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 labour-intensive 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-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.

Peptide mapping by liquid chromatography mass spectrometry (LC-MS) and the related multi-attribute method (MAM) are well-established analytical tools for verification of the primary structure and mapping/quantitation of co-and posttranslational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digestion is a key step in peptide mapping workflows, which traditionally is laborintensive, involving multiple manual steps. Recently, simple hightemperature workflows with automatic digestion were introduced, which facilitate robustness and reproducibility across laboratories. Here, a modified workflow with an automatic digestion step is presented, which includes a two-step digestion at high and low temperatures, as opposed to the original one-step digestion at a high temperature. The new automatic digestion workflow significantly reduces the number of missed cleavages, obtaining a more complete digestion profile. In addition, we describe how chromatographic peak tailing and carry-over is dramatically reduced for hydrophobic peptides by switching from the traditional C18 reversed-phase (RP) column chemistry used for peptide mapping to a less retentive C4 column chemistry. No negative impact is observed on MS/MS-derived sequence coverage when switching to a C4 column chemistry. Overall, the new peptide mapping workflow significantly reduces the number of missed cleavages, yielding more robust and simple data interpretation, while providing dramatically reduced tailing and carry-over of hydrophobic peptides.

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Peptide mapping of antibodies is an essential method to monitor peptide modifications in antibody lots that could affect the safety and efficacy of the product. Conventional protocols rely on protein digestion using proteases, such as trypsin, before mapping with mass spectrometry (MS). However, trypsin digestion may cause incomplete mapping of peptides, especially those that include highly hydrophobic peptides. Here, we show how pepsin can be used as an alternative and complementary protease for digestion that allows for improved sequence coverage, especially in proteins with highly hydrophobic regions. We also show that using guanidine hydrochloride (GuHCl) post-digestion improves peptide mapping results. Overall, these two methods—pepsin digestion and GuHCl post-digestion—can be used to provide more comprehensive antibody peptide maps, thereby enabling more thorough quality checking of biopharmaceutical products.