Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS

Abstract

Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies (mAbs) remains a challenge. Since residual HCPs might affect product stability or safety, constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg. The current standard analytical approach for this procedure is based on ELISA; however, this approach only measures the overall HCP content. Therefore, the use of orthogonal methods, such as liquid chromatography-mass spectrometry (LC-MS), has been established, as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present. In the present study, a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation, in combination with a data-independent acquisition (DIA) LC-MS analysis, was established. Employing the same instrumental setup commonly used for peptide mapping analysis of mAbs allows for its quick and easy implementation into pre-existing workflows, avoiding the need for dedicated instrumentation or personnel. Thereby, quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.

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

“…We [38, 39] and others [33, 66] have previously demonstrated the benefits of using DIA methods and MS2‐based quantification to accurately monitor HCP impurities, and it is clearly confirmed in the current study. While DDA allowed the identification of the highest number of protein groups, namely 135, against 115 in DIA (Figure 1D), DIA allowed quantifying 100 HCPs (222 peptides) against 68 in DDA (121 peptides).…”

“…We [38,39] and others [33,66] have previously demonstrated the benefits of using DIA methods and MS2-based quantification to accurately monitor HCP impurities, and it is clearly confirmed in the current study.…”

Mimotope peptide modified pompon mum-like magnetic microparticles for precise recognition, capture and biotransformation analysis of rituximab in biological fluids

“…S6 and S7 . These critical biotransformation should be carefully monitored during quality control, biosimilar development or treatment cycle to guarantee efficacy and safety 44 , 45 , 46 , 47 , 48 .

Figure 7 Analytical process for non-Hodgkin's lymphoma patient serum and example data of identified and confirmed peptides using ZenoTOF TM 7600 system.

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Espectrometria de massas: uma ferramenta versátil para a caracterização de anticorpos monoclonais terapêuticos

“…Dado que espectrômetros de massa tem a capacidade de detectar quantidades muito reduzidas de analitos de interesse, especialmente quando operando em alta resolução, torna-se viável a identificação dos peptídeos derivados dessas proteínas residuais. Proteínas de células CHO foram identificadas em instrumento com analisador híbrido quadrupolo-orbitrap mostrando proteínas presentes em determinadas condições de cultivo utilizando aquisição independente de dados (STRASSER et al, 2021) e também em aquisição dependente de dados (KREIMER et al, 2017).…”