a,b)
Anionic flocculation PVS Charge interaction with the 6.5–7.5 10–60 (pg/TCD) >90 HCP: <1 LRV DNA: 3–5 LRV Product stability toxicology (Fahrner, 2008; McDonald et al., 2009;
PAA product HMW: none viruses: NT concerns Peram et al., 2010)
CMD
Cationic flocculation pDADMAC Electrostatic hydrogen 4.5–7.5 10–60 (pg/TCD) >90 HCP:<1 LRV DNA: 4–6 LRV scalability variability (Ma et al., 2010; McNerney et al., 2015;
PEI bonding HMW: none viruses: NT toxicology concerns McNerney et al., 2013; Riske et al.,
polyamino acid 2007; Tomic et al., 2015)
Mix-mode AMPS-AB2 Electrostatic hydrophobic 4.5–7.5 10–500 (pg/TCD) >90 HCP: <1 LRV DNA: 3–5 LRV Product stability toxicology (Capito et al., 2013a,b; Jaber et al,
flocculation Copolymer hydrogen bonding HMW: yes viruses: >3LRV concerns 2011; Kang et al., 2013)
Modified
benzyl poly
(allylamme)

pg/TCD, picogram/total cell density; w/v, weight/volume; NT, not tested.

Biotechnology and Bioengineering

Singh et al.: Clarification Technologies for Monoclonal Antibody
701
precipitating the HCP (Westoby et al., 2011). Recently, the addition
of “generally recognized as safe” flocculation reagents such as
calcium chloride and potassium phosphate in combination with
acid precipitation have been studied and demonstrated superior
performance compared to acid precipitation alone (Romero, 2010;
Westoby et al., 2011). The investigators observed reductions in DNA
concentrations of 1–3 log10 reduction value (LRV) and HCP levels of
3–10 fold reduction value (FRV). The mechanism involved in this
type of separation is hypothesized to be related to the
co-precipitation of colloidal calcium phosphate with impurities.
Colloidal calcium phosphate is more insoluble at pH values close to
near neutral pH and with the additional advantage of the higher
density of the calcium phosphate enables easy precipitation. Studies
into the potential of the CaCl2 precipitation step for virus reduction
would be promising as calcium phosphate has been shown to
co-precipitate viruses (Pham et al., 2001).
Caprylic Acid
Caprylic acid, also called octanoic acid (OA), is an eight-carbon
saturated fatty acid. It can be used effectively for the precipitation of
biologics. The use of OA and its sodium salt is well described for
precipitation of proteins from serum, plasma, ascites fluid
(McKinney and Parkinson, 1987; Parkkinen et al., 2006; Russo
et al., 1983; Steinbuch and Audran, 1969). In recent times, it has
also been explored for antibody production (Brodsky et al., 2012;
Herzer et al., 2015; Joseph, 2013; Russo et al., 1983; Temponi et al.,
1989). OA is poorly soluble in water at
4.85 mM and its solubility
decreases even further with decrease in pH driven by its pKa (4.8).
Precipitation equilibrium of proteins by OA is thermodynamically
controlled and proteins can either partition between a OA-water
partially miscible phase or have specific interactions with OA
(Brodsky et al., 2012). At low pH the hydrophobicity of the octyl
moiety of OA dominates and acidic proteins such as albumin and
other HCP tend to precipitate alongwith OA. Antibodies with
relatively basic pI have sufficient charge to counteract that
hydrophobocity and remain in solution.
Addition of OA during low pH treatment in the bioreactor
prior to cell culture fluids (CCF) or post Protein A purification
has been demonstrated recently for purification capability
enabling a two-step purification process for mAb and dimeric
domain antibody fragment (dAB) (Brodsky et al., 2012; Herzer
et al., 2015). Although recovery of the mAb has been observed
in the range of 86–100%, it was found to be lower for dAB
(<60%). The differences are attributed to hydrophobic
interactions with OA due to large exposed hydrophobic region
to the complement detrmining region (CDR) for dAB that would
be buried in full length antibody. When used post Protein A,
Brodsky et al. demonstrated >90% reduction in HCP bringing
their levels down to less than 200 ppm for the six antibodies
tested. In addition, a modest decrease in the high molecular
weight (HMW) aggregates was observed. Finally, OA treatment
has also demonstrated efficient inactivation against enveloped
viruses and moderately efficient against non-enveloped viruses
(Brodsky, 2012; Dichtelmuller et al., 2002; Mpandi et al., 2007;
Vacante and Connell-Crowley, 2014). The viral inactivation by
OA is attributed to the capability in destroying the lipid bilayer
702 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016
of enveloped viruses and has the potential to aid in viral
clearance.
Organic Solvents
Organic solvents are often used for fractional precipitation of proteins
from a complex mixture. Since the 1940s when this process was first
established by Cohn and coworkers, it has undergone several
modifications to improve yield and purity for the manufacture of
human plasma products. (Buchacher and Iberer, 2006; Cai et al.,
2002). Still, the basic principle is decreased solubility of proteins in
presence of organic solvents resulting in selective precipitation.
However, organic solvents are protein destabilizers and can denature
proteins at high concentrations or at high temperatures due to their
favorable interactions with hydrophobic group (Arakawa et al., 1990,
2007; Yoshikawa et al., 2012).
More recently, Tscheliessnig et al., have developed a new strategy
for purification of recombinant human antibodies from CHO cell
culture supernatant based on cold ethanol precipitation (CEP) and
CaCl2 precipitation (Tscheliessnig et al., 2014). The investigators
have shown the applicability of the precipitation process for four
different antibodies (rhAbs) of different pIs resulting in similar
yield and purity as Protein A chromatography. HCP clearance was in
some cases very efficient (80 to 90% reduction). Hammerschmidt
et al. (2015) discussed the use of continuous system based on CEP of
the clarified cell culture and CaCl2 flocculation. This study
compared the potential of using precipitation in continuous mode
and compared findings with bind-and-elute purification using
Protein A chromatography. The continuous operating mode
exhibited the same performance characteristics, in terms of yield
and purity, as the batch mode without any alterations to the
precipitation parameters determined at small batch scale.
Poly(Ethylene Glycol) (PEG)
The fundamentals of protein precipitation by PEG were established
in the early 1960s (Polson et al., 1964) and since then has been
extensively studied to understand the protein behavior (Atha and
Ingham, 1981; Hasko et al., 1982). PEG is a polar uncharged
polymer and can be considered as a viable option for precipitation
of antibodies. Although PEG is an attractive method due to its low-
cost, it lacks selectivity. However transition region of protein
precipitation size can be adjusted by PEG size and PEG
concentration, whereby size has a higher impact with less drastic
viscosity increase. The generally accepted mechanism of precip-
itation of proteins using polar uncharged polymers such as PEG is a
steric exclusion mechanism which provides specificity based on size
(Arakawa and Timasheff, 1985; Atha and Ingham, 1981; Bhat and
Timasheff, 1992; Shulgin and Ruckenstein, 2006). Larger protein
complexes such as HMWs precipitate more readily in the presence
of polar uncharged polymers like PEG due to larger exposed surface
being destabilized by competition with the PEG for binding of water
molecules and the exclusion of the PEG from the protein surface
(Bhat and Timasheff, 1992). The mechanism for HCP removal is
believed to be interaction of HCP with PEG based on hydro-
phobocity of HCPs, rather than size exclusion since HCP are
heterogeneous in size and generally smaller than antibodies.
 Giese et al. tested precipitation of impurities using PEG for a
variety of antibodies over a range of PEG concentrations to
determine the optimum for each antibody (Giese et al., 2013).
Protein A pools were used in this study to determine the effect of
increasing PEG concentration on the yield, HMW reduction and
HCP reduction. With increasing PEG concentration, all three
attributes decreased, characteristic of the classic trade-off between
purity and yield. Direct capture step for the purification of
recombinant antibodies from clarified culture supernatants has also
been evaluated with combination of CaCl2 and PEG precipitation
equivalent to protein A affinity chromatography (Sommer et al.,
2014). All precipitation tests were performed with five different CHO
cell culture supernatants, and showed yields between 80–95%.
Chromatography and electrophoreses data of CaCl2/PEG precip-
itation were roughly comparable to Protein A purification.
Aqueous Two-Phase Systems (ATPS)
ATPS are biphasic systems that have been used for downstream
processing of different biological products such as cells, viruses,
proteins, enzymes, and plasmid DNA by extraction (Albertsson,
1970; Benavides et al., 2006; Bradley and Scott, 2004; Everberg
et al., 2004; Frerix et al., 2005; Gomes et al., 2009; Kumar, et al.,
2001). ATPS extraction is a nondenaturing and nondegrading
technique that exploits the spontaneous formation of two
immiscible aqueous phases usually created when either two
polymers or one polymer and one salt are mixed in water above
certain critical concentrations. The partitioning between both
phases is dependent on the intrinsic and extrinsic propeties of the
target protein and on the composition of the two phase system.
Polymers, such as PEG, and buffering salts, such as phosphates,
sulfates and citrates, are typically used to create phases that result
in the target protein partitioning into one phase while impurities
partition into the other. The extraction of mAbs using ATPS such
as PEG-phosphate, PEG-citrate and PEG-dextran system has been
demonstrated for purification of mAbs with greater than 90%
recovery yield and significant reduction in process and product-
related impurities (Azevedo et al., 2007, 2009; Mao et al., 2010;
Muendges et al., 2015a; Zijlstra et al., 1996). However, in order to
achieve those high recovery yields and purities in just one stage,
usually high salt concentrations are required. This may be a
bottleneck during the scale-up of these systems to a manufacturing
process since these high salt concentrations can potentially shorten
the life time of the equipments and may cause more precipitation of
the target product. Multi-stage equilibrium ATPS extraction has been
successfully evaluated in recent times for the purification of mAbs
while overcoming the limitations of single stage extraction
(Muendges et al., 2015b; Rosa et al., 2009). High recovery yields
and purities can be acheived by optimizing the salt concentration
and/or the number of stages and/or the phase volume ratio. This
opens promising perspectives for the integration of multi-stage
equilibrium ATPS extraction as a powerful, nonchromatographic unit
operation for the downstream processing of mAbs, allowing
simultaneously the clarification, concentration and partial purifica-
tion of the target protein in one step.
Overall, precipitation technologies can offer significant benefits
to biologics processes, including increased clarification efficiency,
impurity removal, and process simplification. Several parameters
(pH, conductivity, precipitant type, and mixing time) must be
tested with definitive screening designs in a product-specific
manner for effective precipitation. Other operating parameters
affecting mass transfer such as temperature and mixing speed also
must be optimized for consistent performance of the precipitation
step during scale-up. Nevertheless, optimized precipitation
processes could allow to overcome the current drawbacks of
limited biologics purification capacity (DePalma, 2009; Th€ommes
and Etzel, 2007).
Affinity Precipitation
Purification through the expression of recombinant proteins as
fusions to carrier domains or peptides followed by affinity based
precipitation has been demonstrated (Kim and Raines, 1993; Smith
et al., 1988, 1998; Su et al., 1992; Tsao et al., 1996). Although useful
at laboratory and medium-scale purification, this tag based
precipitation can be cost prohibitive at large scale preparative
processes. Meyer and Chilkoti proposed the use of phase reversible
elastin like polypeptides (ELPs) for non-chromatographic,
thermally stimulated phase separation for recombinant proteins
(Meyer and Chilkoti, 1999). Urry and colleagues have shown that
synthetic poly (GVGVP), also termed as ELP, is soluble in water
below 25 C, but when the temperature is raised above 25 C,
aggregation occurs followed by settling, and phase separation (Urry,
1997; Urry et al., 1988). Experimental and computational evidence
suggests that the phase transition is a result of conformational
changes in the polypeptide, including formation of beta-turns, and
“beta-spiral” a structure unique to elastin and ELPs and not known
to exist in any other mammalian proteins (Sandberg et al., 1981;
Urry, 1997). Urry has summarized a comprehensive list of
parameters that modulate the hydrophobicity of ELPs which include
temperature, length of polypeptide, composition of the polypeptide,
salt concentration and pH of the solution among many others (Urry,
1997). As the temperature is raised, linear ELPs associate to form
rigid filamentous structures separating from liquid phase and can
be targeted for affinity precipitation.
ELP Fusion Peptide
Though Meyer and Chilkoti were the first to demonstrate the
applicability of ELP to purify target proteins by fusion expression
and precipitation, the procedure required enzymatic treatment to
elute the target protein from the precipitate (Meyer and Chilkoti,
1999). In 2005, Banki et al. eliminated the need for proteolytic
cleavage by fusing the target proteins to ELPs via self splicing intein
domain (Banki et al., 2005). During the same period, Kim et al.,
employed ELP-Protein A fusion as an affinity precipitating agent for
mAbs (Kim et al., 2005). Following precipitation, ELP-Protein A:
antibody complex is re-solubilized, dissociated by low pH treatment
and the ELP-Protein A fusion is re-precipitated leaving the mAb in
solution. More recently Sheth et al., have utilized ELP-Z peptides
(ELP domain fused with an engineered B-domain from staph-
ylococcal Protein A) to selectively precipitate antibodies followed by
resolubilization and elution of the antibody into solution (Sheth
et al., 2013, 2014a,b) as shown in Figure 2. The Z-domain consists
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Figure 2. Schematic overview of the ELP-Z based affinity precipitation mechanism that shows an initial capture and co-precipitation of the mAb by ELP-Z, and mAb elution from
the ELP-Z–mAbcomplex followed by a final ELP-Z precipitation step.
of an antiparallel three-helix bundle containing 58 amino acid
residues that selectively binds to the Fc portion of IgGs with high
affinity (KD ¼ 10–50 nM) (Braisted and Wells, 1996; Starovasnik
et al., 1997). In addition to the high selectivity, the Z domain also
exhibits high tolerance to proteolysis, extremes of pH and high
concentration of denaturants such as urea and guanidine
hydrochloride, which makes it attractive for mAb purification
(Linhult et al., 2004). During this study, the investigators have
shown that an ELP-Z: mAb molar ratio of 4: 1 yields highest
precipitation in cell culture harvest and subsequently good recovery
of the mAb (Sheth et al., 2014b). Without the need for a
chromatography column, this mode of separation resulted in >2
LRV of HCP clearance and >4 LRV of DNA reduction which is
comparable to the numbers normally achieved using Protein A
affinity chromatography. Thus, affinity precipitation by ELP-Z or
ELP-Protein A domains provides an effective alternate method to
rapidly enrich the antibody without the need for a column. However,
to be noted is the fact that ELP fusion peptides based precipitation
is still an emerging technology and cost of production of these
peptides is prohibitive at manufacturing scale for implementation
yet. Progress in large scale expression and commercial availability
of the ELPs would enable implementation of this technology in a
more widespread manner.
Flocculation
Flocculation is widely implemented in waste water treatment, food,
beverage and cosmetics industries (Baets and Coldenhoff, 2007;
Liang et al., 2014; Lopez-Maldonado et al., 2014; Parkkinen et al.,
2006). However it has been recently been explored in the antibody
manufacturing processes. Flocculants can be anionic, cationic or
“multimodal” (Figure 1 and Table II). When the solution pH is less
than the pI of a particular protein, the protein carries a net positive
charge. Under these conditions, a cationic polyelectrolyte may
precipitate impurities and leave the protein of interest in solution.
Conversely, an anionic polyelectrolyte may precipitate the protein of
interest forming a protein-polyelectrolyte precipitate, leaving
impurities in solution. The efficacy of flocculation for any given
process depends on the nature and the amount of insoluble
particulates in the feed stream, the concentration, charge density
and molecular weight (Mw) of the flocculant, the shear rate,
duration of flocculation, properties of the feed stream (e.g., pH and
ionic strength) and the properties of the target products. Finally,
product recovery is dependent on the volume of CCF recovered and
704 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016
any potential interaction of the product with the flocculant should
be minimized. All these factors have been studied extensively in
recent times by various groups to understand the mechanisms that
govern the binding selectivity between negatively charged impurites
and cationic polyelectrolytes.
Cationic Flocculation
Flocculation of negatively charged compounds found in cell culture
with cationic polymers (e.g., polydiallyldimethylammonium
chloride (pDADMAC), polyamines, polyaminoacids, polyacryla-
mides, and chitosan) takes place through ionic interactions that
result in the neutralized particle falling out of solution as shown in
Figure 3. Flocs formed by bridging of the negatively charged
particles produce larger and less stable agglomerates that are prone
to shear stress resulting in irreversible disruption of flocs and
slower settling times. However, flocs formed by charge neutraliza-
tion, where cationic polymers with high charge densities neutralize
anionic patches on particles in suspension are much stronger and
less prone to disruption due to shearing. Careful consideration
should be given to optimize the operating concentration and pH
range for effective flocculation that yields high product recovery and
maximum impurity removal.
Poly-(Diallyldimethylammonium Chloride) (pDADMAC)
pDADMAC (IUPAC: N,N-dimethyl-N-propylpropan-1-aminium
chloride) is a water-soluble quaternary ammonium polymer widely
used as a flocculant in waste water treatment. Flocculation with
pDADMAC in CCF has been shown to be effective in providing an
alternative clarification solution for processing high density cell
culture harvests (McNerney et al., 2015; Tomic et al., 2015). Multiple
antibody feed streams treated with addition of pDADMAC and
subsequently filtered using Clarisolve1 depth filters (EMD
Millipore, Bedford, MA) resulted in improved removal of cells
and colloids, increased clarification throughput, efficient reduction
of DNA and high process yield (Tomic et al., 2015). McNerney et al.
showed that flocculated particle size and the particle size growth
rate were greatly enhanced in the presences of non-ionic polymers
and surfactants resulting in settling times of less than 2 h
(Mcnerney et al., 2013). The mechanism of increasing the flocculant
size in a pDADMAC/non-ionic polymer flocculation reaction is
described by depletion forces, where the non-ionic polymer
concentrates the flocculated cells and cellular debris by volume
Figure 3. Outline of the flocculation mechanism for cationic flocculants. Negatively charged particles such as cells, cell debris, nucleic acids, and HCP proteins bind to the
positively charged polymers, leaving the protein of interest in solution.

