The effect of low intensity ultraviolet-C light on monoclonal antibodies.

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.

A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior.

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.