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: