Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.

This review article with 223 references describes recent developments in capillary electrophoresis (CE) of proteins and covers papers published during last two years, from the previous review (V. Dolnik, Electrophoresis 1999, 20, 3106-3115) through Spring 2001. It describes the topics related to CE of proteins including modeling of the electrophoretic properties of proteins, sample pretreatment, wall coatings, improving selectivity, detection, special electrophoretic techniques, and applications.

An ultrahigh-voltage capillary electrophoresis system was built to demonstrate the possibility of extending the applied potential and thus the separation power of capillary electrophoresis. A commercial 30-kV power supply was extensively modified in order to provide electrical potentials up to 120 kV. A unique electrical shielding system was developed to prevent capillary breakdown and corona or spark discharges. Electrophoretic studies using a mixture of peptide standards, as well as a complex mixture of peptides obtained from a protein digest, showed that the numbers of theoretical plates achieved increase linearly with applied voltage. Theoretical plate counts ranging from 2.7 to 6.1 million plates were obtained for peptides in a separation done at 120 kV. Resolution also increased with the square root of applied voltage, as predicted by theory.

This review article with 244 references describes recent developments in capillary electrophoresis of proteins and covers the two years since the previous review (Dolník, V. Hutterer, K. Electrophoresis 2001, 22, 4163-4178) through Spring 2003. It covers topics related to CE of proteins including modeling of the electrophoretic properties of proteins, sample pretreatment, wall coatings, improving selectivity, detection, special electrophoretic techniques, and applications.