Tracking the Behavior of Monoclonal Antibody Product Quality Attributes Using a Multi-Attribute Method Workflow

Abstract

The multi-attribute method (MAM) is a liquid chromatography–mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.

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

“…[9][10][11][12][13][14][15] The time-course evaluation of trypsin digestion highlights the need of a desalting step for sample preparation in order to achieve fast and complete digestion (Figure 1). 10,15,20 The desalting step in a trypsin digestion may bring another source of variations and prevent high-throughput MAM testing and the full automation of MAM due to the many challenges of implementing desalting steps in an automated workflow. In additional, the desalting tips, plates or spin columns needed for both manual and automatic sample preparation may encounter supply chain issues for timely MAM release testing in commercial quality control labs.…”

“…21 To improve the digestion efficiency and throughput, a desalting step carried out by buffer exchange is often used in the sample preparation of MAM. [9][10][11][12][13][14][15] The extra desalting step brings challenges for high-throughput MAM testing with additional experimental variables, such as potential sample loss. 18 The desalting steps increase the difficulty of fully automating sample preparation for MAM, although recent progress has been made with bufferexchange plates and tips.…”

“…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.…”

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

“…Although MAM is not able to provide information related to conformational changes (e.g., unfolding, partial reduction or protein fragments), that can be obtained with weak anion or strong cation exchangers analysis [ 19 ]. Importantly, MAM has also the ability to search for sequence variants, critical for mAb biological function [ 11 , 20 , 21 ]. Therefore, by streamlining monitoring and quality control analysis, MAM has the potential to accelerate mAbs’ process development and manufacturing, contributing to reduce the risk of failure and costs associated with analytical characterization during bioprocess development, product release, and in-process control [ 6 , 9 , 22 ].…”