A Robust Purity Method for Biotherapeutics Using New Peak Detection in an LC–MS-Based Multi-Attribute Method

Abstract

New peak detection (NPD), as part of the LC–MS-based multi-attribute method (MAM), allows for sensitive and unbiased detection of new or changing site-specific attributes between a sample and reference that is not possible with conventional UV or fluorescence detection-based methods. MAM with NPD can serve as a purity test that can establish whether a sample and the reference are similar. The broad implementation of NPD in the biopharmaceutical industry has been limited by the potential presence of false positives or artifacts, which increase the analysis time and can trigger unnecessary investigations of product quality. Our novel contributions to the success of NPD are the curation of false positives, use of the known peak list concept, pairwise analysis approach, and the development of a NPD system suitability control strategy. In this report, we also introduce a unique experimental design utilizing sequence variant co-mixes to measure NPD performance. We show that NPD has superior performance relative to conventional control system methods in the detection of an unexpected change as compared with the reference. NPD is a new frontier in purity testing that reduces subjectivity, need for analyst intervention, and potential for missing unexpected product quality changes.

Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control

“…Sadek et al proposed an improved NPD strategy to curate false positives, such as adducts, 43 using a “known peak list” concept, and the implementation of an NPD system suitability control strategy. Sequence variant co-mixes were also used to measure the performance of the NPD function.…”

“…Sequence variant co-mixes were also used to measure the performance of the NPD function. 43 Such strategies would reduce or avoid false detection of NPD in QC and mitigate the risk of creating time-intensive discrepancy investigations or unnecessary batch rejection.…”

“…MAM including TAQ for seven attributes and NPD using the parameters described in our recent work 43 was successfully validated. Workflows, infrastructure readiness, expert knowledge, lessons learned, and validation results will be leveraged for future GMP applications of MAM.…”

Targeted CQA analytical control strategy for commercial antibody products: Replacing ion-exchange chromatography methods for charge heterogeneity with multi-attribute monitoring

“… 16–23 Although there has been technical and logistical progress using the multi-attribute method, implementation in commercial good manufacturing practice (GMP) labs is currently limited. 16 , 24–30 Alternative MAM approaches using MS in QC have also been designed and implemented. 31–35 We recently presented a targeted MAM method using the QDa mass spectrometer that was co-validated across 6 labs worldwide to monitor several product-specific CQAs and determine the identity of a bispecific antibody.…”

Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies

“…The most commonly used protease for conventional antibodies is trypsin, because it produces peptides in the size range that is most efficient for peptide identification via MS analysis [ 23 , 26 ]. Our MAM platform method uses trypsin as a digestion enzyme [ 27 ]. However, alternative protein digestion using different enzymes might be more desirable for specific applications, in order to obtain complete sequence coverage [ 28 , 29 , 30 ].…”