exclusion and pDADMAC neutralizes the negative charge found on
the cells and cellular debris, allowing for the formation of large
flocculated particles that settle rapidly. It was also shown that
pDADMAC dosing is dependent on the cell type or size as the large
tetraploid-like CHO cells with a diameter of 22–24 mm, required
approximately double the dose of PDADMAC compared to smaller
diploid-like CHO cells with a diameter of 15–18 mm. This was
attributed to the difference in the total charge density of the cell.
Overall, harvest recovery yields of 90% or greater is consistently
achieved with significantly reduced host DNA, HCPs, and some
HMW species.
(flocculation)–CEX (B/E)–HIC (B/E), they demonstrated that it is
feasible to eliminate the use of the Protein A affinity chromatography
step with acceptable yield (>90%), good impurity reduction, and
comparable product quality. However, in the purification process
with the AEX as the last step, impurity removal was not as robust
probably due to redundant separation mechanism between PAA
flocculation and AEX chromatography in flow-through mode. Based
on the overall results (CEX yield  92%, HIC yield  90%,
HCP  12 ppm, DNA < LOD, Insulin < LOD, and Gentamicin  0.2
ppm), the PAA–CEX–HIC process was more robust and efficient
than the PAA–CEX–AEX process.

Polyamines
Polyamines are another class of cationic polyelectrolytes with
multiple repeating amine functional groups that can be used to
flocculate negatively charged cells, cellular debris, and process
impurities in the CCF. Polyamines are protonated over a wide pH
range due to the basic nature of these amine functional groups and
therefore are positively charged. Ma et al. have evaluated various
polyamines based on high throughput screening (HTS) method such
as polyallylamine, polyvinylamine, polyethyleneimine (PEI) and
poly-N-methylvinylamine (PMVA) as flocculants in harvested cell
culture fluid (HCCF) containing antibody (Ma et al., 2010). Several
purification processes were evaluated to compare employment of
orthogonal separation mechanisms, including both flocculation and
chromatography steps versus flocculation replaced chromatography
steps with respect to the yield of the mAb, impurities such as HCP,
DNA, insulin, gentamicin as well as product quality. By incorporating
Figure 4. Mechanism of particle transport and capture through pre-filtration and
flocculation using polyamines into a mAb purification process polishing zones during depth filtration.
using either PAA (flocculation)–CEX (B/E)–AEX (FT) or PAA

