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One of the basic structural features of human IgG1 is the arrangement of the disulfide bond structure, 4 inter chain disulfide bonds in the hinge region and 12 intra chain disulfide bonds associated with twelve individual domains. Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. However, a complete ranking of the susceptibility of disulfide bonds in IgG1 molecules is lacking. A method including reduction, differential alkylation, and liquid chromatography-mass spectrometry (LC-MS) analysis was developed and employed to investigate the complete ranking order of the susceptibility of disulfide bonds in two recombinant monoclonal antibodies. The results confirmed that inter chain disulfide bonds were more susceptible than intra chain disulfide bonds. In addition, it was observed that the disulfide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains. The upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one. Furthermore, disulfide bonds in the CH2 domain were the most susceptible to reduction. Disulfide bonds in VL, CL, VH, and CH1 domains had similar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction. Interestingly, a difference between IgG1kappa and IgG1lambda was also observed. The difference in the susceptibility of inter light heavy chain disulfide bonds and inter heavy chain disulfide bonds was smaller in IgG1kappa than in IgG1lambda. The intra chain disulfide bonds in the Fab region of IgG1kappa were also less susceptible than disulfide bonds in the Fab region of IgG1lambda.

Host cell proteins (HCPs) are process-related impurities that may copurify with biopharmaceutical drug products. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, biologically active, or enzymatically active with the potential to degrade either product molecules or excipients used in formulation. Some have been shown to be difficult to remove by purification. Why should the biopharmaceutical industry worry about these high-risk HCPs? What approach could be taken to understand the origin of its copurification and address these *Marisa Jones and Nisha Palackal should be considered joint first authors About Biophorum Development Group (BPDG): Since its inception in 2004, BioPhorum has become a trusted environment in which senior leaders of the biopharmaceutical industry come together to share and discuss openly the emerging trends and challenges facing their industry.BioPhorum currently comprises more than 3800 active participants in seven "phorums" covering cell and gene therapy, drug substance, development, fill-finish, a technology roadmap, information technology, and supply partners. The Host Cell Protein (HCP) Workstream is part of the Development Group (BPDG). This article is a composite view of opinions shared by the whole of the BPDG-HCP Workstream and should not be attributed to the individual positions of the participating companies.

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Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4.

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