Targeted Host Cell Protein Quantification by LC–MRM Enables Biologics Processing and Product Characterization

Abstract

Multiple reaction monitoring (MRM) is a liquid chromatography−mass spectrometry (LC−MS) based quantification platform with high sensitivity, specificity, and throughput. It is extensively used across the pharmaceutical industry for the quantitative analysis of therapeutic molecules. The potential of MRM analysis for the quantification of specific host cell proteins (HCPs) in bioprocess, however, has yet to be well established. In this work, we introduce a multiplex LC−MRM assay that simultaneously monitors two high risk lipases known to impact biologics product quality, Phospholipase B-like 2 protein (PLBL2) and Group XV lysosomal phospholipase A2 (LPLA2). Quantitative data generated from the LC−MRM assay were used to monitor the clearance of these lipases during biologics process development. The method is linear over a dynamic range of 1 to 500 ng/ mg. To demonstrate the fitness for use and robustness of this assay, we evaluate a comprehensive method qualification package that includes intra-and inter-run precision and accuracy across all evaluated concentrations, selectivity, recovery and matrix effect, dilution linearity, and carryover. Additionally, we illustrate that this assay provides a rapid and accurate means of monitoring high risk HCP clearance for in-process support and can actively guide process improvement and optimization. Lastly, we compare direct digestion platforms and affinity depletion platforms to demonstrate the impact of HCP−mAb interaction on lipase quantification.

Identification and characterization of a residual host cell protein hexosaminidase B associated with N‐glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product

“…To compliment the shotgun proteomics approach for quantification, a targeted LC–MS‐based MRM approach with spike‐in stable isotope‐labeled internal standards was developed for process and product characterization. 19 The MRM data provide more accurate measurement of the absolute level of HEXB in mAb1 DS. The HEXB level from MRM assay provides reference for acceptance ranges of HEXB in biologics development.…”

“…Although this approach may suffer accuracy and precision limitations due to the nature of the quantification approach, it provides a quick and fit‐for‐purpose readout of HCP identification and relative quantification. To compliment the shotgun proteomics approach for quantification, a targeted LC–MS‐based MRM approach with spike‐in stable isotope‐labeled internal standards was developed for process and product characterization 19 . The MRM data provide more accurate measurement of the absolute level of HEXB in mAb1 DS.…”

“…Process intermediate samples for mAb‐1 were tested by MRM using the same approach for lipase quantification. 19 The total protein concentrations of all the samples were determined by measuring the absorbance at 280 nm using a UV–Vis spectrometer. Samples were all diluted to 5 mg/ml with PBS buffer before sample preparation.…”

Identification and Characterization of a Residual Host Cell Protein Hexosaminidase B Associated with N-glycan Degradation During the Stability Study of a Therapeutic Recombinant Monoclonal Antibody Product

“…To compliment the shotgun proteomics approach for quantification, a targeted LC-MS based MRM approach with spike-in stable isotope-labeled internal standards was developed for process and product characterization. 17 The MRM data provides more accurate measurement of the absolute level of HEXB in mAb1 DS. The HEXB level from MRM assay provides reference for acceptance ranges of HEXB in biologics development.…”

“…A affinity chromatography-purified product (PAP), filtered neutralized viral inactivation product (FNVIP), second column-purified product, third column-purified product, and various DS from Process 1 and Process 2, were tested by MRM as we did for lipase quantification. 17 The total protein concentrations of all the samples were determined by measuring the absorbance at 280 nm using a UV-Vis spectrometer. Samples were all diluted to 5 mg/mL with PBS buffer before sample preparation.…”