Multi attribute method implementation using a High Resolution Mass Spectrometry platform: From sample preparation to batch analysis

Abstract

Quality control of biopharmaceuticals such as monoclonal antibodies (mAbs) has been evolving and becoming more challenging as the requirements of the regulatory agencies increase due to the demanding complexity of products under evaluation. Mass Spectrometry (MS)-based methods such as the multi-attribute method (MAM) are being explored to achieve a deeper understanding of the attributes critical for the safety, efficacy, and quality of these products. MAM uses high mass accuracy/high-resolution MS data that enables the direct and simultaneous monitoring of relevant product quality attributes (PQAs, in particular, chemical modifications) in a single workflow, replacing several orthogonal methods, reducing time and costs associated with these assays. Here we describe a MAM implementation process using a QTOF high resolution platform. Method implementation was accomplished using NIST (National Institute for Standards and Technology) mAb reference material and an in-process mAb sample. PQAs as glycosylation profiles, methionine oxidation, tryptophan dioxidation, asparagine deamidation, pyro-Glu at N-terminal and glycation were monitored. Focusing on applications that require batch analysis and high-throughput, sample preparation and LC-MS parameters troubleshooting are discussed. This MAM workflow was successfully explored as reference analytical tool for comprehensive characterization of a downstream processing (DSP) polishing platform and for a comparability study following technology transfer between different laboratories.

Qualification of Identity, Quality-Attribute Monitoring and New Peak Detection in An Improved Multi-Attribute Method with Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

“…The stability of deamidated and oxidated peptides have been a technical challenge for peptide mapping based MS analysis. 4,19,[25][26] The baseline level of those PTMs are generally low (< 2%).…”

Improvements on Sample Preparation and Peptide Separation for Reduced Peptide Mapping Based Multi-Attribute Method analysis of Therapeutic Monoclonal Antibodies Using Lys-C Digestion

“…24 This critical deamidation site is often detected only on missed cleavage peptide resulting in a larger peptide or often undetected completely by Lys-C or trypsin-based reduced peptide mapping because of the hydrophilic nature of the VSNK peptide. [16][17]25 For example, VSNK was one of the few undetected peptides from an optimized LC-MS/MS peptide mapping protocol for NIST mAb with close to 97% sequence coverage. 25 Additional protein digestion with chymotrypsin or Glu-C was needed to detect this site.…”

“…20 Trypsin or a trypsin/Lys-C mixture are the most frequently used proteolytic enzymes for high-resolution MS based MAM. [9][10][11][12][13][14][15][16][17][18][19] Besides its superior availability and affordability, trypsin digestion also offers some major advantages, such as an optimal average peptide length of ~14 amino acids, 21 rendering tryptic peptides well-suited for MS analysis. Trypsin digestion efficiency, however, is less resistant to denaturants, such as guanidine hydrochloride, which is often used to denature therapeutic proteins.…”

“…[2][3][6][7][8] As such, the multi-attribute method (MAM), including PQA monitoring and purity testing through new peak detection (NPD), serve as an orthogonal method and could eventually replace several traditional HPLC and CE-based assays currently used for characterization and release of biopharmaceuticals. [9][10][11][12][13][14][15][16][17][18][19] One of the challenges for the implementation of MAM in Good Manufacturing Practice (GMP)/ quality control (QC) areas is the streamlined sample preparation, which enables robust highthroughput testing while controlling assay variability. Reduced peptide mapping-based MAM (RPM-MAM) involves enzymatic digestion of proteins into peptides for MS detection.…”

“…21 The applications of using Lys-C digestion for high-resolution MS based MAM for PQA monitoring and NPD has not been reported. [9][10][11][12][13][14][15][16][17][18][19] Beyond sample preparation and protein digestion, peptide separation is another critical aspect of MAM development with the goal of capturing as many PQAs as possible. In the past, the changes observed on heat-stressed mAbs by ion exchange chromatography (IEX) were difficult to trace back to amino acid modifications by RPM.…”