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\begin{document}

\title{Antibody Disulfide Bond Reduction and Recovery during Biopharmaceutical
Process Development - A Review}



\author[1]{Tingwei Ren}%
\author[1]{Zhijun Tan}%
\author[2]{Vivekh Ehamparanathan}%
\author[1]{Angela Lewandowski}%
\author[1]{Sanchayita Ghose}%
\author[3]{Zheng Jian Li}%
\affil[1]{Bristol Myers Squibb}%
\affil[2]{Bristol-Myers Squibb Co}%
\affil[3]{Bristol-Myers Squibb Company}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Disulfide bond reduction has been a challenging issue in antibody
manufacturing, as it leads to reduced product purity, failed product
specifications and more importantly, impacting drug safety and efficacy.
Scientists across industry have been examining the root causes and
developing mitigation strategies to address the challenge. In recent
years, with the development of high-titer mammalian cell culture
processes to meet the rapidly growing demand for antibody
biopharmaceuticals, disulfide bond reduction has been observed more
frequently. Thus, it is necessary to continue evolving the disulfide
reduction mitigation strategy and development of novel approaches to
achieve high product quality. Additionally, in recent years as more
complex molecules emerge such as bispecific and trispecific antibodies,
the molecular heterogeneity due to incomplete formation of the
interchain disulfide bonds becomes a more imperative issue. Given the
disulfide reduction challenges that our industry are facing, in this
review, we provide a comprehensive contemporary scientific insight into
the root cause analysis of disulfide reduction during process
development of antibody therapeutics, mitigation strategies and recovery
based on our expertise in commercial and clinical manufacturing of
biologics. First, this paper intended to highlight different aspects of
the root cause for disulfide reduction. Secondly, to provide a broader
understanding of the disulfide bond reduction in downstream process,
this paper discussed disulfide bond reduction impact to product
stability and process performance, analytical methods for detection and
characterization, process control strategies and their manufacturing
implementation. In addition, brief perspectives on development of future
mitigation strategies will also be reviewed, including platform
alignment, mitigation strategy application for bi- and tri-specific
antibodies and using machine learning to identify molecule
susceptibility of disulfide bond reduction. The data in this review are
originated from both the published papers and our internal development
work.%
\end{abstract}%



\sloppy


\textbf{Antibody Disulfide Bond Reduction and Recovery during
Biopharmaceutical Process Development - A Review}

Tingwei Ren, Zhijun Tan, Vivekh Ehamparanathan, Angela Lewandowski,
Sanchayita Ghose, Zheng Jian Li

Biologics Development, Global Product Development and Supply,
Bristol-Myers Squibb, Devens, MA

\textbf{Correspondence}

Zhijun Tan, Biologics Development, Global Product Development and
Supply, Bristol-Myers Squibb, 38 Jackson Road, Devens, MA 01434.

Email: zhijun.tan@bms.com

\textbf{Abstract}

Disulfide bond reduction has been a challenging issue in antibody
manufacturing, as it leads to reduced product purity, failed to meet
targeted product profile and/or specifications and more importantly,
impacting drug safety and efficacy. Scientists across industry have been
examining the root causes and developing mitigation strategies to
address the challenge. In recent years, with the development of
high-titer mammalian cell culture processes to meet the rapidly growing
demand for antibody biopharmaceuticals, disulfide bond reduction has
been observed more frequently. Thus, it is necessary to continue
evolving the disulfide reduction mitigation strategy and development of
novel approaches to maintain high product quality. Additionally, in
recent years as more complex molecules emerge such as bispecific and
trispecific antibodies, the molecular heterogeneity due to incomplete
formation of the interchain disulfide bonds becomes a more imperative
challenging issue. Given the disulfide reduction challenges that biotech
industry is facing, in this review we provide a comprehensive
contemporary scientific insight into the root cause analysis of
disulfide reduction during process development of antibody therapeutics,
mitigation strategies and its potential remediated recovery based on our
expertise in clinical and commercial manufacturing of biologics. First,
this paper intended to highlight different aspects of the root cause for
disulfide reduction. Secondly, to provide a broader understanding of the
disulfide bond reduction in downstream process, this paper discussed
disulfide bond reduction impact to product stability and process
performance, associated analytical methods for detection and
characterization, process control strategies as well as their
manufacturing implementation. In addition, brief perspectives on
development of future mitigation strategies will also be reviewed,
including platform alignment, mitigation strategy application for the
emerging new modalities such as bi- and tri-specific antibodies as well
as using machine learning to identify molecule susceptibility of
disulfide bond reduction. The data in this review are originated from
both the published papers and our internal development work.

\textbf{KEYWORDS}

Antibody, Disulfide bond, Reduction/Oxidation, Process development

\textbf{1 INTRODUCTION}

Antibodies are proteins produced by immune cells and help to defend the
host when facing foreign invaders (Dowd, Halonen, \& Maier, 2009).
Monoclonal antibodies (mAbs) are antibodies with single specificity
generated from plasma B cells in vitro (Nelson et al., 2000). Compared
with small chemical molecules, mAbs showed advantages such as high
selectivity and potency, which improve therapeutic efficiency and reduce
the toxicity (Cui, Cui, Chen, Li, \& Guan, 2017; Imai \& Takaoka, 2006).
Due to these advantages, commercial mAbs have been developed rapidly
since U.S Food and Drug Administration (FDA) approved the first mAb
product in 1986 (Ecker, Jones, \& Levine, 2015). Based on Nature Reviews
Drug Discovery reports (Hughes, 2010; Mullard, 2011, 2012, 2013, 2014,
2015, 2016, 2017, 2018, 2019, 2020), in the last ten years (2009 -
2019), 61 out of 95 FDA approved Biologics License Applications (BLAs)
were mAb products or mAb related products, and the percentage of mAb (or
mAb related) products in the BLAs increased over time (Figure S1).

Commercial mAbs are generally produced in mammalian cells such as
Chinese hamster ovary (CHO) (Du et al., 2018; Kao, Hewitt,
Trexler-Schmidt, \& Laird, 2010; T. Wang, Liu, Cai, Huang, \& Flynn,
2015). Ideally, the disulfide bonds of antibody should pair correctly
before mAbs are secreted extracellularly. Correct disulfide bonds
pairings are critical during the mAb development process: (1) to meet
ICH Q6B specifications for biotechnological and biological products that
the number and the position of disulfide bridges should be determined
based on the gene sequence for the desired product (Lakbub, Shipman, \&
Desaire, 2018); (2) to ensure antibody drug therapy efficiency and
eliminate the immunogenicity (Kao et al., 2010; Swope et al., 2020). It
is also critically important for bi- and tri- specific antibody (BsAb
and TsAb) stability improvement during the drug product development
(Caravella \& Lugovskoy, 2010; Rossi et al., 2006; Vaks, 2018). Many
research studies have been conducted to analyze and understand the
function of disulfide bond structure on antibody stability, and there
are several reviews on this topic (Correia, 2010; H. Liu \& May, 2012;
Trivedi, Laurence, \& Siahaan, 2009b). While developing the fundamental
understanding of disulfide bond structure and function, great research
efforts have been made to identify the root cause of disulfide bond
reduction happened during mAb manufacturing process, and develop
measures to minimize the reduction as well as to recover the reduced mAb
during the downstream process (Du et al., 2018; Hutterer et al., 2013;
Kao et al., 2010; Mun et al., 2015; Tan et al., 2020; Tang et al., 2020;
Trexler-Schmidt et al., 2010). As far as the authors are aware of, most
of these studies focused on certain downstream process steps instead of
the whole downstream process, with relatively limited discussions on the
effect of disulfide bond reduction on downstream processing. In reality,
it is rather challenging to completely eliminate disulfide bond
reduction in a single downstream process step. Therefore, to minimize
disulfide bond reduction, understanding how to apply the mitigation
strategies across multiple downstream process steps is necessary.

