Exact Sequencing of Novel Therapeutic Antibodies and QC analysis of Commercial Antibodies Using High Resolution Mass Spectrometry and Error-Tolerant Search

David Morgenstern, University of Haifa, Haifa, Israel
Marshall Bern, Protein Metrics, San Carlos, Usa
Yong Kil, Protein Metrics, San Carlos, Usa
Walter Bogdanoff, School Of Engineering , Uc Santa Cruz, Santa Cruz, Usa
Rebecca Dubois, School Of Engineering, Uc Santa Cruz, Santa Cruz, Usa
Chris Becker, Protein Metrics, San Carlos, Usa
Beatrix Ueberheide, Proteomics Resource Center, Nyu Medical Center, New York, Usa

Over the last 30 years, immunotherapy has become one of the pillars of modern medicine. With recent advances in Next-Generation sequencing, identification of the hundreds of antibodies in the pipeline considerably accelerated. This resulted in an increase in treatments aimed at targeting wide array of diseases – autoimmune, cancer, transplant rejection and others, taking an increasing share of the physician’s medicinal toolkit. Unfortunately, the development of therapeutic antibodies is still an expensive and difficult challenge; identification of the accurate sequence of the specific antibodies is expensive, time-consuming and requires a significant investment of man-hours – either for the discovery stage or the validation stage.

Advances in mass spectrometry and processing power provide an opportunity for faster and cheaper antibody identification and product QC. Here we present the sequencing of a rare anti-viral antibody obtained from a defunct white cell population, using a small quantity (<100ug) of starting material. Using an iterative error-tolerant search, we provide an accurate determination of the sequence of the antibody. Moreover, we identify a rare N-glycosylation on the heavy chain of the Fab fragment.

Using the same approach, we also present a QC analysis of Bevacizumab. We have analyzed two commercial versions of the antibodies, by Roche and Genentech, along with three biosimilars. We present the difference between the samples in terms of sequence variations and post translational modifications, both qualitatively and quantitatively. We observe variations in the glycosylation patterns, as well as deamidations, oxidation and glycations. Importantly, we have identified mutations of Cysteines which should have adverse structural effect on the antibodies

Taken together, our results present our ability to provide accurate sequence information of novel antibodies as well as comprehensive QC analysis of biosimilars using protein mass spectrometry, at a fraction of the cost of the current NGS-based analyses, and in greater detail.