Addressing Acid-Catalyzed Deamidation and the Solubility of Hydrophobic Peptides in Multi-Attribute Method Workflows.

Recently, we introduced an optimized and automated Multi-Attribute Method (MAM) workflow, which (a) significantly reduces the number of missed cleavages using an automated two-step digestion procedure and (b) dramatically reduces chromatographic peak tailing and carryover of hydrophobic peptides by implementing less retentive reversed-phase column chemistries. Here, further insights are provided on the impact of postdigest acidification and the importance of maintaining hydrophobic peptides in solution using strong chaotropic agents after digestion. We demonstrate how oxidation can significantly increase the solubility of hydrophobic peptides, a fact that can have a profound impact on quantitation of oxidation levels if care is not taken in MAM workflows. We conclude that (a) postdigestion acidification can result in significant acid-catalyzed deamidation during storage in an autosampler at 5 °C and (b) a strong chaotropic agent, such as guanidine hydrochloride, is critical for preventing loss of hydrophobic peptides through adsorption, which can result in (sometimes extreme) biases in quantitation of tryptophan oxidation levels. An optimized method is presented, which effectively addressed acid-catalyzed deamidation and solubility of hydrophobic peptides in MAM workflows.

Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides.

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 post-translational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digestion is a key step in peptide mapping workflows, which traditionally is labor-intensive, involving multiple manual steps. Recently, simple high-temperature 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.

Characterization of Protein Glycoforms at Intact Level by Orbitrap Mass Spectrometry.

Intact mass analysis of proteins is simple, fast, and specific, and it effectively provides structural insight into the proteoforms or variants of the analyzed protein. For instance, the multiple glycoforms of recombinant monoclonal antibodies can be effectively analyzed by intact mass spectrometry (MS). A recent development in the Orbitrap technology has made this platform particularly well suited for analysis of large intact biomolecules, and here we describe procedures for performing intact mass analysis of intact glycoproteins using the Orbitrap platform, with the aim of identifying and quantitating the glycoforms. Emphasis is placed on the analysis of biopharmaceutical immunoglobulins (IgGs), but the procedures can be extended to other glycoproteins as needed.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes.

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. Graphical abstract.