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Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.

As part of an investigation to identify potential new viral reduction strategies, ultraviolet-C (UV-C) light was examined. Although this technology has been known for decades to possess excellent virus inactivation capabilities, UV-C light can also introduce significant unwanted damage to proteins. To study the effect on monoclonal antibodies, three different antibodies were subjected to varying levels of UV-C light using a novel dosing device from Bayer Technology Services GmbH. The range of fluencies (or doses) covered was between 0 and 300 J/m(2) at a wavelength of 254 nm. Product quality data generated from the processed pools showed only minimal damage done to the antibodies. Aggregate formation was low for two of the three antibodies tested. Acidic and basic variants increased for all three antibodies, with the basic species increasing more than the acidic species. Peptide maps made for the three sets of pools showed no damage to two of the three antibody backbones, whereas the third antibody had very low levels of methionine oxidation evident. Samples held at 2-8 degrees C for 33 days showed no increase in aggregates or charge variants, indicating that the proteins did not degrade and were not damaged further by reactive or catalytic species that may have been created on exposure to UV-C light. Overall, UV-C light was shown to induce very little damage to monoclonal antibodies at lower fluencies and appears to be a viable option for viral inactivation in biotechnology applications.

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