To accommodate the aforementioned need, this paper first reviews the
root causes of disulfide bond reduction, and then discusses how existing
mitigation strategies have been able to address this issue. By
summarizing these mitigation strategies, we are able to provide a work
chart to bridge disulfide bond reduction mitigation strategies during
mAb downstream processing. A case study will then be presented as an
example to illustrate how these approaches were applied to downstream
manufacturing process. In addition, this paper also discusses the
effects of disulfide bond reduction on downstream process, and the
associated analytical methods for disulfide bond analysis, and future
perspectives, based on our own experiences, in addressing the disulfide
bond reduction challenges for mAbs and multi-specific antibodies, all of
which provide a broader view of disulfide bond reduction challenge in
mAbs and multi-specific antibodies downstream processing. This article
provides a useful resource for people in biotech industry who are facing
the challenge of disulfide bond reduction during the antibody process
development.

\textbf{2 DISULFIDE BONDS in Therapeutica Proteins}

\textbf{2.1 Disulfide bond structure in mAbs}

A major advancement in antibody structure was revealed in the 1960s by
Gerald M. Edelman and Rodney R. Porter, who were awarded 1970 Nobel
Prize ``for their discoveries concerning the chemical structure of
antibodies'' (Edelman \& Gall, 1969; Edelman \& Gally, 1962;
Preud'homme, Petit, Barra, Morel, \& Leli\selectlanguage{ngerman}èvre, 2000; Raju, 1999). There
are five subclasses of antibodies (immunoglobulins): IgA, IgD, IgE, IgG
and IgM. Each subclass of antibodies are composed of one or several
immunoglobulin (Ig) monomers. An Ig monomer has two identical heavy
chains and two identical light chains. Figure 1A and 1B showed an
example of the disulfide bonds presented in IgG monomers (Correia, 2010;
H. Liu \& May, 2012). Both heavy chains and light chains are composed of
constant domain and variable domains, which are constructed from two
beta-sheets (\(\beta\)-sheets). Disulfide bonds that connect
the two\(\beta\)-sheets in a single domain are known as
intra-chain disulfide bonds (Figure 1A)(W. Li et al., 2016). Disulfide
bonds that connect two heavy chains or connect a light chain and a heavy
chain are known as inter-chain disulfide bonds. In an IgG monomer
(Figure 1B), there are twelve intra-chain disulfide bonds (one per
domain), two inter-chain disulfide bonds between light chain and heavy
chain, and two to eleven inter-chain disulfide bonds between two heavy
chains. Researchers found that inter-chain disulfide bonds are more
prone to degrade than intra-chain disulfide bonds (Kikuchi, Goto, \&
Hamaguchi, 1986; H. Liu, Chumsae, Gaza-Bulseco, Hurkmans, \&
Radziejewski, 2010). There are two possible reasons for the higher
stability of intra-chain disulfide bond: (1) as shown in Figure 1A,
intra-chain disulfide bond are buried inside the two
\(\beta\)-sheets (Amzel \& Poljak, 1979) and the accessible
area of intra-chain disulfide bonds have been calculated to be zero
(Kikuchi et al., 1986); (2) Molecule Dynamic (MD) simulation found that
the atom distance of sulfur molecules for intra-chain disulfide bond is
shorter than that for inter-chain disulfide bond, which may benefit the
intra-chain disulfide bond higher stability than inter-chain disulfide
bond (X. Wang, Kumar, \& Singh, 2011).

\textbf{2.2 Disulfide bond structure in BsAbs and TsAbs}

BsAbs and TsAbs (also known as multi-specific antibodies) are
artificially designed complex antibodies that are capable of binding two
or more antigens (Brinkmann \& Kontermann, 2017; Kontermann \&
Brinkmann, 2015; Runcie, Budman, John, \& Seetharamu, 2018; Sedykh,
Prinz, Buneva, \& Nevinsky, 2018). They are thought to have improved
therapy efficiency since they have the ability to bind to two or more
different targets simultaneously (Runcie et al., 2018; Tustian,
Endicott, Adams, Mattila, \& Bak, 2016; Wu et al., 2020). Due to this
advantage, multi-specific antibodies development has gained increasing
attention in recent years. By 2019, there are more than 110 BsAbs
reported under clinical development (Nie et al., 2020).

Disulfide bond structures also exist in BsAbs and TsAbs. Since there are
more existing studies on BsAbs than TsAbs, here we use BsAbs as examples
to illustrate disulfide bond function in multi-specific antibodies.
Besides the similar functions that disulfide bonds exhibit in mAbs,
disulfide bonds play a critical role in avoiding BsAb chain mispairing
and keeping BsAb structure stable. Figures 1C-1E show three examples
that illustrated disulfide bonds roles in two subclasses of BsAbs,
immunoglobulin G (IgG)-like BsAb (having an Fc region, Figure 1C and
Figure 1D) and small BsAb (lacking an Fc region, Figure 1E)(Elgundi,
Reslan, Cruz, Sifniotis, \& Kayser, 2017; Kontermann \& Brinkmann,
2015).

First example shows how the disulfide bonds improve the stability of KIH
IgG-like BsAb (Figure 1C). In an IgG-like BsAb design, one way to
correctly pair the heavy chains from different antibodies is the
``Knobs-into-holes'' (KIH) approach: in one heavy chain where a small
amino acid in the CH3 domain is replaced by a large amino acid
(functioned as ``knobs'') and in the other heavy chain where a large
amino acid is replaced by a small amino acid (functioned as
``holes'')(Brinkmann \& Kontermann, 2017). The stability of this
heterodimeric region is further improved by forming additional disulfide
bonds in this region. Some research reported that inducing the disulfide
bond to the KIH design can improve functional BsAb yield by 10\%, and
overall higher than 90\% functional BsAb yield (Carter, 2001; Klein et
al., 2012).

In the second example, disulfide bonds improve the rate and efficiency
of correctly pairing heavy chain with light chain in IgG-like BsAb
(Figure 1D). Besides forming heterodimeric heavy chain pair, two light
chains also need to correctly pair with the corresponding heavy chains.
One way to improve the correct light chain-heavy chain pairing is to
introduce an artificial disulfide bond in one arm. Researchers mutated
the pair of cysteines, forming a disulfide bond between heavy chain and
light chain constant regions, to valines, and introduced a new pair of
cysteines in different location in heavy chain and light chain constant
regions to form an engineered disulfide bond. In this way, the mutated
light chain can only pair with the mutated heavy chain and vice versa,
the un-mutated light chain can only pair with the un-mutated heavy chain
(Mazor et al., 2015).

Last example illustrates the disulfide bonds link the two heavy chain
variable region in Dual-affinity Re-targeting (DART) small BsAb design
(Figure 1E). DART BsAb is another type of the BsAb designs (Nie et al.,
2020). As shown in Figure 1E, in DART design, light chain variable
region from one antibody was linked with heavy chain variable region
from the other antibody by small peptide, and the two heavy chain
variable regions were linked by disulfide bond. The short linker sizes
of DART design can improve small BsAb stability and reduce
immunogenicity (Johnson et al., 2010).

\textbf{3 IMPACT OF DISULFIDE REDUCTION ON DOWNSTREAM PROCESSING}

As discussed above, correct disulfide bond formation is required for
proper antibody folding and maintaining their bioactivity and stability
(Lakbub et al., 2016). Researchers found that the unpaired free thiols
could form incorrect disulfide bonds and result in covalent aggregation
(Andya, Hsu, \& Shire, 2015; Cromwell, Hilario, \& Jacobson, 2006;
Vázquez-Rey \& Lang, 2011; W. Zhang \& Czupryn, 2002). Protein
aggregates in the final drug products could induce adverse immune
responses in patients and cause immunogenicity (Moussa et al., 2016).
The mechanisms of how protein aggregations cause immunogenicity is a
separate but interesting research topic (Bessa et al., 2015; Moussa et
al., 2016; Ratanji, Derrick, Dearman, \& Kimber, 2014; Rosenberg, 2006)
and out of the scope of this paper. Instead, to the best of our
knowledge, there are limited studies reporting disulfide bond reduction
impact on whole downstream processing performance. As such, this section
we discuss disulfide bond reduction impact on downstream processing,
including the challenge of identifying reduced mAbs and disulfide bond
reduction effects on mAbs stability based on our own commercial antibody
process development experiences.

\textbf{3.1 Identification of reduced mAb during the downstream
processing}

Besides the incorrect disulfide bond formation, the unpaired free thiols
may change the antibody's surface charge distribution and
hydrophobicity, which consequently will result in different
chromatographic profiles during mAb process development. For example, in
cation exchange chromatography - high performance liquid chromatography
(CEX-HPLC) and anion exchange chromatography (AEX-HPLC) analysis, mAbs
with and without free thiols showed separate peaks at different elution
times (Chen, Nguyen, Jacobson, \& Ouyang, 2009; Cheng et al., 2017;
Pristatsky et al., 2009; T. Zhang et al., 2012). However, to the best of
our knowledge, there are very few papers that reported the effects of
disulfide bond reduction on the performance of the downstream
processing, particularly whether the disulfide reduced mAbs can be
identified during downstream processing. Here, we use three monoclonal
antibodies (mAb1 (IgG1, pI 8.2), mAb2 (IgG4, pI 8.0), mAb3 (IgG4, pI
6.8)) purified in our in-house studies as examples to show the impact of
disulfide bond reduction on individual downstream purification
processes, including Protein A chromatography, low pH viral inactivation
(VI) and ion exchange chromatography (IEX) respectively.

\textbf{(1)} \textbf{Protein A chromatography} . For Protein A
chromatography separation, the affinity between mAb and Protein A ligand
is primarily through the Fc region of the antibody. Since the high-order
structure of the disulfide-reduced mAb is intact, the affinity between
reduced mAb and Protein A resin would remain unchanged (Tan et al.,
2020). As presented in Figure 2A, the Protein A chromatography profile
of the low level disulfide reduced mAb3 sample (containing 89.5\% intact
mAb3) and the high level disulfide reduced mAb3 sample (containing
10.6\% intact mAb3) were identical. Additionally, no noticeable process
yield difference was observed between the reduced mAb and intact mAb on
Protein A chromatography step. These results confirmed that disulfide
reduction had no major impact on the Protein A chromatography step.

\textbf{(2)} \textbf{Low pH viral inactivation (VI)} . A previous study
showed that high level of disulfide bond reduction may increase the
aggregation level after low pH viral inactivation (Chung et al., 2017).
However, a clear trend was not observed for disulfide reduction effects
on protein aggregation based on mAb1 and mAb2 low pH viral inactivation
results. As shown in Figure 3A, after low pH viral inactivation the high
molecular weight (HMW) level increased by 0.6\% and 0.5\% for intact
mAb1 pool and reduced mAb1 pool, respectively. In contrast, the HMW
level decreased by 1.7\% for the intact mAb2 pool verses a decrease of
0.3\% for the reduced mAb2 pool. The lack of consistent trend for
disulfide reduction impacts on aggregation at the low pH VI step
indicates that there may be other more critical contributing factors to
the aggregation formation, such as intrinsic molecular properties (e.g.
mAb type and hydrophobicity).

\textbf{(3) IEX} . As discussed above, disulfide reduction may change
mAb surface charge distribution, which subsequently lead to the
appearance of separate peaks in CEX-HPLC and AEX-HPLC analysis. Here, we
compared AEX profiles and CEX profiles respectively for two runs of mAb1
with different levels of disulfide reduction (Figure 2B and 2C). AEX
step of mAb1 was operated in flow through (F/T) mode, and the results in
Figure 2B showed that the overall patterns of AEX are similar between
the low level disulfide reduced mAb sample (\textgreater{}95\% intact
mAb) and the high level disulfide reduced mAb sample (67\% intact mAb).
The differences in the F/T volumes during the loading step were due to
differences in the initial loading sample volumes. Similarly for CEX
step (bind elute mode, performed after AEX), disulfide reduction level
did not show significant impact on the overall CEX profile (Figure 2C).
However, it was noticed that the mAb1 sample having had a high level of
disulfide reduction showed longer peak tailing than the ones that had a
low level of disulfide reduction at the end of the AEX loading step
(Figure 2B) and at the end of the CEX elution step (Figure 2C),
respectively. The differences in the peak tailing for both AEX (F/T) and
CEX (B/E) could be due to the difference of disulfide reduction levels.
However, it is not clear whether this type of chromatographic difference
generally exists in all types of mAbs. In fact, the difference in the
chromatographic profiles between the intact mAb and reduced mAb is so
subtle that it would be very challenging to identify the highly reduced
mAb just based on chromatography profiles alone. Thus, fast but accurate
analytical methods should be used in the downstream purification process
for disulfide bond reduction monitoring and determination. These methods
will be discussed in more detail in Section 4.

Besides comparing intact mAb and highly reduced mAb profiles at
different downstream process unit operation step, it was also noticed
that for the highly reduced sample of mAb1 and mAb2, the purity
increased from 64\% and 35\% in the Protein A chromatography pools to
94\% and 92.9\% in IEX pools respectively (Figure 3B). The increase of
mAb purity during downstream purification process may be due to removal
of reduced mAbs species during downstream purification, and/or
reoxidation of reduced mAbs during downstream purification. To confirm
this, we did conduct a study that used the disulfide re-oxidation
strategy to recover the previously reduced mAb in downstream process
(Tan et al., 2020; Tang et al., 2020).

\textbf{3.2 Disulfide bond reduction impact mAb stability in downstream
processing}

Disulfide bonds enhance protein stability (Bulleid \& Ellgaard, 2011;
Chung et al., 2017; Zhu, Dupureur, Zhang, \& Tsai, 1995). It is
reasonable to expect that improper disulfide bond formation and
disulfide bond reduction would impact process performance, protein
stability and biological functionality. (Chung et al., 2017; Fass, 2012;
H. Liu et al., 2014; Manteca et al., 2017; Trivedi, Laurence, \&
Siahaan, 2009a; T. Wang et al., 2015). Disulfide reduction generates
free sulfhydryl groups on the cysteine residue, potentially resulting in
decreased thermal stability, pH stability, and potency as well as
elevated aggregation (Harris, 2005; Huh, White, Brych, Franey, \&
Matsumura, 2013; Lacy, Baker, \& Brigham-Burke, 2008). However, in our
internal low pH viral inactivation (VI) study, we did not observe a
general correlation between the aggregation level at the post-low pH VI
step and the initial low molecular weight percentage resulting from
disulfide bond reduction (as discussed in Section 3). It was suggested
that the sensitivity of antibody aggregation to disulfide bond reduction
may be dependent on the characteristics of the molecule (Manteca et al.,
2017). In our study, we performed thermal and photostability studies
using samples containing different levels of starting LMW species
(resulting from disulfide reduction). As shown in Figure 4, light
exposure led to significant increase of aggregate formation in
comparison to the high temperature and room temperature exposures.
Specifically, a higher aggregation rate was observed for the sample
containing initial higher level of LMW species under the light exposure
condition. In contrast, both initial high and low levels of LMW samples
did not show significant difference of aggregation rate at room
temperature. At high temperature (40 °C), the overall aggregation levels
trended higher over time and the initial LMW level also influenced the
aggregate formation rate, but at a much slower rate compared to the
light exposure condition. The significant impact of the LMW level on the
sample photostability highlighted the importance of controlling the
disulfide bond reduction in the manufacturing process.

