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Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: benefits of FAIMS separation and gas-phase fractionation DIA

ABSTRACT: In this study, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation prior to data dependent acquisition (DDA) and gas phase fractionation (GPF) prior to data independent acquisition (DIA) LC-MS/MS analysis. Native digestion followed by LC-MS/MS with FAIMS allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been applied to commercial DPs and demonstrate their ability to dig deeper into the HCP landscape with the identification of 60 and 67 HCPs, and accurate quantification of 29 and 31 of these impurities in nivolumab and trastuzumab respectively, with sensitivity down to the sub-ng/mg of mAb level

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus) Mus Musculus (mouse)

SUBMITTER: Corentin Beaumal

LAB HEAD: Christine Carapito

PROVIDER: PXD039585 | Pride | 2023-05-07

REPOSITORIES: Pride

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Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: benefits of FAIMS separation and gas-phase fractionation DIA

Project description:In this study, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation prior to data dependent acquisition (DDA) and gas phase fractionation (GPF) prior to data independent acquisition (DIA) LC-MS/MS analysis. Native digestion followed by LC-MS/MS with FAIMS allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been applied to commercial DPs and demonstrate their ability to dig deeper into the HCP landscape with the identification of 60 and 67 HCPs, and accurate quantification of 29 and 31 of these impurities in nivolumab and trastuzumab respectively, with sensitivity down to the sub-ng/mg of mAb level.

2023-05-07 | PXD039582 | Pride

Host Cell Protein Quantification Workflow Using Optimized Standards combined with Data-Independent Acquisition Mass Spectrometry

Project description:Monitoring of host cell proteins (HCPs) during the manufacture of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug product. ELISA assays are still the current gold standard for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) has emerged as an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography (LC)-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. In this study, we developed a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performance of the HCP Profiler solution was compared to more conventional standard protein spikes and the DIA approach was benchmarked against classical data-dependent acquisition (DDA) using samples collected at various stages of the manufacturing process. Finally, we further explored the possibility to use spectral library-free DIA. As a result, our method showed accurate and reproducible (coefficients of variation (CVs) < 10%) quantification of HCPs while reaching a sensitivity down to the sub-ng/mg mAb level.

2023-07-20 | PXD029305 | Pride

A Sensitive, Rapid, Robust and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development

Project description:There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels.1-6 In some cases, regulatory agencies require the identification as well as the quantification of HCPs in drug products for risk assessment, and this is an active and growing topic of conversation in industry and amongst regulators. In this study, we developed a sensitive, robust, and reproducible workflow for HCP detection and quantification in the significantly shorter turnaround time than previously reported using an Evosep ONE LC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Due to its fast turnaround time, this HCP workflow can be integrated into process development for the high-throughput (60 samples analyzed per day) identification of HCPs. The ability to rapidly measure HCPs and follow their clearance throughout the downstream process can be used to pinpoint sources of HCP contamination, which can be used to optimize biopharmaceutical production to minimize HCP levels. Analysis of the NIST monoclonal antibody (mAb) reference material using the rapid HCP profiling workflow detected the largest number of HCPs reported to date, underscoring an improvement in performance along with an increased throughput. The HCP workflow can be readily implemented and adapted for different purposes to guide biopharmaceutical process development and enable better risk assessment of HCPs in drug substances (DS) and drug products (DP).

2020-06-02 | PXD018427 | Pride

Optimized sample preparation, data processing of data-independent acquisition methods for the robust quantification of trace-level host cell protein impurities in drug products

Project description:In this study, we developed powerful and reproducible MS-based analytical workflows coupling optimized and efficient sample preparations, library-free DIA acquisition method and stringent validation criteria. The performances of several preparation protocols and DIA versus classical DDA were evaluated using a series of four commercially available Drug Products. Depending on the selected protocols, the user has access to different information: on the one hand, a deep profiling of tens of identified HCPs, and on the other hand an accurate and reproducible (CV<12%) quantification of major HCPs. Overall, a final global HCP amount of a few tens of ppm in these mAb samples was measured, while reaching a sensitivity down to the sub-ppm level. Thus, this straightforward and robust approach can be intended as a routine quality control for whichever Drug Products’ analysis.

2020-10-19 | PXD019668 | Pride

Coupling high-field asymmetric ion mobility spectrometry with capillary electrophoresis-electrospray ionization-tandem mass spectrometry improves protein identifications in bottom-up proteomic analysis of low nanogram samples

Project description:In this work, we pioneered the assessment of coupling high-field asymmetric waveform ion mobility spectrometry (FAIMS) with ultra-sensitive capillary electrophoresis hyphenated with tandem mass spectrometry (CE-MS/MS) to achieve deeper proteome coverage of low nanogram amounts of digested cell lysates. An internal stepping strategy using three or four compensation voltages (CVs) per analytical run with varied cycle times was tested to determine optimal FAIMS settings and MS parameters for the CE-FAIMS-MS/MS method. The optimized method applied to bottom-up proteomic analysis of 1 ng HeLa protein digest standard identified 1,314±30 proteins, 4,829±200 peptide groups, and 7,577±163 peptide spectrum matches (PSMs) corresponding to a 16%, 25%, and 22% increase, respectively, over CE-MS/MS alone, without FAIMS. Furthermore, the percentage of acquired MS/MS spectra that resulted in PSMs increased nearly 2-fold with CE-FAIMS-MS/MS. Our results also identified from 1 ng HeLa protein digest without any prior enrichment, 76±9 phosphopeptides, 18% of which were multiphosphorylated. These results represent a 46% increase in phosphopeptide identifications over the control experiments without FAIMS yielding 2.5-fold more multiphosphorylated peptides.

2023-05-10 | PXD034255 | Pride

Dilute-and-Shoot Analysis of Therapeutic Monoclonal Antibody Variants in Fermentation Broth: A Method Capability Study

Project description:Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient thera-peutic agents for various diseases, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drug. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N-glycosylation and truncation variants of the expressed mAb can be identified at the intact protein level. The molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Simultaneously, absolute quantification of mAb content in fermentation broth yielded concentrations of 67 and 2578 ng.mL-1 at the beginning and end of fermentation, respectively. The whole workflow features excellent robustness as well as retention time and peak area stability of 0.3% and 4.9% RSD. Additional enzymatic removal of N-glycans enables determination of mAb glycation levels, which are subsequently considered in relative N-glycoform quantification to correct for isobaric galactosylation. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production as well as targeted biosimilar development.

2019-11-12 | PXD011017 | Pride

Multiplex secretome engineering enhances recombinant protein production and purity

Project description:Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

2020-03-19 | GSE144624 | GEO

Probing the Sensitivity of the Lumos Mass Spectrometer using a Standard Reference Protein in a Complex Background

Project description:we determined the sensitivity of the Lumos mass spectrometer using the NIST monoclonal antibody reference material at various concentrations to detect its peptides in a background of S. cerevisiae whole cell lysate, which was kept at a constant concentration.

2018-09-12 | PXD009441 | Pride

Reutilizing MetaMorpheus as an open tool for monoclonal antibody characterization

Project description:NIST mAB standard in intact, top down and multi-protease digested form for the testing of new tools for mAB characterization.

2023-03-14 | MSV000091487 | MassIVE

MAM Nature Protocol 2022-chimeric IgG1mAb DP spiked with HCPs

Project description:Peptide mapping experiment on chimeric Ig1 mAb drug product spiked with spiked with different levels of HCPs, cathepsin and rHLPL, at 3 different levels, 10, 100, and 1000ppm (three replicates are provided for each spiked level as 1, 2, and 3)

2022-02-03 | MSV000088775 | MassIVE

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