Singh et al.: Clarification Technologies for Monoclonal Antibody 705

Biotechnology and Bioengineering
Anionic Flocculation
One of the earliest phase separation studies involving anionic polymers
was carried out by Michaels and Miekka (Michaels and Miekka, 1961)
where oppositely charged strong polyelectrolytes sodium poly
(styrenesulfonate) and poly (vinylbenzyl trimethyl ammonium
chloride) displayed irreversible precipitation. On the basis of this
observation, different polyanions were evaluated for protein
fractionation. These included polymethacrylic acid (PMA), polyacrylic
acid (PAA), carboxymethylcellulose (CMC), polyvinylsulfonate (PVS),
polyacrylamidomethyl propane sulfonate (PAMPS). Target proteins
were bovine serum albumin (BSA), human serum albumin (HAS),
ribonuclease (RNAse), ovalbumin, lysozyme, and hemoglobin
(Anufrieva et al., 1987; Clark and Glatz, 1987; Kaibara, 2000;
Kuramoto et al., 1984; Morawetz and Hughes, 1953; Park, 1992;
Sternberg and Hershberger, 1974; Xia, 1993). In all cited cases, the
intermolecular association between the proteins and the polyelec-
trolytes was shown to be a result of a combination of electrostatic
interactions, hydrogen bonding and/or hydrophobic interactions
(Cooper, 2005; Dubin, 1994; Hattori, 2000). Through the manipulation
of solution pH, ionic strength, as well as polyelectrolyte properties such
as type of polyelectrolyte and Mw, a protein–polyelectrolyte complex
results in phase separation. This phase separation can either precipitate
or coacervate proteins depending on key effectors listed above. Figure 4
shows a schematic of anionic flocculation in a cell culture harvest
process. Specific classes of polyanions for protein are discussed in
greater detail in subsequent sections.
Polyvinyl Sulfonic Acid (PVS), Polyacrylic Acid (PAA) and
Carboxymethyl Dextran Sulfate (CMD)
McDonald and colleagues have evaluated the use of anionic
polyelectrolytes, polyvinylsulfonic acid (PVS), polyacrylic acid
(PAA) and carboxymethyl dextran sulfate (CMD) for enrichment,
and selective precipitation of mAbs from CHO cell harvests leaving
impurities in the supernatant (Fahrner, 2008; McDonald et al., 2009;
Zhang et al., 2005). All three polymers have similar structural
properties where they are polymeric and contain linear carbon
backbones. However, PVS has a sulfonic acid functional group
giving it a pKa of
1.0 whereas PAA has a carboxylic acid
functional group with a pKa of
5.0. PAA is richer in carboxyl
groups compared to CMD. CMD is structurally less flexible. The
mAbs tested in all of these studies have basic pIs. Below neutral pH,
the selected antibodies are predominantly positively charged and
McDonald et al., showed that they exhibit strong electrostatic
interactions with the polyanionic polymers leading to formation of
larger aggregates and precipitate (McDonald et al., 2009). However,
as the solution pH is increased, the net positive charge on the
antibody decreases and ionic strength plays a very significant role at
these higher pH values to shield the somewhat weaker positive
charge on the antibody and prevent a formation of an antibody-
polyelectrolyte complex. Thus, the solution ionic strength has to be
reduced if a higher pH is to be used for successful implementation
of anionic flocculation. Also, increasing the concentration of
polyelectrolytes results in increased phase separation; however
increasing it past a point of minimum antibody solubility results
in resolubilizing the complex. Charge repulsion prevents the
706 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016
formation of further aggregates and forces the complexes into
solution (Carlsson et al., 2003). Lastly, excess polyelectrolyte
reactant can also impair the removal of the polyelectrolyte from
the final drug substance.
The design space is bounded by conductivity (<6 mS/cm) as well as
antibody titer (<3 g/Lit). This limits utility as advances in cell culture
have driven titers as high as 10 g/L while harvest material ranges from
15–20 mS/cm conductivity. As shown by McDonald et al., starting
materials at high titer and conductivity hampered effectiveness and
required feed manipulation to achieve product precipitation. Alstine
et al. overcome these limitations through use of a modified sodium salt
of PAA and CMD at much higher concentrations (5–30% w/w) to
achieve full precipiation (Van Alstine, 2012).
In both of the above studies impurities in the supernatant were
removed by clarification and re-suspension followed by resolubiliza-
tion of the precipitated antibody. Recoveries of the antibody in both
cases have been reported to be as high as 95%. Impurity reduction
was low for HCP at about 1 LRV but high for DNA at more than 5 LRV.
In comparison, Protein A chromatography on HCCF yielded a
recovery of greater than 90% at higher HCP (>2.5 LRV) but lower
DNA (>4.0 LRV) clearance. However, comparison of PVS
flocculation with Protein A chromatography followed by anion-
exchange chromatography demonstrated a less favorable overall
performance of the PVS train in terms of impurity reduction. PVS
proved valuable after Protein A chromatography as it was as effective
as CEX chromatography. HCP levels of less than 0.7 ppm for the
Protein A-CEX-AEX process train were comparable to the Protein A-
PVS-AEX train at 1 ppm (McDonald et al., 2009). These results
suggest that, while not a substitute for Protein A based purification,
PVS can effectively take the place of CEX chromatography. As CEX
often follows Protein A chromatography where an isoelectric
precipitation and subsequent clarification is already necessitated, it
would fit fairly seamlessly into the process flow while alleviating the
need for both column packing, and resin life time studies.
Multimodal Ionic Flocculation
A very recent approach is the use of flocculants with multimodal
functionalities that can be applied to a broad range of
antibodies without the demand for customization (Capito et al.,
2013a,b; Jaber et al., 2011; Kang et al., 2013; Moya and Jaber,
2013). Limitations have been demonstrated for traditional
anionic and cationic flocculants, that includes CCF dilution
prior to polymer addition and unsatisfactory pellet re-
dissolution behavior after precipitation for anionic flocculants,
excess residual polymer, and narrow operating windows
(Carlsson et al., 2003; Cooper et al., 2005; McDonald et al.,
2009; Tomic et al., 2015). To circumvent these problems, recent
efforts have been devoted towards development of multimodal
polyelectrolytes which can interact with the target molecules
through a combination of electrostatic interactions, hydrogen
bonds and/or hydrophobic interactions.
AMPS-ABZ Copolymer
Researchers have designed copolymers with carefully adjusted
hydrophobic and electrostatic properties to allow for precipitation
at high ionic strength, eliminating the need for dilution as well as
showing good redissolution behavior after precipitation (Capito
et al., 2013a). Capito et.al., evaluated the capability of copolymers
consisting of 2-acrylamido-2-methylpropane sulfonic acid (AMPS)
and 4-(acryloylamino) benzoic acid (ABZ), respectively, for
secondary clarification of mAbs in HCCF. In contrast to AMPS-
ABZ copolymers, AMPS homopolymer lacks hydrophobic proper-
ties. HCP clearance and mAb yields were compared to flocculation
using anionic homopolymers (McDonald et al., 2009) as well as to
conventional Protein A chromatography. Results at pH 5.0 and an
ionic strength of 15 mS/cm showed an optimum ratio of mAb to
polymer within the range of 0.35–0.9 (w/w; mg polymer/mg mAb),
where highest precipitation yields were obtained. Comparing the
AMPS-ABZ copolymer against AMPS-homopolymer, the former
showed recovery yields of >80%, whereas the pure AMPS
homopolymer yielded only 10–40%. Additionally, when comparing
different compositions of the AMPS-ABZ copolymer, it was
discovered that the ABZ content directly influences the mAb
precipitation yield. Together, these results strongly suggest that the
interplay of both hydrophobic and electrostatic interactions play a
significant role in the mAb precipitation process. Precipitation of
mAb (pI  7.0–9.0) decreased from >90% at pH 5.0 to
30% at
pH 5.7. This is likely caused by the reduced positive charge on the
mAb when approaching its pI, which consequently reduces
electrostatic interaction between the mAb and the anionic
copolymer (Izumrudov et al., 1998; McDonald et al., 2009).
When varying ionic strength at constant pH (pH 5.0), no significant
influence of salt conditions were observed up to an ionic strength of
22.5 mS/cm. As the ionic strength was further increased,
precipitation yield of mAb decreased from >90% to around 30%
at 32mS/cm. This effect is well known for polyelectrolytes showing
non-monotonic ionic strength precipitation dependence such that
precipitation yield decreases at both, very low and very high ionic
strength (Antonov et al., 2010; Capito et al., 2013b; Carlsson et al.,
2003; Dobrynin and Rubinstein, 2003; Hattori et al., 2000; Marky
and Manning, 2000).
Modified Benzyl Poly(Allylamine)
Modified Benzyl Poly(allylamine) is a salt tolerant, partially
benzylated poly(allylamine) cationic polymer athat can operate over
a wide range of pH and conductivity (Jaber et al., 2011). Kang et al.
investigated this stimulus responsive polymer, termed smart
polymer (SmP) as a potential flocculant to address the mAb
clarification and purification challenges for multiple antibodies
including bi-specific antibodies (Kang et al., 2013). SmP undergoes
a soluble to insoluble transition when exposed to multivalent anions
such as phosphate ions. Strong interactions between multivalent
anions and the closely arranged amine repeat units present on the
polymer result in collapse and subsequent aggregation of polymer
chains rendering the polymer insoluble. This unique property
allows highly efficient binding to negatively charged impurities such
as HCP and DNA under typical cell culture conditions while
enhancing the removal of cells and cell debris and controlling the
level of residual polymer.
In comparison to cationic flocculants like pDADMAC and PEI,
SmP flocculation demonstrated high step recovery, significantly
improved clearance of cell debris and efficient reduction of process
and product related impurities for four different mAbs (Kang et al.,
