Impact of Glycosylation on the Comparability of the Higher-Order Structures in Idursulfase by Hydrogen-Deuterium Exchange Mass Spectrometry.

Characterization of the higher-order structures in idursulfase (iduronate-2-sulfatase, I2S) has been accomplished through the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). The method has over 97% sequence coverage, including seven of the eight glycosylation sites, and has been used to study the impact of glycosylation on backbone proton exchange. In addition, the method adapted a well-used biophysical spectra comparison method (similarity scoring) to define quantitative acceptance criteria for analytical comparability of different batches of drug substance as well as samples with modulated glycans. Differences in the HDX profile were induced by enzymatic removal of terminal sialic and phosphate groups on negatively charged glycans. These differences were mapped to the crystal structure and demonstrated synergistic HDX changes focused around the N221 and N255 glycosylation sites, which contain mannose-6-phosphate motifs important for I2S uptake into cells.

Assessing in vivo dynamics of multiple quality attributes from a therapeutic IgG4 monoclonal antibody circulating in cynomolgus monkey.

Characterization of biopharmaceutical proteins and assessment and understanding of the critical quality attributes (CQAs) is a significant part of biopharmaceutical product development and is routinely performed in vitro. In contrast, systematic analysis of the quality attributes in vivo is not as widespread, although metabolism and clearance of multiple variants of therapeutic proteins administered to non-human primates and human subjects may have a different impact on safety, efficacy and exposure. The major hurdles of such studies are usually sample availability and technical capability. In this study, we used affinity purification coupled with liquid chromatography and mass spectrometric analysis of the digested protein for consistent and simultaneous detection of the full amino acid sequence of a therapeutic IgG4 monoclonal antibody, MAB1. This methodology allowed us to assess in vivo changes of all sequence-related modifications and quality attributes of MAB1 over the duration of a preclinical pharmacokinetic study in cynomolgus monkeys.