Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclo
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RESEARCH PAPER
Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes Silvia Millán-Martín 1 & Craig Jakes 1,2 & Sara Carillo 1 & Tom Buchanan 3 & Marc Guender 4 & Dan Bach Kristensen 5 & Trine Meiborg Sloth 5 & Martin Ørgaard 5 & Ken Cook 6 & Jonathan Bones 1,2 Received: 5 May 2020 / Revised: 9 June 2020 / Accepted: 7 July 2020 # The Author(s) 2020
Abstract Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labourintensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-tocharge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LCMS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.
Keywords Inter-laboratory study . Peptide mapping . Monoclonal antibody . Post-translational modifications (PTMs) . Trypsin digestion . Method transferability
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02809-z) contains supplementary material, which is available to authorized users. * Jonathan Bones [email protected] 1
Characterisation and Comparability Laboratory, National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co., Dublin A94 X099, Ireland
2
School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4 D04 V1W8, Ireland
3
Thermo Fisher Scientific, Tudor Rd, Runcorn WA7 1TA, UK
4
Thermo Fisher Scientific, Reinach TechCenter, Neuhofstrasse 11, 4153 Basel, Switzerland
5
Symphogen, Pederstrupvej 93, 2750 Ballerup, Denmark
6
Thermo Fisher Scientific, Stafford House, 1 Boundary Park, Hemel Hempstead HP2 7GE, UK
Peptide mapping is commonly used in the biopharmaceutical industry to confirm that the d
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