Impact of S-sulfocysteine on fragments and trisulfide bond linkages in monoclonal antibodies

Abstract

The quality of recombinant proteins such as monoclonal antibodies produced using Chinese hamster ovary cell-based mammalian systems is dependent on many factors, including cell line, process and cell culture media. Due to these factors, the generated product is heterogeneous and may have chemically-induced modifications or post-translational modifications that affect antibody stability, functionality and, in some cases, patient safety. This study demonstrates that S-sulfocysteine, a cysteine derivative, can increase the antibody specific productivity in different cell lines cultivated with different processes while minimizing trisulfide linkages in generated mAbs, mainly between heavy and light chain. The supplementation of a cell culture feed with S-sulfocysteine also proved to be useful to reduce the percentage of antibody fragments generated from the monoclonal antibody. Overall, this new component used in the upstream process allows a reduction of product heterogeneity.

Cysteine in cell culture media induces acidic IgG1 species by disrupting the disulfide bond network

“…A defective disulfide bond network has been reported to introduce different charge to mAbs (Bloom et al, 1997; Gevondyan et al, 2006; Gu et al, 2010; Schauenstein et al, 1986; Seibel et al, 2017; Wang et al, 2018). Although mAb charge variants have been characterized and described in the literature, insights to the underlying mechanisms of their generation would be beneficial in controlling mAb charge variants in industrial production processes.…”

“…Samples were subjected to a combined Lys‐C/tryptic digest as described previously (Seibel et al, 2017). For analysis of free thiols, half of the Lys‐C/tryptic digest was reduced by addition of 4.4 µl 0.64 M Tris‐(2‐carboxyethyl)‐phosphin (Roth) and incubation for 30 min at 37°C.…”

“…Sample measurement was conducted as described previously (Seibel et al, 2017). For analysis of disulfide connectivity, reduced and nonreduced data sets from the same sample were analyzed using PepFinder TM (Thermo Fisher Scientific Inc.) and manually reviewed ( n = 1).…”

“…Modifications which result in acidic elution on CEX include trisulfide bonds, disulfide bond scrambling, and unbound cysteines resulting in free thiol (sulfhydryl) groups (Dillon et al, 2008; Schuurman et al, 2001; Wypych et al, 2008; Yoo et al, 2003). The IgG2 and IgG4 subclasses are most frequently affected by these modifications, but some have also been reported for IgG1 (Bloom et al, 1997; Gevondyan et al, 2006; Gu et al, 2010; Schauenstein et al, 1986; Seibel et al, 2017; Wang et al, 2018). On average, one IgG1 molecule contains 0.1–1.1 free cysteines (Gevondyan et al, 2006).…”

Oxidative stress‐alleviating strategies to improve recombinant protein production in CHO cells

“…The use of S‐sulfocysteine in feed solutions was hypothesized to reduce the production of hydrogen sulfide by protecting thiol groups against oxidation. In an additional study, the same lab reported that this compound could also lower antibody low molecular weight species and trisulfide bond levels by decreasing free hydrogen sulfide in the medium (Seibel et al, ).…”