Abstract
Bispecific antibodies (bsAbs) are considered as an important class of biopharmaceutical drugs, with about 160 products in clinical trials. From an analytical point of view, the correct chain-association is one of the most critical challenge to monitor during bsAbs development and production. In the present study, a full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatography (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydrophilic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS). This analytical strategy was applied to Hemlibra® (emicizumab), which is certainly the most successful commercial bsAb to date. Using this strategy, it was possible to monitor the presence of mispaired bsAb species and detect and identify additional post-translational modifications (PTMs).
Highlights
With recent successes of therapeutic monoclonal antibodies for treatment of diseases such as arthritis, cancer, diabetes and cardio vascular diseases, the field of protein biopharmaceuticals has become one of the fastest growing and most innovative class of human thera peutics
A full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatog raphy (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydro philic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS)
One of the main goal of the CEX-MS analysis was to confirm the correct hetero-dimerization of the heavy chains (LC-HCFIXa-HCFX-LC format, having pI 6.9) and the absence of homo-dimers traces (LC- HCFXHCFX-LC, having pI 6.1, or LC-HCFIXa- HCFIXa-LC, having pI 8.3) [7]
Summary
With recent successes of therapeutic monoclonal antibodies (mAbs) for treatment of diseases such as arthritis, cancer, diabetes and cardio vascular diseases, the field of protein biopharmaceuticals has become one of the fastest growing and most innovative class of human thera peutics. The vast number of therapeutic applications results in a vast diversity within the bsAb family, literally referred as a zoo by Brinkmann & Kontermann, including more than 100 different combi nation of antigen-binding moieties and (homo/hetero) dimerization modules, classified based on their format (fragment-based, symmetric, and asymmetric) and their valency (number of binding sites, generally 1 + 1, 1 + 2, or 2 + 2) [2,3] This molecular (complex) variability arises from the need to develop bsAbs with desired specificity and functionality to serve diverse thera peutic applications, and from the need to meet developability criteria for fitting production and upstream/downstream processing [2].
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