Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing

Abstract

In the biopharmaceutical industry, therapeutic monoclonal antibodies are primarily produced in mammalian cell culture systems. During the scale-up of a monoclonal antibody production process, we observed excessive mechanical cell shear as well as significant reduction of the antibody's interchain disulfide bonds during harvest operations. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Subsequent laboratory studies have demonstrated that cells subjected to mechanical shear release cellular enzymes that contribute to this antibody reduction phenomenon (manuscript submitted; Kao et al., 2009). Several methods to prevent this antibody reduction event were developed using a lab-scale model to reproduce the lysis and reduction events. These methods included modifications to the cell culture media with chemicals (e.g., cupric sulfate (CuSO(4))), pre- and post-harvest chemical additions to the cell culture fluid (CCF) (e.g., CuSO(4), EDTA, L-cystine), as well as lowering the pH and air sparging of the harvested CCF (HCCF). These methods were evaluated for their effectiveness in preventing disulfide bond reduction and their impact to product quality. Effective prevention methods, which yielded acceptable product quality were evaluated for their potential to be implemented at manufacturing-scale. The work described here identifies numerous effective reduction prevention measures from lab-scale studies; several of these methods were then successfully translated into manufacturing processes.

Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability

“…L‐cystine was reported to be a potential competitive inhibitor of the reducing enzymes or to act as a surrogate substrate for the enzyme in place of the mAb product (Trexler‐Schmidt et al, 2010). …”

“…They hypothesized that the mechanical forces in the centrifuge were responsible for reducing the molecule into half‐antibodies (Hutchinson et al, 2006). Trexler‐Schmidt and co‐workers further demonstrated that antibody disulfide reduction can be attributed to the cell lysis and the release of intra‐cellular reducing enzymes (primarily thio‐redoxin reductase/thioredoxin) as a result of harsh centrifugation conditions (Trexler‐Schmidt et al, 2010). Hutterer and co‐workers attributed the extent of antibody reduction to be dependent on the cell line and cell culture process (Hutterer et al, 2013).…”

“…Maintaining a highly oxidative environment through air sparging was proposed as a solution to shift the equilibrium of the reversible redox reaction toward oxidation (Mun et al, 2015). Addition of chemical inhibitors (e.g., cystine, copper sulfate, EDTA) to act as a competitive inhibitor, directly inhibit responsible enzymes, or remove the metal ions required in the enzymatic pathway was suggested as means of minimizing enzymatic activity (Trexler‐Schmidt et al, 2010). However, while the manufacturing process conditions that cause the antibody reduction (Hutterer et al, 2013), the impact on the molecular structure, as well the susceptibility of various mAb isoforms to reduction (Magnusson et al, 1997; Wang et al, 2015) were well‐characterized, the impact of antibody reduction on purification process performance and long term drug substance stability has not been reported.…”

Tailoring recombinant protein quality by rational media design

“…Many authors have published extensive reviews, depicting the current state of the art and strategies, which focus on the cell line 1,7,16,45 and the cell culture parameters 18,51 . Furthermore, through the concentration adjustment of selected media components, and in some cases by supplementing the medium with specific co-factors, it is possible to adjust the glycosylation profile 52 , the charge variants 53 , the aggregation level 46,54 , and the abundance of LMW species 55 . Figure 1.1 outlines the three main strategies allowing to affect cell culture process performance and to tailor the quality attributes of therapeutic molecules.…”

“…Dehydroepiandrosterone, epiandrosterone, pyridoxal 5'-phosphate, 1-fluoro-2,4-dinitrobenzene have effectively reduced G6PD activity and the addition of chelating agents (EDTA & EGTA), citrate, distinct types of phosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose, or lyxose inhibit hexokinase 186 . L-cystine exhibited chemical inhibitory capacities for a recombinant monoclonal antibody due to its function as competitive inhibitor for reducing enzymes 55 . A mixture of protease inhibitors including E64, leupeptin, benzamidine, E-amino caproic acid, pepstatin A, and EDTA was tested.…”