\textbf{4 ROOT CAUSE ANALYSIS FOR DISULFIDE BOND REDUCTION}

MAb disulfide bond reduction is essentially an oxidation-reduction
(redox) reaction that involves redox enzyme. Glutathione and thioredoxin
system (comprising thioredoxin, thioredoxin reductase and nicotinamide
adenine dinucleotide phosphate (NADPH)) are the known enzymes, and
enzyme systems, that contribute to disulfide reduction (Chakravarthi \&
Bulleid, 2004; Handlogten, Zhu, \& Ahuja, 2017; Koterba, Borgschulte, \&
Laird, 2012; O'Mara et al., 2019). NADPH is generated from the pentose
phosphate pathway and serves as an electron source in the disulfide bond
reduction reaction (Arne Holmgren \& Lu, 2010). Electrons first transfer
from NADPH to thioredoxin reductase (TrxR) and reduce the TrxR disulfide
bond, then move to the oxidized thioredoxin (Trx) to form the reduced
thioredoxin, and finally reduce the mAb disulfide bond. Glutathione
(GSH) catalyzes the disulfide bond reduction in a similar way as Trx
system (Gilbert, 1995; Handlogten et al., 2017; Meister \& Anderson,
1983) (Figure S2). Therefore, during manufacturing processing including
both upstream and downstream, factors that promote these
enzyme-catalyzed reactions can lead to disulfide bond reduction. With
this root cause analysis, we categorized the factors that influence mAb
disulfide bond reduction into the following types:

\textbf{4.1 Enzymatic factors.}

Researchers found that during the protein harvest operation, high
mechanical shear force could cause cell lysis. Consequently, more
intracellular enzymes are released to the Harvest Cell Culture Fluid
(HCCF) thus lead to a higher level of disulfide bond reduction (Hutterer
et al., 2013; Trexler-Schmidt et al., 2010). Alternatively, low
dissolved oxygen (DO) levels in cell culture or HCCF can also cause
disulfide bond reduction since there is a limited amount of oxygen
molecules to consume NADPH, thus leaving enough NADPH as ``electron
fuel'' for the disulfide bond reduction reaction (Mun et al., 2015;
O'Mara et al., 2019). These aspects of root causes have been the focus
of recent industrial studies.

\textbf{4.2 Physical factors.}

Environment pH and temperature are two best known factors that influence
enzyme reaction rate. As a consequence, mAb disulfide bond reduction
level is higher when cell culture pH is closer to neutral pH since the
NADPH oxidization rate was higher under a neutral pH condition (pH 7.0)
than acidic pH condition (e.g. pH 6.5) (A. Holmgren, 1979; P. Xie et
al., 2016). In addition, Low temperature could also slow the enzyme
reaction rate and eventually reduced the disulfide bond reduction level
(Hutterer et al., 2013; H. Liu, Nowak, Shao, Ponniah, \& Neill, 2016;
O'Mara et al., 2019). Furthermore, short HCCF storage time can eliminate
the disulfide bond reduction level as the reaction time was reduced (Mun
et al., 2015; O'Mara et al., 2019). However, it should be noted that if
the storage time is long enough, the reduced mAb can re-oxidize and form
intact mAb in HCCF as the enzymes lose their activities (Mun et al.,
2015).

\textbf{4.3 Intrinsic factors.}

While disulfide bond reduction is generally observed during the mAb
manufacturing process, the reduction level depends on the mAb subclass
and the light chain type (Hutterer et al., 2013). It was found that mAb
disulfide bond susceptibility to enzymatic activity was as
follows:\(IgG1\lambda>IgG1\kappa>IgG2\lambda>IgG2k\), which could be related to differences in the
NADPH availability with different cell lines and molecular structure
differences (e.g. IgG2 is more compact than IgG1) (Magnusson,
Björnstedt, \& Holmgren, 1997; Q. Zhang \& Flynn, 2013).

\textbf{5 ANALYTICAL METHODS FOR DISULFIDE BOND REDUCTION MONITORING AND
ANALYSIS}

As discussed in Section 3, identifying potential disulfide reduction
based on downstream processing unit operation profiles could be
challenging, since highly reduced mAb samples and intact mAb samples
showed similar peak profiles. In addition, protein properties can change
drastically due to peptide bond cleavage (Magnusson et al., 1997). Thus,
reliable analytical methods are essential to detect and quantify protein
modifications. Based on the mechanisms of separation, these methods can
be divided into two groups: size-based and chemistry of amino acid side
chains. Size-based separation methods include size-exclusion
chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and capillary electrophoresis with SDS
(CE-SDS) (Dada, Rao, Jones, Jaya, \& Salas-Solano, 2017; Davagnino,
Wong, Shelton, \& Mankarious, 1995; H. Liu, Gaza-Bulseco, \& Chumsae,
2009; Rao \& Kroon). Other separation methods typically include various
types of chromatography based on the charge and hydrophobicity
differences of these species. In addition to the above methods for
monitoring and quantitation of protein fragmentation during process
development and quality release testing, the identification of the exact
cleavage site and further characterization of these fragments are
accomplished using mass spectrometry (MS) (H. Liu, Gaza-Bulseco, \&
Lundell, 2008).

Size-based separation methods are often straightforward due to the
physical size differences, while other chemical degradations of amino
acid side chains are not easily detected. Peptide bond cleavage becomes
detectable only after two fragments are separated. In addition,
non-covalent interactions such as hydrophobic interactions between these
disulfide-reduced species may prevent separation of the two fragments
under native conditions. Thus, denaturation may be required to detect
the cleavages in a folded immunoglobulin domain. In contrast, the
cleavage in the hinge region is readily detectable by SEC under a native
condition. SEC and non-reduced CE-SDS results of a partially reduced
sample showed that its native structure was intact although the majority
of inter chain disulfide bonds were broken (Figure S3).

In recent years, CE-SDS method has emerged as a valuable alternative to
conventional SDS-PAGE method for the characterization and automatic
quantitation of antibodies (Krylov \& Dovichi, 2000). By providing an
excellent resolution of fragments, CE-SDS methods have been widely used
in biotech industry to monitor overall fragmentation during process
development and is now commonly used for final drug substance and drug
product GMP release and in-process testing due to the straightforward
quantification and improved sensitivity with fluorescence detection
(Cherkaoui et al., 2010). Additionally, in recent years chip-based
protein characterization system has been utilized to offer an automated
alternative to traditional methods by streamlining slab gel
electrophoresis, while also providing high throughput and data quality
required by bio-therapeutics and genomics workflows (Wagner et al.,
2020).

SEC is usually considered as the gold standard method for quantitation
of aggregates, but it is rarely used to quantify fragmentation since
fragments may co-elute with the monomer peak and lead to poor resolution
(Ricker \& Sandoval, 1996). For this reason, SEC can be run under
denaturing conditions (dSEC), such as in the presence of guanidine
hydrochloride, SDS, or an organic solvent in mobile phases or during
sample preparation (H. Liu et al., 2009). Denaturing SEC is an
alternative separation method to quantitatively determine protein
fragments. Similar to SDS-based methods, the protein is denatured using
denaturing reagent such as 6M guanidine at 50 °C for 60 minutes and
injected onto a SEC column with mobile phase containing guanidine. The
denaturing reagent will detach fragments from association before
separation on SEC column. The separation resolution of denature SEC may
not be as good as SDS-based methods. However, due to its relative high
loading amount, the denaturing SEC can be a useful tool to separate and
isolate fragment species for further characterization. Denaturing SEC
coupled with MS is a very powerful tool to characterize protein
fragments (García, 2005; H. Xie et al., 2010). The denaturing SEC-MS can
be operated as an in-line method that uses organic solvent in the SEC
mobile phase followed by MS detection. The organic solvent serves as a
denaturing reagent to detach IgG light chains from heavy chains before
SEC separation, allowing the mass spectrometer to measure the
dissociated IgG heavy and light chains. For example, the improved
SE-UPLC coupled with MS employing mobile phases containing acetonitrile
(ACN), trifluoroacetic acid (TFA), and formic acid allowed the
separation of antibody light chain and heavy chain, and to obtain a
direct molecular weight measurement (H. Liu et al., 2009; R. Yang et
al., 2015).

In recent years, significant progress has been made in process
analytical technology (PAT), which enables real-time monitoring and
control, to maintain consistent product quality through better process
understanding (Jenzsch et al., 2018; Read, Park, et al., 2010; Read,
Shah, et al., 2010). Real time monitoring of bioprocess with the
integration of analytics at critical unit operations of processes is one
of the paramount necessities for implementing real time release of
biopharmaceuticals (Chemmalil et al., 2020; Wasalathanthri et al.,
2020). For example, protein purity, a critical quality attribute (CQA),
can be monitored using a variety of PAT tools during downstream
operations (Großhans et al., 2018; Patel et al., 2018). Another example,
a PAT application using the denaturing SEC, can be integrated with the
Protein A chromatography to provide real-time data of product purity and
to enforce necessary process control strategies to ensure product
quality (Chemmalil et al., 2020).

\textbf{6 APPROACHES TO MINIMIZE DISULFIDE BOND REDUCTION AND RECOVER of
REDUCED-MAB}

\textbf{6.1 Overview}

As discussed above, disulfide bond reduction exists during mAb
manufacturing process and impacts mAb stability, thus minimizing the
disulfide bond reduction is critical to ensure high process yield and
purity of the final drug substance. Since enzyme catalyzed redox
reaction is primarily the root cause of disulfide bond reduction,
different approaches that inhibit or slow down the redox reaction,
directly or indirectly, have been applied in the manufacturing process
to eliminate the disulfide bond reduction. Generally, these prevention
approaches are applied at relatively upstream manufacture steps, such as
cell culture, cell culture fluid harvest and storage (Figure 5, Table
1). However, the disulfide bond reduction prevention approaches do have
some limitations such as prolonging operation time, increasing equipment
cost and decreasing operational flexibility. To keep the flexibility and
efficiency of the manufacturing process, approaches to rescue the
reduced mAbs and to savage ``waste'' batches should also be considered
to address the disulfide bond reduction challenges. Redox systems can be
introduced to chromatography steps during the manufacturing process to
rescue previously reduced mAbs (Figure 5, Table 1), and the rescued mAbs
showed comparable properties as intact mAb with no negative impact on
final drug product quality (Tan et al., 2020).

\textbf{6.2 Development of scale-down models for disulfide bond
reduction study}

It was reported that the disulfide bond reduction was mostly observed
during the manufacturing-scale process, but not very often observed
during the lab-scale process (Du et al., 2018; Kao et al., 2010). Also
the disulfide bond reduction level could vary significantly during the
manufacturing process (Du et al., 2018; Kao et al., 2010; O'Mara et al.,
2019; Trexler-Schmidt et al., 2010). Thus, it is critical to develop a
reliable scale-down model to demonstrate disulfide bond reduction for
lab-scale studies. A general approach is to create a worst-case
disulfide bond reduction sample, where HCCF is completely homogenized to
release intracellular enzymes that cause disulfide bond reduction. Then
the lysed HCCF can mix with non-lysed HCCF at different ratios to create
samples with different levels of disulfide bond reduction (Du et al.,
2018; Trexler-Schmidt et al., 2010). Furthermore, to prevent free thiol
re-oxidization, clarified bulks (CB) were purged with
N\textsubscript{2}gas and stored at room temperature under anaerobic
environment (Du et al., 2018).

\textbf{6.3 Disulfide bond reduction prevention methods}

As shown in Figure 5, multiple approaches can be applied to prevent (or
eliminate) the disulfide bond reduction at different manufacturing
steps. Based on their impact on the enzyme catalyzed redox reaction, we
categorized these approaches into four types:

\textbf{6.3.1 Inhibit enzyme over expression in the cells.}

Trx is the terminal enzyme that is responsible for disulfide bond
reduction. If the cells cannot overexpress Trx, the enzyme system
activity is significantly inhibited. Koterba et al. demonstrated the
reduced disulfide reduction by using Trx knock out CHO cells. Their
research showed that inhibiting Trx overexpression did not affect the
overall cell growth (Koterba et al., 2012). By incubating cell lysate
with intact IgG for 24 hours, there is still higher than 50\% intact IgG
remaining in engineered cell lysate (without Trx overexpression) pool,
while in the control cell lysate (with Trx overexpressed) the intact IgG
is close to 0\%. This result showed that it is possible to inhibit the
Trx overexpression in cells and minimize disulfide bond reduction caused
by cell lysis. However, this way of controlling disulfide reduction
depends on concentrations of other enzymes: if concentrations of other
enzymes are high, the redox reaction still can be catalyzed efficiently
and result in high level of disulfide bond reduction.

\textbf{6.3.2 Inhibit enzyme activity}

Metal ions and chelation can inhibit different steps of enzyme catalyzed
redox reaction. For instance, Ethylenediaminetetraacetic acid (EDTA) can
inhibit hexokinase activity and reduce the formation of glucose-6-P.
Cu\textsuperscript{2+,} Zn\textsuperscript{2+,}
Hg\textsuperscript{2+}and Co\textsuperscript{2+} can inhibit Trx
activity. Aurothioglucose (ATG) and aurothiomalate (ATM) can inhibit
TrxR activity (Kao et al., 2010). Inhibitors can be introduced at
earlier manufacture steps, such as cell culture media or HCCF (before
and after filtration) with the final concentration of inhibitors are at
micro-molar to milli-molar levels. Previous research showed that if the
inhibitor efficiency is high, it can completely inhibit disulfide bond
reduction during 2-3 days HCCF storage time (Trexler-Schmidt et al.,
2010). However, studies on the inhibitor performances when HCCF storage
time is longer were limited and may need further investigation.

\textbf{6.3.3 Reduce the amount of enzyme present}

Disulfide bond reduction can also be minimized if there is less enzyme
available to catalyze the disulfide bond redox reaction. One approach is
to introduce components that can react with the enzyme as competing
reaction pathways to consume the enzyme so it becomes unavailable for
disulfide bond reduction. As discussed in root-cause analysis section,
O2 can react with NADPH; thus improving DO levels in HCCF can minimize
disulfide bond reduction (Handlogten, Wang, \& Ahuja, 2020; Mun et al.,
2015). For instance, researchers found that keeping a minimum 30\% DO
level by air sparging can prevent disulfide bond reduction and maintain
higher than 90\% intact mAb. Besides O\textsubscript{2},
H\textsubscript{2}O\textsubscript{2} and L-cystine with optimal
concentration can also react with enzyme and reduce the available enzyme
for disulfide bond reduction. Consequently, researchers induced
micro-molar level H\textsubscript{2}O\textsubscript{2} and L-cystine to
the HCCF to eliminate disulfide bond reduction (Chung et al., 2017; Du
et al., 2018). Another approach is to minimize cell lysis during CCF
harvest step, consequently there will be less enzyme secreted into CCF.
This can be achieved by minimizing cell shear force in harvest step,
such as using a hermetic style centrifuge (Trexler-Schmidt et al.,
2010), or controlling the depth filtration differential pressure lower
than certain values (O'Mara et al., 2019).

