Quantitative analysis of glycation and its impact on antigen binding

Abstract

Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.

Robust Neutralizing Antibodies to SARS-CoV-2 Develop and Persist in Subjects with Diabetes and COVID-19 Pneumonia

“…In spite of this, the presence of valid neutralizing activity was not obvious. Antibody glycation has been observed in subjects with diabetes ( 25 ) and glycation can affect the function by weakening antigen binding ( 36 ). Moreover, although antibodies are generally beneficial and protective, sometimes virus-specific antibodies can promote pathology, a phenomenon defined as antibody-dependent enhancement (ADE).…”

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry

“…In fact, techniques such as boronate affinity chromatography, capillary isoelectric focusing and liquid chromatography, commonly used for the analysis of glycated mAbs at an intact level, only allow the separation of nonglycated from glycated structures, without isomeric resolution. 9,11,45,46 Thus, although intact mass analysis provides information on the number of sugar molecules attached to a BsAb, the contribution of the different isomeric structures (i.e., combinations of glycated BsAb subunits) to the glycation level cannot be determined. 10,45 In addition, the presence of glycosylated structures affects the quantification of glycation levels.…”

“…9,11,45,46 Thus, although intact mass analysis provides information on the number of sugar molecules attached to a BsAb, the contribution of the different isomeric structures (i.e., combinations of glycated BsAb subunits) to the glycation level cannot be determined. 10,45 In addition, the presence of glycosylated structures affects the quantification of glycation levels. This limitation can be overcome by removal of the glycans prior to MS analysis (e.g., using Peptide:N-glycosidase F (PNGase F)).…”

Quantification of glycated IgG in CHO supernatants: A practical approach

“… 1 In this respect, nonenzymatic chemical modification of proteins (the glycation of N‐terminus and each potential lysine sidechain, respectively) in the presence of reducing sugars (aldose or ketose) leads, in the final phase, to the formation of irreversible advanced glycation products. 2 , 3 In the food and pharmaceutical industries, glycation has become of great significance in relation to several therapeutic candidates, especially recombinant monoclonal antibodies (mAbs). 4 Although the glycation cascade has been thoroughly investigated, few studies have explored bioprocess‐relevant protein glycation.…”

“…Besides glycosylation (the most prominent intracellularly processed modification), extracellular events also lead to significant post‐translational modifications of the protein 1 . In this respect, nonenzymatic chemical modification of proteins (the glycation of N‐terminus and each potential lysine sidechain, respectively) in the presence of reducing sugars (aldose or ketose) leads, in the final phase, to the formation of irreversible advanced glycation products 2,3 . In the food and pharmaceutical industries, glycation has become of great significance in relation to several therapeutic candidates, especially recombinant monoclonal antibodies (mAbs) 4 .…”