Multi-attribute method performance profile for quality control of monoclonal antibody therapeutics

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

“…Trypsin is the most widely used enzyme for RPM-MAM using high-resolution MS in the literature thus far. [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.…”

“…[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.…”

“…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.…”

“…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.…”

“…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.…”