\textbf{6.3.4 Decrease enzymatic reaction rate and reduce reaction time}

Lowering pH and temperature of HCCF or reducing HCCF storage time can
slow down the enzymatic reaction. These approaches can be coupled with
other methods to further eliminate disulfide bond reduction. It should
be noted that lowing pH is less frequently used since it changes
microenvironment around mAb and may cause mAb aggregation
(Trexler-Schmidt et al., 2010).

While there are different approaches to minimize disulfide bond
reduction in manufacturing process, these approaches have some
limitations: (1) inhibition of enzyme overexpression genetically in
cells may increase the mAb development time, also this may increase the
risk of not overexpressing the mAb correctly since the genetically
modified mutant clone may not survive in antibody selection process at
the first stage; (2) the type and the concentration of the inhibitors
that eliminate disulfide bond reduction may vary with each mAbs, thus
optimizing the inhibitor selection could be time-consuming; (3) the
final inhibitor concentration present in drug substance needs be
monitored and controlled, and it may require extra manufacturing steps
to remove the inhibitors to meet the drug substance GMP release
specifications for clearances; (4) air sparging, lowering the storage
temperature and reducing the storage time may require extra financial
investment for equipment thus increase the cost of the manufacturing
process and reduce the flexibility of manufacturing process.
Consequently, developing approaches to recover (or ``savage'') the
reduced disulfide bond should be considered in the manufacturing
development process.

\textbf{6.4 Rescue reduced mAb during downstream processing}

Due to the limitations of operation flexibility and efficiency of
controlling disulfide bond reduction, saving and recovering the already
reduced mAb becomes a compelling alternative approach. Compared to the
elimination approach, the recovery approach has advantages including:
(1) saving the ``waste'' reduced mAbs, (2) further lowing the risks of
mAb disulfide bond reduction in downstream process steps, (3) more
flexible and economically favorable since this approach requires neither
extra equipment nor extra operation steps.

Reduced mAb recovery involves the thiol-disulfide exchange reaction
between the reduced mAb and a redox reagent (Bulleid \& Ellgaard, 2011;
Mamathambika \& Bardwell, 2008). The thiol-disulfide exchange reaction
includes two steps (Figure S4). In the first step, the nucleophilic
thiolate group (S-) formed by free thiol deprotonation attacks one of
the sulfur atoms of the redox reagent, and a disulfide bond forms
between the redox reagent and the mAb (Figure S4A); in the second step,
the remaining thiol group in mAb attacks the newly formed disulfide
bond, releases the redox reagent and forms disulfide bond in mAb (Figure
S4B). While the thiol-disulfide exchange reaction has been widely
studied to provide deeper understanding of disulfide bond formation
relations in cells (Cappel \& Gilbert, 1988; Østergaard, Tachibana, \&
Winther, 2004; Tu, Ho-Schleyer, Travers, \& Weissman, 2000), there are
very few research in literature that applied this reaction to recover
reduced mAb in the manufacturing process. In our recent in-house
studies, we successfully recovered the reduced mAb by using redox
reagent buffer cysteine/cystine as wash buffer in Protein A
chromatography step (Figure S5, data not shown) (Tan et al., 2020; Tang
et al., 2020). Based on the kinetic model developed in this study, the
cysteine/cystine concentration, pH and mAb/redox buffer contact time
were optimized to recover the disulfide reduced mAb. The results showed
that mAb purity was improved from \textless{}5\% in the load to
\textgreater{} 90\% in the elution. Cystine concentration and pH were
found to be the most critical factors that affected reduced mAb
recovery. To achieve a high mAb recovery ratio, buffer pH 8 to 10 and a
minimum of 60 minutes mAb/redox buffer contact time was recommended from
this study. The recovered mAb showed comparable properties to original
intact mAb based on a variety of biochemical and biophysical analytical
characterization. The studies demonstrated the feasibilities of applying
recovery methods in downstream process to produce acceptable quality mAb
product for potential clinical uses.

\textbf{6.5 Disulfide bond reduction prediction and monitoring during
manufacturing process}

Besides disulfide bond reduction elimination approaches and recovering
already reduced mAb approaches summarized above, proactively applying
the process analytical technology (PAT) for disulfide bond reduction
prediction and on-line/at-line monitoring during the manufacturing
development process is also important (Chemmalil et al., 2020). PAT
enables the detection of the disulfide bond reduction in early process
steps thus allows the proper monitoring, and the prevention methods or
rescue methods to be applied in time to ensure final product quality. In
addition, the real-time analytical results of disulfide bond reduction
can provide a quantitative understanding of the root cause of the
disulfide bond reduction in manufacturing process and allow more
efficient control to improve process design (Read, Park, et al., 2010;
Read, Shah, et al., 2010; Simon et al., 2015).

It was also reported that, the correlation between cell culture redox
potential and antibody reduction can be applied to predict sulfide bond
reduction levels in early manufacturing process steps (Handlogten et
al., 2020). Through online cell culture redox potential measurements and
analyzing the amount of intact mAb at late stage of cell culture such as
on Day 12 and Day 14, a redox potential of -70mV was found to be used as
a cell culture redox potential threshold for an IgG2. Above this redox
potential, there was minimal level of disulfide bond reduction while
below this redox potential, the disulfide bond reduction level could
vary significantly. These researchers further designed a redox reduction
control system to maintain the cell culture redox potential higher than
the threshold by adding CuSO4 and /or improving DO level, and
demonstrated the success in keeping the disulfide bond reduction level
of cell culture at a minimal level.

In addition to using redox potential as a predictive tool during cell
culture step, free thiol levels in HCCF can also be controlled to
predict disulfide bond reduction level and appropriate measures may be
taken to mitigate the risk. In a typical monoclonal antibody
manufacturing process with the Chinese Hamster Ovary (CHO) cell line, we
have established three critical thresholds of free thiols in HCCF based
on the disulfide reduction risk: \textless{} 100 \selectlanguage{greek}μ\selectlanguage{english}M (low risk); 100 -
200 \selectlanguage{greek}μ\selectlanguage{english}M (medium risk) and \textgreater{} 200 \selectlanguage{greek}μ\selectlanguage{english}M (high risk) (Du et al.,
2018). For the high risk HCCF, in addition to low temperature storage
and maintaining aeration, the harvested material is processed through
Protein A chromatography as early as possible. To further de-risk the
possible disulfide reduction, each elution pools are kept separately if
multiple Protein A run cycles are performed.

