Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process

Abstract

During large-scale monoclonal antibody manufacturing, disulfide bond reduction of antibodies, which results in generation of low molecule weight species, is occasionally observed. When this happens, the drug substance does not meet specifications. Many investigations have been conducted across the biopharmaceutical industry to identify the root causes, and multiple strategies have been proposed to mitigate the problem. The reduction is correlated with the release of cellular reducing components and depletion of dissolved oxygen before, during, and after harvest. Consequently, these factors can lead to disulfide reduction over long-duration storage at room temperature prior to Protein A chromatography. Several strategies have been developed to minimize antibody reduction, including chemical inhibition of reducing components, maintaining aeration before and after harvest, and chilling clarified harvest during holding. Here, we explore the use of hydrogen peroxide in clarified harvest bulk or cell culture fluid as a strategy to prevent disulfide reduction. A lab-scale study was performed to demonstrate the effectiveness of hydrogen peroxide in preventing antibody reduction using multiple IgG molecules. Studies were done to define the optimal concentration of hydrogen peroxide needed to avoid unnecessary oxidization of the antibody products. We show that adding a controlled amount of hydrogen peroxide does not change product quality attributes of the protein. Since hydrogen peroxide is soluble in aqueous solutions and decomposes into water and oxygen, there is no additional burden involved in removing it during the downstream purification steps. Due to its ease of use and minimal product impact, we demonstrate that hydrogen peroxide treatment is a powerful, simple tool to quench reducing potential by simply mixing it with harvested cell culture fluid.

On-column disulfide bond formation of monoclonal antibodies during Protein A chromatography eliminates low molecular weight species and rescues reduced antibodies

“…All eight pairs of interchain disulfide bonds were fully identified. In addition, both the rescued mAb-4 and reference material showed 0.2–0.3 free thiols per mAb molecule, 11 consistent with observations for a typical drug substance. Relatively lower thiol observed for the rescued mAb-4 than the reduced mAb (2.5 free thiols per mAb) further confirmed the effectiveness of the on-column disulfide reoxidation.…”

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

“…Disulfide-bonds have an impact on protein stability as well as on activities [33]. Du et al stated that during the manufacturing process, extensive reduction of antibodies has been observed after harvest operation or Protein A affinity chromatography and multiple process parameters correlate to the extent of the reduction [34]. The topic "disulfide bonds of therapeutic proteins" is in depth discussed by Lakbub et al [35].…”

Antibody disulfide bond reduction and recovery during biopharmaceutical process development—A review

“…Based on Nature Reviews Drug Discovery reports (Hughes, 2010;Mullard, 2011Mullard, , 2012Mullard, , 2013Mullard, ,2014Mullard, , 2015Mullard, , 2016Mullard, , 2017Mullard, , 2018Mullard, , 2019Mullard, , 2020, in the last ten years (2009-2019), 61 of 95 FDA approved Biologics License Applications (BLAs) were mAb products or mAb-related products, and the percentage of mAb (or mAb related) products in the BLAs increased over time (Figure 1). Commercial mAbs are generally produced in mammalian cells such as Chinese hamster ovary (CHO) (Du et al, 2018;Kao et al, 2010;T. Wang et al, 2015).…”