2013). Changes in conductivity did not significantly affect the
flocculation suggesting a salt-tolerant flocculation process with
SmP. Compared to the control treatments, SmP treatment resulted
in decreasing the residual HCP by 20–50% and >5 LRV of DNA
clearance. Given that SmP is also a hydrophobic polymer; the
authors attributed the possible role of hydrophobic interactions
between SmP and HCP that led to a high level clearance in the
Protein A eluate step. In addition, the SmP flocculation process
reduced HMW species, including aggregated mAb to less than 1%
in the Protein A eluate step. All these findings suggest a synergistic
effect on HCP removal in SmP flocculation process due to
hydrophobic interaction, charge interaction, and precipitation.
Overall, mixed mode flocculants have been shown to overcome
the limitations of cationic or anionic flocculants and can be
effectively implemented for the purification of antibodies.
However, there are several technical and regulatory issues that
need to be addressed including poor recovery in bind and elute
mode as well as the potential for denaturation of the product as
interactions are too strong.
Considerations for Implementing Flocculation
and Precipitation Technologies in Biologics
Manufacturing Processes
As the biologic drugs are mostly parenteral, a thorough risk analysis
should be conducted in regards to cytotoxicity of the polymers/
flocculants used. Although an intravenous route of administration
capitalizes on systemic circulation and therefore ensures most rapid
delivery, serious adverse affects such as hemolysis, precipitation of
the drug, phlebitis and pain may occur due to residual process
related impurities such as polymers. When injected subcutaneously,
the cytotoxicity associated with polymers may occur locally at the
site of injection or distal to the site of injection within the
musculature. Depending on the nature of the polymer, some
flocculants can aggravate these symptoms. Hemolysis, the release of
hemoglobin from red blood cells due to cell lysis may be caused by a
hypotonic environment which may disrupt the erythrocyte’s
membrane. A number of investigators have reported in-vitro
hemolysis with various polymers used in pharmaceuticals. Overall,
these studies have revealed that as the concentration and Mw of a
polymer increases, (e.g., PEG or pDADMAC) so does the hemolytic
activity of the compound (Fort et al., 1984; Kameneva et al., 2003;
McNerney et al., 2015).
Because of the potential cytotoxicity, antibody manufacturing
processes employing flocculants and precipitants need to
demonstrate effective clearance to an acceptable residual level in
the final drug substance to ensure safety of the drug product
(Coffman et al., 2010). To establish a safe residual level however, a
sensitive and accurate analytical method needs to be in place to
assess the residual flocculant level. Researchers have developed
various assays for the detection of the residual polymers; these
include surface plasmon resonance spectroscopy (SPR), quantita-
tive polymerase chain reaction (QPCR), high performance liquid
chromatography (HPLC), enzyme-linked immunosorbent assay
Singh et al.: Clarification Technologies for Monoclonal Antibody 707
Biotechnology and Bioengineering
(ELISA) or fluorescence tagged techniques that can achieve
sensitivity of less than 1 ppm level for the analyte of interest (Kang
et al., 2013; McNerney et al., 2015; Singh and Beattie, 2015). In
general, depending on the charge of the polymer, the CEX, AEX or
adsorptive filters could further remove polymers to a safe residual
level (McNerney et al., 2015; Tomic et al., 2015; Van Alstine, 2012).
Safety factors are of course not only driven by the total achievable
clearance but also by flocculants inherent toxicity. McNerney et al.
have recently ranked four flocculants based on their hemolytic and
toxic behavior: Polyethylene amine (PEI) > Chitosan > pDADMAC
> DEAE Dextran (Mcnerney et al., 2013). In general, polymer
toxicity correlates directly with an increase in Mw, charge density,
and hydrophobocity and a decrease in order of amines (primary >
quaternary) (Dincer et al., 2005; Fischer et al., 2003; Zern et al.,
2011). Therefore, it is recommended to determine the relative risk
of hemolysis and other local toxicity associated with the use of
flocculants in biotechnology-derived drug products.
Polymer flocculants need to be developed with better control on
residual monomer, narrower polydispersity index (PDI) and
bioburden claims for biopharmaceutical applications. Commer-
cially available polymer flocculants contain the monomer and
additives in significant concentrations and may come with high
bioburden. Recent developments such as biopharmaceutical grade
flocculants such as pDADMAC and SmP (EMD Millipore, Bedford,
MA) that have reduced levels of low Mw polymer, <0.1% monomer
and <10 CFU/mL microbial count will result in lesser variability
and meet GMP requirements. Finally, several other factors such as
scalability, impact on product quality attributes such as mAb
charged variants and clipped species, effects on a resin’s lifetime
especially Protein A, step yield, HCP removal, and extent of protein
carryover into the subsequent elution cycles should also be assessed
for successful implementation and scale-up.
Depth Filtration
Depth filtration is commonly used in primary recovery along with
sterilizing membrane filtration before passing the clarified bulk for
chromatographic capture. With new improvements in filter
morphology and surface chemistry, significant improvement in
throughputs are being demonstrated along with efficient removal of
impurities thereby reducing the dependence on chromatography
and paving the way for potential downstream process compression.
Depth filtration has traditionally been used after centrifugation or
tangential flow microfiltration to further clarify the centrate (or
permeate) (Roush and Lu, 2008). This mode of filtration is different
from conventional filtration in that, the filter media is multilayered
with different filtration zones and a tortuous flow path (Bolton et al.,
2005; Roush and Lu, 2008). Owing to the high cell densities in
excess of 20  106 cells/mL for CHO cell lines, depth filtration is
becoming the preferred first step in harvesting cell-culture broth
(Gottschalk, 2008; Roush and Lu, 2008; Th€ommes and Etzel, 2007).
Depth filtration offers the advantage of direct application of feed
material, minimal shear and clearance of impurities like HCP, DNA
and endotoxins during the clarification process (Yigzaw et al.,
2006). Moreover, improvements in depth filtration technology have
led to superior filters which could completely replace centrifugation
(Singh et al., 2013b; Tomic et al., 2015) while boosting viral
708 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016
clearance (Metzger et al., 2015; Venkiteshwaran et al., 2015; Zhou
et al., 2008). In this section, a review of different options available
for filtration is presented along with a review of mechanism of the
filtration, filter chemistry and relevant products produced and
marketed for the cGMP manufacture of antibodies.
Design of Depth Filters and Filtration Mechanism
Depth filters are mostly composed of three essential components:
layers of fibrous material such as cellulose or polypropylene, a filter
aid such as diatomaceous earth and a positively charged binder to
bond the filter aid and the fibers together. The mechanism of
particle retention is based on a combination of steric exclusion
(size-based sieving), interception, particle settling in the niches of
the tortuous path and adsorption of particles based on hydro-
phobic, electrostatic and multi-modal interactions (Figure 4). There
are usually different filtration zones in a gradient density depth
filter: the pre-filtration zone, the primary filtration zone and the
polishing zone that capture particles with a broad particle size
distribution efficiently. The presence of a broad and efficient
gradient density structure enables the filter to retain the particles
(via size-based sieving, adsorption and interception) as opposed to
the forming a deposit (i.e., cake layer) that plugs rapidly. The
different materials for constructing these different zones in a
gradient density depth filter may be found in several recent patents
(Singh, 2013; Yavorsky et al., 2006 Yavorsky, 2010).
An essential component of depth filters is the binder that bonds
the fibers together and imparts a charge to the depth filter. Table III
shows various different depth filters by pore size range commonly
used in the purification of antibodies. The binders used in the depth
filters consist of a polymeric primary charge modifying agent that
contains an epoxide group to bond the molecule to the filter
elements. Commonly preferred primary charge modifiers are
polyamido-polyamine epichlorohydrin or polyamine epichlorohy-
drin. More details on the chemistry of these binders can be found in
Ref. Hou and Ostreicher (1982). At neutral or near neutral pH (pH
6–7), the polyamines carry a net positive charge and therefore, the
anionic impurities in the cell-culture fluid bind to the positively
charged binder of the depth filter. In addition, the presence of
aliphatic groups and benzyl groups in the binder provide avenues
for hydrophobic adsorption. To summarize, depth filters trap
particles by two mechanisms (i) particle entrapment by
interception and sieving (ii) adsorptive binding via charged and
hydrophobic moieties (van Reis and Zydney, 2007).
Depth filtration for Process Related Impurities Clearance
Depth filtration serves a critical role in removal of insoluble
particulates during antibody purification. However, this mode of
filtration is known to demonstrate removal of soluble impurities as
well. Some of the earlier studies have reported the use of depth
filtration to clear endotoxins from water (Gerba, 1985) and for
clearance of DNA from cell culture supernatants (Charlton et al.,
1999). Yigzaw et al. thoroughly investigated the ability of depth
filters to reduce the amount of HCP and DNA through optimization
of the harvest clarification step (Yigzaw, 2006). Historically, depth
filters are readily qualified for downstream filter sizing and
 Strong positive charge for 20MS and 40MS; No
protection but, soluble impurity removal remained a welcome yet

