Quantification of glycated IgG in CHO supernatants: A practical approach

Abstract

Post-translational, nonenzymatic glycation of monoclonal antibodies (mAbs) in the presence of reducing sugars (in bioprocesses) is a widely known phenomenon, which affects protein heterogeneity and potentially has an impact on quality, safety, and efficacy of the end product. Quantification of individual glycation levels is compulsory for each mAb therapeutically applied in humans. We therefore propose an analytical method for monitoring glycation levels of mAb products during the bioprocess. This is a useful tool for process-design considerations, especially concerning glucose-feed strategies and temperature as major driving factors of protein glycation. In this study, boronate affinity chromatography (BAC) was optimized for determination of the glycation level of mAbs in supernatants. In fact, the complex matrix found in supernatants is an underlying obstacle to use BAC, but with a simple clean-up step, we found that the elution profile could be significantly improved so that qualitative and quantitative determination could be reached. Complementary analytical methods confirmed the performance quality, including the correctness and specificity of the results. For quantitative determination of mAb glycation in supernatants, we established a calibration procedure for the retained mAb peak, identified as glycated antibody monomers. For this approach, an available fully characterized mAb standard, Humira®, was successfully applied, and continuous monitoring of mAbs across three repetitive fed-batch processes was finally performed. With this practical, novel approach, an insight was obtained into glycation levels during bioprocessing, in conjunction with glucose levels and product titer over time, facilitating efficient process development and batch-consistency monitoring.

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

“…To obtain better insights on the effect of glycation on ENDO-I, we developed a BAC method to selectively enrich for these proteoforms. The specific interaction between glycated proteins with the boronic acid stationary phase is most stable under alkaline conditions (pH > 8.2) [23,28]. Therefore, the BAC separation is preferably performed at this pH or higher.…”

“…The separation of BAC is based on the specific and reversible interaction between the boronic ligands and cis-diol structures (i.e., glycated proteoforms) [9,[19][20][21][22]. Currently, BAC is a widely applied technique to determine the glycation levels of monoclonal antibodies [23], endogenous proteins [24], and milk proteins [25]. Besides the efficient separation of these specific proteoforms, an advantage of BAC is the possibility to work at a preparative scale enabling the collection of sufficient material for further functional tests in a single run.…”

A Novel 3D-Printed and Miniaturized Periodic Counter Current Chromatography System for Continuous Purification of Monoclonal Antibodies

“…Monoclonal antibodies (mAb) are proteins that specifically recognize antigenic epitopes [ 1 ] and thus have great potential for targeted therapeutic uses [ 2 ]. For instance, mAbs are used to alleviate autoimmune diseases such as multiple sclerosis [ 3 , 4 ] and rheumatoid arthritis [ 5 , 6 , 7 ] or for the treatment of cancer [ 8 , 9 ]. mAbs are preferably produced in mammalian cells such as Chinese hamster ovary (CHO) cells, since these cells generate the most human-like glycosylation patterns and thereby support immunogenic safety and efficacy [ 10 ].…”