Therapeutic Antibody Glycosylation Analysis: A Contract Research Organization Perspective in the Frame of Batch Release or Comparability Support

Abstract

Glycosylation of the Fc moiety of a monoclonal antibody is a heterogeneous posttranslational process considered as a critical quality attribute of the purified drug substance due to its major impact on safety and efficacy (i.e., immunogenicity, CDC or ADCC effector functions, etc.). Glycosylation should thus be addressed for batch-to-batch comparability and for drug substance characterization, in terms of identity and/or purity testing. We present below a set of efficient, performing and complementary analytical tests that can be used alone or in combination, depending on the information needed and available laboratory instrumentation. The results obtained using these techniques for "global" glycosylation profile, N-glycans profiling, monosaccharides, and sialic acids determination are presented for the Trastuzumab (Herceptin)-humanized mAb produced in CHO.

Batch-to-batch N-glycosylation study of infliximab, trastuzumab and bevacizumab, and stability study of bevacizumab

“…To conclude, differences between 12 batches of infliximab, nine batches of trastuzumab and seven batches of bevacizumab were studied as changes in glycosylation profiles influence the safety, efficacy and quality of therapeutic glycoproteins 5. For example, changes in terminal galactosylation and/or sialylation affect the half-life and the efficacy (anti-inflammatory effect) of therapeutic glycoproteins,24 changes in core fucosylation may alter their anti-inflammatory effects,25 and changes in mannosylation can affect their half-life and cause off-target hepatic toxicity 26.…”

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

“…Given the potential of recombinant glycoproteins as therapeutic drugs and diagnostic reagents, the analysis of protein glycosylation has attracted much interest in recent years. − Glycotyping, a comprehensive and detailed characterization of the various glycoforms present in a glycoprotein, has been used to detect subtle yet biologically relevant differences in glycan composition of different batches of glycoprotein pharmaceuticals. , Such differences can affect their biological activities and influence drug safety, efficacy, and stability . The common practice to determine the glycan composition of recombinant or plasma-purified protein therapeutics involves the enzymatic release of the carbohydrate moiety with endoglycosidases (peptide N-glycosidase F or A), followed by the analysis and detection of either labeled or underivatized glycans. ,, …”

Sensitive, High Throughput Detection of Proteins in Individual, Surfactant-Stabilized Picoliter Droplets Using Nanoelectrospray Ionization Mass Spectrometry

“…We therefore addressed this challenge by studying the antibody Trastuzumab (Herceptin) in droplets. Trastuzumab (hIgG1) exhibits a degree of microheterogeneity as a result of post-translational glycosylation, however, a raw molecular weight has recently been calculated at 148 057 Da . This microheterogeneity of the glycoprotein sample should make the derivation of a molecular weight even more challenging from a single picodroplet.…”

“…Trastuzumab (hIgG1) exhibits a degree of microheterogeneity as a result of posttranslational glycosylation, however, a raw molecular weight has recently been calculated at 148 057 Da. 27 This microheterogeneity of the glycoprotein sample should make the derivation of a molecular weight even more challenging from a single picodroplet. Trastuzumab-containing droplets (12.5 μM, 410 pL, 92 μm diameter, ca.…”