Positive charge type, functionality, notes

unqualified aspect of the unit operation.
Recent efforts have been pursued to understand and characterize

Strong, quaternary amine

Strong, quaternary amine
HCP adsorption by depth filter media and media components where

Strong positive charge

Strong positive charge

Strong positive charge

Strong positive charge

Strong positive charge

Strong positive charge

Weak, primary amine
Weak, primary amine

charge for 60HX

it has been indicated that the choice of the primary clarification step
can affect downstream performance in terms of the specific HCPs
removed. For example, researchers have used 2D-PAGE to detect the
HCPs removed at different stages of primary clarification (Hogwood
et al., 2013; Tait et al., 2012). The results indicate that the abundance
of certain HCPs increase and those of others decrease during primary
clarification suggesting some level of selectivity in adsorption
behavior. Because of this selectivity, filters with superior adsorptive
Secondary clarification of cell
properties are being actively developed (Woo et al., 2015). This
Primary clarification of cell

includes a newly developed all synthetic hybrid filter, EmphazeTM

For precipitation and

AEX Hybrid Purifier (3M, St. Paul, MN) that relies on a high capacity

flocculation
Q-functional hydrogel and a 0.2 mm polyamide membrane to provide
Purpose

culture

culture

a high degree of soluble and insoluble impurity reduction. By

implementing this new generation chromatographic clarifier, the
investigators have demonstrated significant decrease in HCPs (30%)
and DNA (>4 LRV) during harvest clarification and this resulted in
vastly improved product quality post Protein A neutralized eluate
Functionalized polypropylene non- woven, and polyamide

(Castro-Forero et al., 2015). Gagnon et al. have shown that majority of

20MS: 0.8–20; 40MS: 0.8–40; 60HX: 8–60 Polypropylene non-woven fibers, cellulose, resin, and

the DNA impurities present in the CCF exist in the form of chromatin
and this will increase the burden on cleaning regimen and hence life
time for the costly Protein A chromatography resin (Gagnon et al.,
cellulose, perlite, and diatomaceous earth
cellulose, perlite, and diatomaceous earth

cellulose, perlite, and diatomaceous earth

cellulose, resin, and diatomaceous earth

cellulose, resin, and diatomaceous earth

cellulose, resin, and diatomaceous earth

cellulose, resin, and diatomaceous earth

cellulose, resin, and diatomaceous earth

cellulose, resin, and diatomaceous earth

2014). The investigators have attributed this improvement in

performance of Protein A to the removal of these chromatin species
Material
Commercially available depth filters and their properties used in clarification of biologics processes.

by the Emphaze filters thus helping in protecting and extending the

life time of Protein A resins. However, it should be noted that the
diatomaceous earth

observation of chromatin in CCF is an important advance but may be

limited to low viability processing conditions and hence cannot be
generalized to all processing scenarios.
membrane

When implemented post Protein A chromatography, these

adsorptive depth filters can again offer robust impurity clearance.
Typically, neutralization of the viral inactivated low pH elution pools
are associated with increase in turbidity as the pH is raised (Brodsky,
2012; Chollangi et al., 2015) and this demands usage of depth filters
or larger areas of sterilizing grade filters before a polishing
Retention range (mm)

chromatography step can be implemented. Components that are

generally responsible for precipitation during the low pH adjustment
and neutralization include product-related species such as HMW as
well as process-related molecular impurities like HCPs and DNA
1.0–7.0
0.8–5.0
0.8–9.0

0.2–2.5

5.0–7.0
0.2–0.8
0.2–0.8

0.05–0.1

0.05–0.7

0.2–2.0

(Kandula et al., 2009). Schreffler et al. demonstrated the utility of

adsorptive depth filters after Protein A viral inactivation (PAVIB)
using Millistakþ D0HC and X0HC (EMD Millipore, Bedford, MA) for
two mAb molecules and showed that these depth filters offered good
Manufacturer

impurity clearance and could serve as a replacement for an AEX FT

Millipore

Millipore

Millipore

Millipore

Millipore
EMD

EMD

EMD

EMD

EMD
Pall

Pall

chromatography step after Protein A (Schreffler et al., 2015).

3M
3M

3M
3M

Incorporating chromatographic clarification tools or an adsorptive

depth filter into a mAb process/platform can enable significant train
simplification, afford a decrease in column size, enhance process
Clarisolve1 20MS, 40MS,

robustness, and overall reduce process cost.

Zeta plus 90ZB05A
Zeta plus 10SP02A
Zeta plus 30SP02A

SUPRA disk K700

SUPRA disk KS50

Milli stak DOHC

Milli stak XOHC

Milli stak COHC

Milli stak B1HC

and 60HX

Depth Filtration for Virus Clearance

EmphazeTM
Depth filter
Table III.

