Enhancing the multi-attribute method through an automated and high-throughput sample preparation

Abstract

The multi-attribute method (MAM), a recent advance in the application of liquid chromatography-mass spectrometry within the pharmaceutical industry, enables the simultaneous monitoring of multiple product quality attributes in a single analytical method. While MAM is coupled with automated data processing and reporting, the sample preparation, based on proteolytic peptide mapping, remains cumbersome and low throughput. The standard sample preparation for MAM relies on protein denaturation, reduction, and alkylation prior to proteolytic digestion, but often a desalting step is required to maintain enzymatic activity. While most of the sample preparation can be automated on a standard robotic liquid handling system, a streamlined approach for protein desalting and temperature modulation is required for a viable, fully automated digestion. In this work, for the first time, a complete tip-based MAM sample preparation is automated on a single robotic liquid handling system, leveraging a deck layout that integrates both heating and cooling functionalities. The fully automated method documented herein achieves a high-throughput sample preparation for MAM, while maintaining superior method performance. Abbreviations: MAM: multi-attribute method; PQAs: product quality attributes; CE: capillary electrophoresis; IEX: ion-exchange chromatography; HILIC-FLR: hydrophilic interaction liquid chromatography coupled to a fluorescence detector; RP-LC/UV: reversed-phase liquid chromatography coupled to a UV detector; MS: mass spectrometry; NPD: new peak detection; GdnHCl: guanidine hydrochloride; TIC: total ion current; pAb: polyclonal antibody; IgG: immunoglobulin G; DTT: dithiothreitol; IAA: iodoacetic acid; TFA: trifluoroacetic acid; A280: absorbance at 280 nm wavelength; 96MPH: 96-channel multi-probe head; CPAC: Cold Plate Air Cooled; HHS: Hamilton Heater Shaker; DWP: Deep-Well Plate; PCR: Polymerase Chain Reaction; NTR: Nested Tip Rack; Met: methionine; Trp: tryptophan; N-term pQ: N-terminal glutamine cyclization; Lys: lysine; PAM: peptidylglycine α-amidating monooxygenase; G0F: asialo-, agalacto-, bi-antennary, core substituted with fucose; G1F: asialo-, mono-galactosylated bi-antennary, core substituted with fucose; G2F: asialo-, bi-galactosylated bi-antennary, core substituted with fucose; G0: asialo-, agalacto-, bi-antennary; Man5: oligomannose 5; Man8: oligomannose 8; TriF: asialo-, tri-galactosylated tri-antennary, core substituted with fucose.

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.…”

“…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. [9][10]13 Additionally, trypsin is reported to have side activities, such as trace level of chymotrypsin activity, which may result in many unidentified peaks in the MAM. 22 Collectively, the trypsin digestion efficiency, specificity and extra buffer exchange step bring additional complexity and variability for MAM.…”

“…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.…”