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.
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 <5% in the load to
> 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.
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: < 100 μM (low risk); 100 -
200 μM (medium risk) and > 200 μM (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® 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.
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
(19~25 °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
(< 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).
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:
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).
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 100~200 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.
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.