Viral reduction in antibody purification is typically achieved using

membrane adsorbers that are functionalized with a positively

Singh et al.: Clarification Technologies for Monoclonal Antibody 709

Biotechnology and Bioengineering
charged ligand (Riordan et al., 2009) and nominal virus grade
filters. The use of depth filters to clear HCP and DNA in several
studies indicates that the positively charged depth filters could also
perform viral clearance. It should be mentioned here that
requirements for at least two orthogonal steps for clearance
should still be met according to the EMEA and ICH guidelines
(EMEA, 2008; ICH, 1997). The actual strategy of viral clearance
claims as well as evaluation of robustness is therefore critical in
pursuing this approach. Previous work has demonstrated that
depth filters can remove viruses by either electrostatic adsorption
or a combination of adsorption and mechanical entrapment
(Metzger et al., 2015; Tipton et al., 2002; Venkiteshwaran et al.,
2015; Zhou et al., 2008). Tipton et al. examined the use of
older generation ZetaPlus VR depth filters from 3M for viral
clearance (Tipton et al., 2002). However, less than 1 LRV of
parvovirus was achieved while it was 5 LRV for retrovirus. Recently,
Venkiteshwaran et al. varied feedstream pH and conductivity in a
systematic manner alongwith careful selection of depth filters in
terms of ionic capacities (IEX) and pore sizes to understand
different mechanisms involved in virus clearance (Venkiteshwaran
et al., 2015). It was demonstrated that difference in ionic capacity
correlates with the effectiveness of virus clearance although
there are involvement of mechanisms other than just electrostatic
interactions. Use of newer generation strong depth filters
(Emphaze, 3 M), demonstrated significant virus removal with
samples reaching LRV of 5.0–5.8 for X-MuLV and 4.6–7.2 for
MVM – comparable to values achieved by classic AEX-based
polishing steps (Metzger et al., 2015). Although viruses globally are
negative, positive, or neutral electrostatically depending on pH,
they likely contain negatively, positively charged, and hydrophobioc
regions at any given pH as virus particles are made of scores of
capsid proteins. It is likely that the local charge characteristics and
hydrophobic moeties are directly involved in the electrostatic
interactions with the depth filters.
Case Study: Low pH Flocculation and Depth
Filtration
An in-house study was performed to asses the effect of low pH
flocculation and multiple depth filters to assess their impact on
impurity clearance (HCP, DNA, and high molecular weight
aggregates) on downstream purification. The harvest treatment
included low pH only and low pH adjustment followed by
addition of the flocculant, which was a sodium salt of dextran
sulfate (DS) with a molecular weight of 500 kDa (Sigma–Aldrich).
The antibodies (mAbA and mAbB) used in this study were
produced in a Chinese hamster ovary (CHO) cell line by Bristol–
Myers Squibb. The CCF producing mAb A (IgG4, pI  7.8) and
mAb B (IgG4, pI  6.5) had cell densities of 1.4–1.8  107 cells/
mL, The CCF had the following properties: viability at harvest of
50–70%, antibody titer of approximately 3–3.5 g/L, an initial
turbidity of approximately 3000 NTU. Untreated and harvest
treated CCF were clarified using depth filtration for processing the
HCCF for downstream purification. All the HCCF that were
purified by affinity chromatography (Protein A) and the Protein A
elution pools (PAE) containing the purified mAbs were further
filtered comparing two depth filters with different levels of
710 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016
anionic charges (60SP02A, 90ZB05A, and Emphaze from 3M,
St. Paul, MN). The HCCF samples from the primary clarification
(depth filtration), Protein A eluates and depth filtration post
Protein A were analyzed for titer, HCP, DNA, and HMW. Specific
details on the experimental design and methods may be found in
the supplementary information.
Results and Discussion
Effect of Harvest Treatment on Impurity Clearance: This case-study
examined the use of flocculation and depth filtration for
simultaneous clarification and purification of mammalian cell
culture for producing monoclonal antibodies. Low pH treatment in
addition to dextran sulfate (DS) resulted in the aggregation of
particulates and impurities in the CCF into larger particulates. DS
appears to interact with particle population through intermolecular
bridging effect to increase the average size of particles. Removal of
process and product related impurities due to electrostatic and
hydrophobic interactions was facilitated. However, there was a
trade-off between purity and recovery for the product: as conditions
which favored yield were somewhat juxtaposed to conditions which
favored impurity reduction: increasing the concentration of DS
increased impurity clearance but also resulted in yield loss. DS
concentration appeared to have more pronounced impact on DNA
removal, with greater than 3 LRV for DNA when 0.1 g/L of DS was
used in the flocculation process, while HCP reduction was 40% in
the HCCF. It was found that reducing the pH to between 4.8  0.2
and adjusting the final concentration of DS to 0.1 g/L of CCF
achieved the best compromise of impurity reduction at maximal
product recovery. It was further demonstrated that low pH
treatment with DS addition to CCF can effectively reduce
precipitation and impurities in eluate for two mAbs as shown in
Figure 5. A precipitation of the Protein A eluate is likely caused by
product-HCP aggregates and by HCPs that have a pI close to the pH
of the eluate at the end of viral inactivation, leading to their
isoelectric precipitation. Addition of DS at low pH to CCF effectively
reduces the process related impurities even further that results in
decrease of turbidity of PAE and PAVIB pools. In addition, post
harvest treated samples were also evaluated for the viral clearance
as it has been suggested in literature that DS binds strongly and can
dissociate certain viruses (Christensen et al., 2001). A preliminary
study was designed to use PCR to detect endogeneous retrovirus in
bioreactor samples for mAb A which showed robust clearance of
LRV of endogeneous retrovirus (3.3  0.1). The potential
hypothesis is the charge interaction and co-flocculation between
positive-charged virus particles and anionic DS at pH 4.8.
As explained earlier in this review, it is important to monitor the
levels of DS prior to the final concentration and buffer exchange to
make sure that its levels are acceptable in the bulk drug substance.
Preliminary data shows residual polymer in eluate is less than
1 ppm measured by an ELISA method. The flow-through AEX
incorporating a Q column or membrane adsorber (e.g., Sartobind
Q) is expected to further remove residual DS in eluate. However,
clearance of the residual DS needs to be carefully examined as even
1 ppm could negatively impact AEX capacity as it is large,
hypercharged chain and directly competes for binding with other
impurities, e.g., virus.
 Figure 5. Comparison of HCP, DNA and HMW data for flocculated versus unflocculated feed streams at different stages of purification for two different mAbs.

Post Protein A Depth Filtration for Impurity Clearance: The study
was also intended to gain the understanding of the impurity
clearance with commercially available depth filters of different
charge densities. For this purpose, the feed streams used for the
study included untreated CCF, treated CCF, Protein A eluate from
untreated CCF, and Protein A eluate from treated CCF. All filtrates
were analyzed for HCP, DNA and aggregates. Figure 5 summarizes
impurity levels in the HCCF and subsequent eluates. As shown in
Table IV, strong positively charged 90ZB05A depth filter offered a 2–
3 FRV whereas Emphaze hybrid purifier filter provided a 5–7 FRV,
giving a final filtrate that had an HCP concentration of less than
500 ppm for both mAbs with no harvest treatment. The HCP
concentration did not exhibit a breakthrough and stayed constant
with increase of throughput with Emphaze whereas the HCP content
in the filtrate increased linearly with increase of throughput for
90ZB05A (Data in supplement, Fig. S1). This difference in the FRV
between the 90ZB05A and Emphaze is attributed to binding
capacity because the pore-size rating of both these filters is very
similar (Table III). Figure 6 compares the HCP FRV for 90ZB05A
(n ¼ 3) and Emphaze (n ¼ 14) for mAb A (when there was no
harvest treatment during the primary clarification). It is observed
that the Emphaze depth filters offered a 6.4X reduction in HCP on
average over 14 runs. The data from the HCP fold reduction values
correlated well with the ionic capacity of the depth filters. The ionic
capacity was measured by flushing the depth filters with five bed
volumes of 0.1M NaOH to bind the OH with the quarternary
ammonium moiety. The drained depth filter was then flushed with
1M NaCl to displace the OH ions. The hydroxide ions were then
titrated against 0.1M HCl to a phenolphthalein end point (pink to
colorless). The ionic capacity was measured as: (VHClCHCl)/(VMedia).
The Emphaze Hybrid purifier had the highest ionic capacity and the
highest FRV as shown in Figure 6. It should also be noted that HCP
FRV and ionic capacity data presented in Figure 6 was determined
from three lots and demonstrated the higher variability in ionic
capacity for 90ZB05A as opposed to new generation Emphaze
hybrid depth filter. This is expected as traditional depth filters such
as 90ZB05A usually have a higher lot to lot variabilty due to less
stringent methods used to construct the media.

Table IV. Impurity levels for two different monoclonal antibodies during different stages of recovery. Data is shown with and without harvest treatment
for post Protein A viral inactivation (PAVIB) and post depth filtration of the PAVIB pool

Precipitant Protein A viral inactivation (PAVIB) 90ZB05A Emphaze

pH 4.8 þ 0.1% HCP DNA HMW Yield HCP DNA HMW HCP DNA HMW
mAb (pH þ DS) (ppm) (ppb) (%) (%) (FRV) (LRV) (%) (FRV) (LRV) (%)

mAb A No 4100 1800 6.8 >95 2.7 3.0 5.2 4.2 3.2 5.4
mAb A Yes 170 10 3.8 >95 2.0 2.1 3.7 2.2 2.2 3.7
mAbB No 2100 700 5.9 >95 2.1 2.9 5.8 5.8 2.8 5.4
mAb B Yes 120 14 5.3 >95 1.5 1.0 5.2 2.6 1.1 4.9

