KEYWORDS
Antibody, Disulfide bond, Reduction/Oxidation, Process development
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.
2 DISULFIDE BONDS in
Therapeutica Proteins
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, & Lelièvre, 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).
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).
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.
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.
(1) 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.
(2) 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).
(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 (>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).
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.
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: