Monoclonal antibody interchain disulfide bond reduction was observed in a ChineseHamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452-461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (ΔP) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of ΔP and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP + )/NADPH-dependent manner. K E Y W O R D Sair overlay, antibody, cell lysis, depth filtration, differential pressure, dissolved oxygen, disulfide reduction 1 | INTRODUCTION Monoclonal antibodies (mAbs) are commonly manufactured with fed-batch or perfusion mammalian cell culture processes. In such manufacturing processes, the mAb, which is expressed and secreted from the cell, accumulates in the cell culture fluid and is recovered using centrifugation and/or filtration. With the advent of high productivity (>5 g/L) cell culture processes and the availability of improved disposable technologies, the implementation of single-use systems, such as bioreactors, depth filters, columns, membranes, and bioprocess storage bags in the production of biologics has become standard industry practice (Liu, 2005).

The burgeoning pipeline for new biologic drugs has increased the need for high-throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250-mL automated mini bioreactor (ambr250™) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24-bioreactor ambr250™ system with 10-factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory-scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late-stage characterization.

The regulatory approval of a biosimilar product is contingent on the favorable comparability of its safety and efficacy to that of the innovator product. As such, it is important to match the critical quality attributes of the biosimilar product to that of the innovator product. The N‐glycosylation profile of a monoclonal antibody (mAb) can influence effector function activities such as antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity. In this study, we describe efforts to modulate the high‐mannose (HM) levels of a biosimilar mAb produced in a Chinese hamster ovary cell fed‐batch process. Because the HM level of the mAb was observed to impact ADCC activity, it was desirable to match it to the innovator mAb's levels. Several cell culture process related factors known to modulate the HM content of N‐glycosylation were investigated, including osmolality, ammonium chloride (NH4Cl) addition, glutamine concentration, monensin addition, and the addition of alternate sugars and amino sugars to the feed medium. The process conditions evaluated varied in impact on HM levels, process performance and product quality. One condition, the addition of alternate sugars and amino sugars to feed medium, was identified as the preferred method for increasing HM levels with minimal disruptions to process performance or other product quality attributes. Interestingly, a secondary interaction between sugar and amino sugar supplemented feeds and osmolality was observed during process scale‐up. These studies demonstrate sugar and amino sugar concentrations and osmolality are critical variables to evaluate to match HM content in biosimilar and their innovator mAbs.