For the downstream process steps such as Protein A chromatography and
ion exchange chromatography, currently there are no reported research on
applying on-line analysis methods for disulfide bond reduction. However,
the success of incorporating on-line liquid chromatography (LC) for
charge variant (Alvarez et al., 2011) and size variant analysis
(Chemmalil et al., 2020) showed the potential of on-line disulfide bond
reduction monitoring and analysis. Alvarez et al designed an in-house
on-line liquid chromatography (LC)--mass spectrometry (MS) system that
can directly trap fractions of interests from ion exchange
chromatography (IEC) and size exclusion chromatography (SEC) to the
reversed phase (RP) trap cartridges for desalting then subsequently
analyze the fraction sample in MS (Alvarez et al., 2011). They
successfully identified mAb charge variants due to different levels of
glycosylation with this system. In recent years, commercial on-line
process analysis systems have been developed. Patel and coworkers
developed an on-line ion exchange LC using commercial PATROL\selectlanguage{ngerman}® UPLC
Process Analysis System (Patel et al., 2017), where sample pooling
during downstream operations were guided based on near real time charge
variant analysis. The commercial PATROL UPLC Process Analysis System
used in Patel's study can also be applied to develop the on-line SEC
detection. In our recent in-house study an on-line SEC setup showed the
capability of investigating IEC fraction size variant compositions (LMW,
monomer, and HMW) under both native condition and denaturing condition
(Chemmalil et al., 2020), and may be developed for disulfide reduced mAb
identification in the future.

\textbf{7 CASE STUDY ON DISULFIDE BOND REDUCTION ELIMINATION AND
DISULFIDE REDUCED MAB RECOVERY}

This case study illustrates an example of controlling disulfide bond
reduction by using both a preventive strategy and a rescue strategy in
manufacturing process, as detailed in Section 6 and illustrated in
Figure 5. In brief, the preventive strategy essentially applied the
temperature control as well as oxygen control to prevent disulfide
reduction, and the rescue strategy demonstrated that the ``waste''
disulfide reduced antibody could still be rescued and recovered by
re-oxidizing the reduced disulfide bonds during Protein A
chromatography.

Figure 6A illustrates a comprehensive study plan, in which HCCFs of
three mAbs (mAb-1, mAb-2, and mAb-3) were used for the study (Tan et
al., 2020). Each HCCF was divided into two pools, which underwent two
treatments and storage conditions: ``good HCCF'' (air sparging + 4 °C)
and ``bad HCCF'' (nitrogen sparging + room temperature
(19\textasciitilde{}25 °C)). Both HCCFs were processed through Protein A
purification using two Wash 2 buffers (Control buffer and Redox buffer),
respectively. Figures 6B and 6C compare product purity and aggregation
using two Protein A wash arms for the three mAbs in both intact and
reduced forms. It was observed that ``good HCCFs'' for all three mAbs
maintained high product purity using both wash arms, suggesting that the
preventive strategy (chilled storage temperature of harvested bulk with
dissolved oxygen) was able to prevent the disulfide bond reduction prior
to Protein A step. In contrast, ``bad HCCFs'' using the control wash
condition (arm 1: high pH without redox wash) showed low purity
(\textless{} 50\%) for all three mAbs. However, high product purity was
obtained by using the redox wash (arm 2), which demonstrated the
effectiveness of redox wash to enhance the disulfide bond reformation on
the Protein A column.

Implementation of either preventive strategy or rescue strategy or both
clearly demonstrated that disulfide reduction issue could be resolved to
achieve high-purity antibody product. Furthermore, this case study
demonstrated that the redox wash has no negative impact on process yield
or product quality. Extensive characterization of the recused antibody
confirmed a complete formation of interchain disulfide bonds and
comparable biophysical properties to the reference material. The
detailed study was published in a separate paper (Tan et al., 2020).

\textbf{8 CONCLUSIONS AND FUTURE PERSPECTIVES}

The disulfide bond reduction during the biologics manufacturing process
are getting more and more attention currently in the biotech industry as
it is a frequently encountered technical challenge and its effective
management will help meet critical requirement for protein quality
specifications and therapy efficiency. In this paper, we reviewed the
root cause of disulfide bond reduction, analytical methods for disulfide
bond reduction analysis, as well as disulfide bond reduction elimination
and recovery approaches. Furthermore, we suggest additional aspects of
disulfide bond reduction issue worth being addressed in future antibody
manufacturing development:

\textbf{8.1 Minimizing disulfide bond reduction and rescue disulfide
bond in purification platform}

While it is difficult to adapt a truly pre-defined process for different
individual mAb purification, the platform development approaches based
on common unit operations have been proposed by many biopharmaceutical
companies (Kelley, Blank, \& Lee, 2009; Shukla, Hubbard, Tressel, Guhan,
\& Low, 2007). Since disulfide bond reduction mainly happens at the
beginning of the purification process, the cell culture harvest and
Protein A chromatography could be the main unit operations to align the
strategy for disulfide bond reduction elimination and recovery
approaches. For the harvest step, disk-stack continuous centrifuge is
applied as a standard operation for large scale cell culture harvest and
clearance. However, as discussed in previous sections, the cell lysis
during the cell culture harvest may release intracellular enzymes, and
lead to disulfide bond reduction. To address this issue, fully hermetic
centrifuge may be used as platform harvest equipment. Compared with the
disk-stack continuous centrifuge, the hermetic centrifuge machine can be
fully filled with liquid to eliminate the air-liquid surface, which is
known to be one of the root causes for cell lysis (H. F. Liu, Ma,
Winter, \& Bayer, 2010). Hermetic centrifuge machine is commercially
available (Rose, 2008), however, operation parameters such as
throughput, process speed and time need to be further optimized from
facility fit perspective. Another direction to align the strategy of the
disulfide bond reduction elimination approaches during cell culture
harvest is using computational fluid dynamic simulation (CFD) to
optimize centrifuge parameters. CFD can simulate the shear stress and
support the estimation of cell lysis level under certain centrifuge
operation conditions (Boychyn et al., 2004; Megson, Wilson, \&
MacGregor, 2002; Molina-Miras, Sánchez-Mirón, García-Camacho, \&
Molina-Grima, 2018). For Protein A chromatography step, using redox
reagent buffer during wash step can successfully recover the reduced
mAb. This could be aligned with platform wash buffer to recover the
disulfide-reduced mAb or further reduce risk of the disulfide bond
reduction for the intact mAb (Tan et al., 2020; Tang et al., 2020).

\textbf{8.2 Identifying disulfide bond reduction risks using machine
learning algorithms}

In recent years, machine learning algorithms have been developed rapidly
and started to gain more and more attention in the pharmaceutical
industry. Several machine learning algorithms have been applied to
predict mAb pH stability, thermal stability and chemical stability (Jia
\& Sun, 2017; King et al., 2011; Sankar et al., 2018). For instance,
Sankar et al predicted mAb methionine oxidation risk based on a random
forest prediction model (Sankar et al., 2018). In their studies, mAb
structure-based features, sequence-based features and dynamic-based
features were extracted as input data, and mAb peptide mapping results
on oxidation were extracted as output data for model training. Similar
machine learning algorithms were also developed recently for mAb
deamidation risks prediction (Delmar, Wang, Choi, Martins, \& Mikhail,
2019; Jia \& Sun, 2017). The success of machine learning in predicting
the mAb biophysical and biochemical stabilities provides the
possibilities of applying machine learning algorithm to predict mAb
disulfide bond reduction risks. One proposed approach is to use
different mAb sequence-based features, 3-D structure-based features as
data input, and CE NR-SDS analytical results as output features for
model training. One of advantages of the machine learning prediction is
to identify, in very early development stage, the mAbs that show high
disulfide bond reduction risks. If the ``easily reduced'' mAb can be
recognized early, an alternative mAb candidate can be selected to avoid
the disulfide bond reduction, or the downstream development researchers
can start to optimize the manufacturing process early enough to address
the challenge. Another advantage of the machine learning approach is
that it could identify the features such as structure-based features,
sequence-based features and dynamic-based features that have higher
impacts on mAbs disulfide bond reduction, and provide insights on the
disulfide bond reduction level differences observed in different
molecules. However, machine learning algorithm has its own challenges at
this early stage of its development, and people should be aware of: (1)
the data could be unbalanced, for instance, if there are more mAbs that
show low disulfide bond reduction risk than mAbs that show high risks.
This could increase the false positive and false negative prediction
rate; (2) the data sets may be not large enough. The number of data sets
are around 100\textasciitilde{}200 in existing research mentioned
earlier. Thus, some advanced machine learning algorithms such as neural
network or gradient boosting may not be able to be applied, otherwise it
may lead to overfitting issues.

