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Abstract
Immunoglobulin characterization is of great interest due to their crucial role in the human immune response and their ability to be recombinantly produced and used as therapeutics. Their flexibility, particularly that of Immunoglobulin G (IgG), is enabled by the structural region known as the variable domain: a region for which each antibody clone has a different primary sequence that dictates the antigen and epitope the antibody will bind to. Immunoglobulin G also gains functional flexibility from the N-glycosylation site on the crystallizable fragment (Fc) domain on the constant region of the molecule. While the primary amino acid sequence of this domain is the same for all IgGs (of the same subtype), the N-glycans present at this site have been implicated in modulating the affect of that IgG on the immune response, specifically activation of the different cytotoxic pathways of the immune system. Here liquid chromatography mass spectrometry techniques for quantitation of IgG N-glycosylation, monitoring changes in IgG N-glycosylation, testing purity of biotherapeutic IgGs, and recombinant IgG translation fidelity are examined. Each technique seeks to improve upon current techniques by increasing the depth of information obtained from experiments, increasing reproducibility, and reducing the rate of false positives. These are facilitated by use of internal standards for quantitation, targeted acquisition methods, and leveraging predictive models for protein expression and chromatographic retention.