Interlaboratory Evaluation of a User-Friendly Benchtop Mass Spectrometer for Multiple-Attribute Monitoring Studies of a Monoclonal Antibody

Abstract

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC–MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC–MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC–MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC–MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC–MS platforms for high-throughput determination of major CQAs in a regulated environment.

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

“…Between 0.1 and 2% TFA is routinely used for acidification after digestion in MAM workflows. 13,15,18,19 Avoidance of high pHcatalyzed deamidation is a focus area the MAM community often use to support the use of a postdigest acidification step. However, deamidation may also take place at low pH, a topic much less covered in the MAM literature.…”

“…Between 0.1 and 2% TFA is routinely used for acidification after digestion in MAM workflows. ,,, Avoidance of high pH-catalyzed deamidation is a focus area the MAM community often use to support the use of a postdigest acidification step. However, deamidation may also take place at low pH, a topic much less covered in the MAM literature. , We investigated the in-use sample stability (up to 40 h in autosampler at 5 °C) after acidification with 1% TFA as described previously, as well as acidification with 1% formic acid or no acidification (pH 6.5).…”

“…16,17 Typically from 0.1 to 2% trifluoroacetic acid (TFA) is used for acidification in MAM workflows. 13,15,18,19 At neutral and basic pH, Asn deamidation proceeds through a succinimide intermediate to form Asp and isoAsp, a well known and major degradation pathway for many biopharmaceutical proteins and a typical focus area in MAM sample preparation workflows. 20,21 However, at low pH (<3), deamidation can occur through direct acid-catalyzed hydrolysis to form Asp only, so excessive acidification can potentially be an issue in MAM workflows.…”

“…Chemical degradation of proteins, such as deamidation, is not per se a focus area of proteomics, and as such, detailed focus has not been on avoiding these modifications during sample preparation. One example of a commonly used legacy step is the acidification of samples after proteolytic digestion, to inactive protease and prevent high pH-induced deamidation, the latter being a key focus area of MAM communities. , Typically from 0.1 to 2% trifluoroacetic acid (TFA) is used for acidification in MAM workflows. ,,, At neutral and basic pH, Asn deamidation proceeds through a succinimide intermediate to form Asp and isoAsp, a well known and major degradation pathway for many biopharmaceutical proteins and a typical focus area in MAM sample preparation workflows. , However, at low pH (<3), deamidation can occur through direct acid-catalyzed hydrolysis to form Asp only, so excessive acidification can potentially be an issue in MAM workflows. , In our laboratory, observations indicated potential deamidation stability issues with acidified samples after prolonged storage in an autosampler (unpublished observations). For the above reasons and because our current digestion workflow employs an enzyme immobilized on magnetic beads, which are removed after digestion (i.e., no enzyme inactivation is required after digestion), we decided to do an in-depth evaluation of the impact and necessity of the post-digest acidification step.…”

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

“…The latter method is now commonly referred to as multi-attribute method (MAM). This innovative approach allows for the monitoring of multiple CQAs in a single assay, streamlining biopharmaceutical production, from research and development to the QC environment [25,26]. It stands out as one of the most sensitive methods, offering site-specific identification and quantitation of post-translational modifications (PTMs) in biotherapeutics [27,28].…”