\textbf{8.3 Disulfide bond reduction during multi-specific antibody
purification}

Though multi-specific antibodies (BsAbs and TsAbs) have not been as
widely manufactured as monoclonal antibodies, there is a growing
interest in multi-specific antibody purification in the
biopharmaceutical industry in recent years (Brinkmann \& Kontermann,
2017; Y. Li, 2019; Swope et al., 2020; Tustian et al., 2016; X. Yang et
al., 2015). Taking BsAb as examples, during bispecific antibody
purification process, the inter-chain disulfide bond between the heavy
chains in two parental mAbs need to be reduced first to generate
half-mers and followed by free thiol oxidization to form bispecific
antibodies. For instance, one method for bispecific antibody
purification is to capture parental mAb using Protein A chromatography
separately followed by incubating mixed parental mAbs in
phosphate-buffered saline (PBS) buffer that contains 2-MEA (reducing
agent) for disulfide bond reduction. Then the mixture is re-oxidized by
dialysis against PBS buffer without 2-MEA (Paul et al., 2016). In
addition to purification process, disulfide bond reduction and
re-oxidization also influence bispecific antibody stabilities and
impurities level. Researchers found that intact bispecific antibody are
susceptible to the reduction condition. Compared with mAbs, disulfide
bond reduction for BsAbs is much more severe, since it could lead to
both aggregation and mispairing. Higher HMW levels have also been
observed for the bispecific antibody when the bispecific antibody and
the parental mAb were reduced and re-oxidized under similar conditions.
In addition, disulfide bond mispairing may lead to homodimer
formation(Kuglstatter et al., 2017). Thus, both disulfide bond reduction
elimination and its recovery are critical for bispecific antibody
processing, and the learnings on mAb disulfide bond reduction could also
benefit bispecific antibody development.

\textbf{Acknowledgements}

The authors would like to acknowledge Process Development Analytics for
their contributions to this work. The authors also thank Dr. Magfur
Alam, Dr. Dhanuka Wasalathanthri and Dana Brandariz for providing
thorough review and invaluable comments. All financial support was
provided by BMS: T.R., Z.T., V.E., A.L., S.G., and Z.L. are employees of
BMS. The authors have no conflicts of interest to declare.

\textbf{Disclosure of potential conflicts of interest}

No potential conflicts of interest were disclosed.

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\textbf{Table 1:} Reported approaches to prevent disulfide bond
reduction and rescue reduced mAb during manufacturing process\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Prevention and rescue direction}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Prevention and rescue direction}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Approach examples}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{References}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Prevention\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Inhibit enzyme overexpression\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Knocked down the expression of thioredoxin (Trx) in CHO cells\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Koterba, Borgschulte, \& Laird, 2012\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Inhibit enzyme activity during cell culture and cell harvest\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Induce Ethylenediaminetetraacetic acid (EDTA) to inhibit hexokinase
activity and reduce the formation of glucose-6-P; Induce
Cu\textsuperscript{2+}, Zn\textsuperscript{2+}, Hg\textsuperscript{2+}
and Co\textsuperscript{2+} to inhibit Trx activity; Induce
Aurothioglucose (ATG) and aurothiomalate (ATM) to inhibit thioredoxin
reductase activity\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Kao, Hewitt, Trexler-Schmidt, \& Laird, 2010; Trexler-Schmidt et al.,
2010\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Reduce enzyme amount for disulfide bond reduction during cell culture,
cell harvest and clarified bulk (CB) storage\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Induce O\textsubscript{2}, H\textsubscript{2}O\textsubscript{2} and
cysteine as enzyme competitors;\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Du et al., 2018; Mun et al., 2015; Chung et al., 2017; Handlogten, Wang,
\& Ahuja, 2020\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Control centrifuge force during harvest to minimize cell lysis; Control
depth filtration pressure to minimize cell lysis;\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Trexler-Schmidt et al., 2010; O'Mara et al., 2019\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Slowdown enzyme reaction rate during cell harvest and CB storage\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Lower pH and temperature\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Xie et al., 2016; Hutterer et al., 2013; Liu, Nowak, Shao, Ponniah, \&
Neill, 2016\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Reduce reaction time\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Shorten CB storage time\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Mun et al., 2015; O'Mara et al., 2019\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rescue\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Reoxidize reduced mAb during Protein A chromatography\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Introduce redox wash to reoxidize reduced mAb\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Tan et al., 2020; Tang et al., 2020\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{FIGURE 1} Illustrations of disulfide bond structure. (A)
Intra-chain disulfide bond connects two beta-sheets in Ig domain (Li,
Prabakaran et al. 2016). (B) Intra-chain and inter-chain disulfide bonds
in a IgG monomer; (C) ``knob-into-holes (Kih)'' IgG-like bispecific
antibody (BsAb) stability can be improved by having additional disulfide
bonds (red line) formed in the Kih region; (D) Disulfide bond can
improve correctly pairing IgG-like BsAb light chain and heavy chain: in
one arm, the cysteine residues in C\textsubscript{H} and
C\textsubscript{L} that originally formed disulfide were mutated into
valine, and introduced an addition disulfide bond (marked as red) during
the mutation; the other arm kept unmutated; (E) Dual Affinity
Retargeting Molecules (DART), light chain variable region from one
antibody is linked to the heavy chain variable region from the other
antibody through small peptide (black dash line), and the two variable
regions are linked through disulfide bond (red solid line).

\textbf{FIGURE 2} (A) Protein A chromatography profile from mAb3
purification process (B) AEX (Flow through mode) from mAb1 purification
process; (C) CEX (bind and elute mode) from mAb1 purification process.
Blue line represented the load sample that had no significant level
disulfide bond reduction, red line represented the load sample that had
disulfide bond reduction. The tail part for each step were normalized
and enlarged for comparison purpose.

\textbf{FIGURE 3} Qualifications of product quality attributes for mAb1
and mAb2 during downstream processing. (A) During the low pH viral
inactivation step, for mAb1, the HMW\% for intact mAb pool and reduced
mAb pool increased at similar level (0.5\textasciitilde{}0.6\%); for
mAb2, the HMW\% decreased more for intact mAb pool (-1.7\%) than reduced
mAb pool (-0.3\%); (B) For both mAb1 and mAb2, reduced mAb pool purity
increased during downstream process, showed the possibilities that
reduced mAb can re-oxidize.

\textbf{FIGURE 4} Stability of antibody samples with varied initial
disulfide reduction levels under the conditions of room temperature (25
\selectlanguage{ngerman}ºC), high temperature (40 ºC), and light exposure at 25 ºC for total 14
days. A, Aggregation level as a function of time for all studied
conditions; B, Zoom-in profile of aggregation level as a function of
time for 25 C and 40 C.

\textbf{FIGURE 5} Different approaches have been applied in mAb
manufacturing process to prevent disulfide bond reduction and recover
reduced mAb.

\textbf{FIGURE 6} Comprehensive evaluation of using redox wash system in
the platform Protein A chromatography (Tan, Ehamparanathan et al. 2020).
The study was performed using three mAb harvest cell cultures according
to the design including three arms. The protein A pools from each run
was tested for product quality attributes. (A) Comprehensive study: Arm
1, control; Arm 2, combined wash step; (B) Intact mAb impurity; (C)
Aggregates (HMW\%).

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