"High-risk" host cell proteins (HCPs): A multi-company collaborative view.

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 high-risk HCPs? To answer these questions, the BioPhorum Development Group HCP Workstream initiated a collaboration among its 26-company team with the goal of industry alignment around high-risk HCPs. The information gathered through literature searches, company experiences, and surveys were used to compile a list of frequently seen problematic/high-risk HCPs. These high-risk HCPs were further classified based on their potential impact into different risk categories. A step-by-step recommendation is provided for establishing a comprehensive control strategy based on risk assessments for monitoring and/or eliminating the known impurity from the process that would be beneficial to the biopharmaceutical industry.

Ranking the susceptibility of disulfide bonds in human IgG1 antibodies by reduction, differential alkylation, and LC-MS analysis.

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.

Effect of the conserved oligosaccharides of recombinant monoclonal antibodies on the separation by protein A and protein G chromatography.

Glycosylation of the conserved asparagine residue in CH2 domains of IgG molecules is an important post-translational modification. The presence of oligosaccharides is critical for structure, stability and biological function of IgG antibodies. Effect of the glycosylation states of recombinant monoclonal antibodies on protein A and protein G chromatography was evaluated. Antibodies lacking oligosaccharides eluted later from protein A and earlier from protein G columns than antibodies with oligosaccharides using a gradient of decreasing pH. Interestingly, different types of oligosaccharides also affected the elution of the antibodies. Antibodies with high mannose type oligosaccharides were enriched in later eluting fractions from protein A and earlier eluting fractions from protein G. While antibodies with more mature oligosaccharides, such as core fucosylated biantennary complex oligosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal galactoses, were enriched in earlier eluting fractions from protein A and in the later eluting fractions from protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody binding affinity to protein A and protein G was not affected by the absence or presence of oligosaccharides. It was thus concluded that the elution difference of antibodies with or without oligosaccharides and antibodies with different types of oligosaccharides were due to differential structural changes around the CH2-CH3 domain interface under the low pH conditions used for protein A and protein G chromatography.

Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G.

Oxidation of methionine (Met) residues is one of the most common protein degradation pathways. Two Met residues, Met256 and Met432, of a recombinant fully human monoclonal IgG1 antibody have been shown to be susceptible to oxidation. Met256 and Met432 are located in the antibody CH2-CH3 interface and in close proximity to protein A and protein G binding sites. The effect of oxidation of these susceptible Met residues on the binding to protein A and protein G was investigated in the current study. Incubation of the antibody with 5% tert-butyl hydroperoxide (tBHP) resulted in a nearly complete oxidation of Met256 and Met432, while incubation with 1% tBHP resulted in mixed populations of the antibody with different degrees of Met oxidation. Oxidation of Met256 and Met432 resulted in earlier elution of the antibody from protein A and protein G columns when eluted with a gradient of decreasing pH. Analysis by ELISA and surface plasmon resonance (SPR) revealed decreased binding affinity of the oxidized antibody to protein A and protein G. It is therefore concluded that oxidation of the Met256 and Met432 residues of the recombinant monoclonal antibody altered its interaction with protein A and protein G resulting in a decrease in binding affinity.