Characterization of the basic charge variants of a human IgG1: effect of copper concentration in cell culture media.

We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu ( 2+) ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.

DNA spike studies for demonstrating improved clearance on chromatographic media.

DNA spike clearance methods were used to demonstrate improved clearance factors on anion exchange and hydrophobic interaction columns used in the production of human therapeutic proteins. DNA clearance at large-scale was first measured for a monoclonal antibody expressed in Chinese Hamster Ovary (CHO) cells and an antibody fragment expressed in Escherichia coli. Small-scale spike experiments were then performed on individual chromatographic steps using host-specific DNA paired with TaqMan PCR assay methods. This approach has advantages of improved specificity, sensitivity, cost and throughput compared to other types of spike clearance methods. The anion exchange column used in the monoclonal antibody process was shown to have very high capacity for CHO DNA, resulting in greater than 7.1 log reduction. The anion exchange and hydrophobic interaction columns used in the antibody fragment process were shown to have high E. coli DNA clearance capability, with greater than 5.1 and 5.3 logs clearance, respectively. Compared to the large-scale process, higher log reduction values were achieved in small-scale spike clearance studies by challenging the chromatographic steps with load DNA levels 2-5 logs higher than the large-scale process levels. Using highly specific and sensitive spike clearance methods, we demonstrated consistently high DNA clearance factors for each of the production processes that meet industry and regulatory standards for human therapeutics. The method is applicable to a broad range of industrial scale processes where demonstration of the robustness of DNA clearance is necessary to support development or licensure of biopharmaceutical products.