Singh et al.: Clarification Technologies for Monoclonal Antibody 711

Biotechnology and Bioengineering

Figure 6. Comparison of HCP FRV for two depth filters (90ZB05A (n ¼ 3) and
Emphaze (n ¼ 14)) for mAb A with different ionic exchange capacities.
As shown in Table IV, harvest treatment had the effect of
reducing the HCP content to less than 200 ppm at the PAVIB stage
for mAb A and mAb B. Depth filtration using the strong positively
charged 90ZB05A and Emphaze hybrid filters resulted in a FRV of
two. This is promising because with harvest treatment, Protein A
chromatography and depth filtration post Protein A alone were able
to reduce the HCP, DNA and residual Protein A (rPA) levels to drug
substance specifications. Overall, it has been shown that new
clarification strategies such as use of low pH flocculation harvest
treatment and high capacity hybrid purifier can certainly reduce
process related impurities and thus ease the burden on the
downstream process. Low pH in the presence of DS provided a
synergistic effect by inducing the reduction of solubility of
impuritiesthereby resulting in significant reduction of these
impurities not only in the harvest but also in downstream steps.
Conclusions and Future Perspectives
New clarification technologies are slowly gaining acceptance for
mammalian cell culture processes in biologics manufacturing as
they can offer significant benefits to biologics processes, improving
efficiency, impurity removal and simplifying the process. However,
there are several technical issues that need to be fully addressed.
Despite these challenges, industry and market needs are driving the
incorporation of advanced flocculation and filtration technologies
into current biologics manufacturing processes.
The success of flocculation or precipitation unit operations is
determined by a large number of variables and factors that need to
be carefully evaluated. Insight into the most important effectors of
process robustness can be obtained if a flocculant is incorporated in
a quality by design (QbD) approach as a defined combination of
continuous and discrete parameters (e.g., a parameter set
consisting of flocculants with various modalities, charge densities
and molecular mass) are considered to enrich data for flocculant
properties that affect particle aggregation and impurity removal.
Shear stress impact must be carefully evaluated as well; although
some degree of mixing is essential to bring the flocculant in contact
with dispersed particles, prolonged mixing and high shear rates can
result in irreversible disruption of flocs or a reduction in the average
712 Biotechnology and Bioengineering, Vol. 113, No. 4, April, 2016
floc size, again affecting the performance of downstream filters
(Brostow et al., 2008). Thus, shear resistant flocculent particles such
as SmP are highly desirable from a commercial manufacturing
standpoint.
Flocculants may bind preferentially to impurities, such as HCPs,
DNA and contaminating virus particles resulting in their selective
removal and thus improving the purification performance and
safety profile of the process. However, the polymers themselves
must also be removed during downstream purification to comply
with regulatory guidelines. Because of their molecular structure and
degree of polymerization, it is challenging to detect flocculants
using spectroscopic methods or enzymatic assays. This can make it
difficult to detect residual flocculants in the product. Poor or lack of
detectability is particularly disconcerting if the selected flocculant is
toxic. Therefore, flocculants should be carefully selected from both a
toxicity and regulatory view point to minimize the risks associated
with non-detection (Buyel and Fischer, 2014).
Charged flocculants can hamper other charge based downstream
purification steps. Ion exchange chromatography is particularly
susceptible to the presence of polymers. For example, applying
residual levels of a positively charged polymer to a cation exchanger
may quickly saturated binding sites and begin filling interstitial
pores by virtue of mass, increasing back pressure and may require a
column repack. If polymers of the same charge as the next step are
to be used it can compete for binding of product as they may carry a
higher charge density resuting in lesser recovery. Therefore, the
process strategy should carefully consider step sequencing and
potential impact of subsequent steps. For example, addition of
charged flocculants to a primary recovery step will have least impact
on the mostly hydrophobically driven Protein A capture, but would
negatively impact an ion exchange based capture step. To reap the
full benefits of cost reduction and process simplification and to
ensure regulatory compliance, process sequencing is critical.
Although placing flocculant addition at the start of the process is
most attractive from a residual reduction through the remainder of
the process, it also exposes the process to an increase in input
variability as cell culture conditions are bound to fluctuate and
impact recovery performance. The latter point should be addressed
thoroughly by establishing reliable models that can predict the
impact of a polymer on critical process parameters, ideally
demonstrating the absence of negative effects on the critical quality
attributes of target proteins under a variety of process inputs. At a
minimum impact of cell viability, cell density, product titer, key
indicator impurity levels and ionic species type and concentration
impact should be assessed from a robustness perspective.
For full implementation of flocculation and precipitation in
antibody purification process, several other issues still need to be
addressed. Low pH hold post Protein A chromatography and virus
filters currently offer robust viral clearance/inactivation in a
monoclonal antibody purification process. In addition, AEX
chromatography usually completes the requirement for at least
two orthogonal steps for robust virus removal or inactivation in
antibody processes. The ability of flocculants and precipitants to
remove viruses, however, as consistently and to the same level
remains to be evaluated. Even though both literature and our data
show promise for flocculants as viral removal steps, a more
thorough evaluation needs to be carried out to understand the
mechanism so that it can be considered as a dedicated, orthogonal
virus clearance step. Secondly, clearance of the residual flocculant or
precipitant through subsequent chromatography steps to below the
level of toxicity needs to be demonstrated and proven to be robust.
To understand the implications of the residuals, a dosage model
should be built with input from drug safety evaluation experts to
understand the long term risk. Further steps will then need to be
taken to demonstrate value of this approach as a platform as
individual processes will differ in terms of dosage and admin-
istration. If consistent clearance can be shown for multiple
antibodies to levels which would fulfill the most stringent
requirements, the value of this approach for a platform can be
established. This is of course, still contingent, as mentioned before,
upon demonstrating that the flocculation and precipitation process
can deal with antibodies and feed streams of varying properties.
Depth filtration dominates in cell and cellular debris clarification
as well as the isoelectric precipitation which frequently follows
acidic virus inactivation. In spite of some of the obvious benefits of
adsorptive depth filters, the disavantages are defined by limited
binding capacities. Salt tolerance of electrostatic interactions alone
is understandably low, which can be limiting for cell culture broths
which span a range of conductivity from 15–40 mS/cm. Impurity
clearance, including viral clearance can be reduced however, suffer
from lack of robustness. For charge based adsorption, the vendors
also need to certify and achieve a target charge density range on the
surface of the filters. More work needs to be done to develop the salt
tolerant chemistries, improve the binding capacity and provide the
integrity testing to claim it a robust step for the virus clearance and
replacemnt for AEX polishing step. Moreover, the risk of
introducing leachables and extractables (organic carbon, metals,
inorganic salts, and fibers) into product streams that have been
characterized for depth filtration should be considered especially for
their use in downstream steps. Also, new filter types will at least
initially require a careful case by case evaluation to ensure robust
performance over the breadth of a biologics product portfolio. As
previously discussed, AEX steps often serve to ensure viral clearance.
On the one hand, traditional chromatography based separation,
whether membrane or bead type, carry their own requirement for
functional testing such as height equivalent to a theoretical plate
(HETP) and asymmetry (As). Membrane filters, on the other hand,
have a corresponding post-use integrity test. If filters were to be
claimed in a viral-safety strategy, a similar approach may be required
for any viral clearance claims. Current manufacturing technology
ensures that the filters can remove large particulates from relatively
impure streams, where the effects of variability among device lots are
minimal. Manufacturing technologies for virus-removal media are
designed to much higher standards, however, and include rigorous
lot release testing specific to viral clearance. By contrast, no virus
removal claims are made for other filter types in the process train.
Suppliers of filtration media would need to develop a testing strategy
to ensure consistent virus clearance. The potential utility of depth
filtration to remove viruses in antibody purificationprocess has been
demonstrated, but these challenges burden early adopters with the
development of acceptable strategies to test, validate, and confirm
integrity within their own processes.
If pre-treatment technologies are being considered, diligent
consideration must be made to verify its impact on downstream
chromatography operations as well as ensuring removal of the
precipitants or flocculation agents to acceptable levels in the Drug
Product. General guidelines on the removal of process related
impurities can be found in the FDA’s “Points to Consider in the
Manufacture and Testing of Monoclonal Antibody Products for
Human Use’ and ‘Guidance for Industry Q3A Impurities in New
Drug Substances.” Regulatory compliance also requires that
manufacturers understand and control problems associated with
the high variability of synthetic polyelectrolytes (i.e., residual
unreacted monomers or other chemicals). Most generic purification
platforms for biologics including monoclonal antibodies and Fc
fusion proteins include a Protein A affinity chromatography step
and either one or two polishing steps that typically employ cation
and anion exchange chromatography, or membrane adsorption.
Incorporating these new generation clarification techniques can
enable process compression for antibody manufacturing that
capitalizes on this multifunctionality.
The authors wish to thank Dr. Sibylle Herzer, Dr. Nesredin Mussa, and
Dr. Amanda Lewis for their insightful comments and discussion. In addition
they like to acknowledge the contributions from Bristol–Myers Squibb
(BMS) Process Analytics for support with various assays and the upstream
department for providing us with the harvest material. The authors who
have been employees by BMS at the time of project execution, furthermore,
declare no competing financial interests.
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