Toward a Better Understanding of Bioassays for the Development of Biopharmaceuticals by Exploring the Structure-Antibody-Dependent Cellular Cytotoxicity Relationship in Human Primary Cells.

Pharmaceutical manufacturing relies on rigorous methods of quality control of drugs and in particular of the physico-chemical and functional characterizations of monoclonal antibodies. To that end, robust bioassays are very often limited to reporter gene assays and the use of immortalized cell lines that are supposed to mimic immune cells such as natural killer (NK) cells to the detriment of primary materials, which are appreciated for their biological validity but are also difficult to exploit due to the great diversity between individuals. Here, we characterized the phenotype of the peripheral blood circulating cytotoxic cells of 30 healthy donors, in particular the repertoire of cytotoxic markers, using flow cytometry.

The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange.

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure-function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g.

Effects of sialic acid linkage on antibody-fragment crystallizable receptor binding and antibody dependent cytotoxicity depend on levels of fucosylation/bisecting.

 Fragment crystallizable (Fc) glycosylation of immunoglobulin G-type monoclonal antibodies applied to therapeutic applications is regarded a critical quality attribute and can influence bioactivity, pharmacokinetics and/or immunogenicity/safety. Investigating the impact of certain Fc N-glycans is therefore of importance to assess its criticality for a therapeutic product. This has been done for N-glycan types like fucosylation, galactosylation or sialylation.

Oxidation of M252 but not M428 in hu-IgG1 is responsible for decreased binding to and activation of hu-FcγRIIa (His131).

Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP).

In vitro glycoengineering of IgG1 and its effect on Fc receptor binding and ADCC activity.

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species.