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).
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).
6 APPROACHES TO MINIMIZE
DISULFIDE BOND REDUCTION AND RECOVER of REDUCED-MAB
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).
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 N2gas and stored at room temperature under anaerobic environment (Du et
al., 2018).
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: