Recombinant Antibody Color Resulting from Advanced Glycation End Product Modifications

Abstract

Recombinant antibodies produced in Chinese hamster ovary (CHO) cells often exhibit a slight yellow-brown color, but the molecular basis for this color has remained elusive. We predicted that the color results from post-translational modifications on the antibody, because colored species were found to coelute with antibody products during size exclusion chromatography. Previously, modification by oxidation and advanced glycation end products (AGEs) had been shown to give rise to colored and fluorescent species whose spectral properties are in agreement with the spectral properties of CHO-derived recombinant antibodies. To test whether these modifications give rise to the color exhibited in our CHO-derived antibodies, we searched for 8 different oxidation and 28 different glycation and AGE modifications by mass spectrometry in a variety of samples exhibiting varying color intensities. Oxidation and glycation modifications correlated weakly with color in a subset of samples, but several AGEs exhibited a strong correlation with product color in all samples tested. This strong correlation with sample color was verified for a specific AGE, carboxymethyllysine, by ELISA, thus validating the mass spectrometry data. These data indicate that AGEs are at least partially responsible for the color seen in CHO-derived recombinant antibodies.

Glycation of antibodies: Modification, methods and potential effects on biological functions

“…Since there are multiple AGEs products, each kind of AGEs product needs to be quantitated separately. 16,21,60 In addition to lysine, arginine is a potential spot where AGEs modifications may occur in recombinant antibodies. 17 Isotope labeling has also been applied to antibody glycation analysis.…”

“…63 NBT analysis was applied to glycated antibody by Ahmad, et al 39 in 2012. 37 Recently, Butko, et al 21 reported that several AGEs found in manufactured recombinant mAb were strongly related to a color change in the antibody solution, and that AGEs are partially responsible for the color of mAb products. 19 Additionally, an enzymelinked immunosorbent assay (ELISA) format has been applied to study the AGEs in mAbs, utilizing the binding between a sample and a biotin-conjugated primary antibody that targets a specific kind of AGEs.…”

“…19 Additionally, an enzymelinked immunosorbent assay (ELISA) format has been applied to study the AGEs in mAbs, utilizing the binding between a sample and a biotin-conjugated primary antibody that targets a specific kind of AGEs. 21 …”

“…20 However, with optimal cell culture conditions, AGEs are very low or not observed for therapeutic antibodies. 21 Antibody aggregation is an important CQA related to immunogenicity and safety. Reports correlating glycation levels with aggregation have been inconsistent, with one study showing a correlation, 32 but another indicating that there was no significant difference in the level of aggregation observed between glycated and control mAb stored at 40 C for 1/week.…”

“…[25][26][27][28] AGEs-damaged proteins are highly reactive and become cross-linked, structurally altered, and ultimately aggregated, and these non-native states are toxic for many cell types (e.g., endothelial, neuronal, retinal, leukocytes) and associated with advanced diseases. 29 However, in the manufacturing of therapeutic recombinant antibody products, AGEs formation has not been observed at significant amounts, even with low 21 or high levels of glycation. 8 For therapeutic mAbs, the potential effects of glycation, such as blocking the biologically functional site or further degradation that induces aggregation, make glycation a potential critical quality attribute (CQA).…”