Quantitative Host Cell Protein Analysis Using Two Dimensional Data Independent LC–MS^E

Abstract

Host cell proteins (HCPs) are bioprocess-related impurities arising from cell-death or secretion from nonhuman cells used for recombinant protein production. Clearance of HCPs through downstream purification (DSP) is required to produce safe and efficacious therapeutic proteins. While traditionally measured using anti-HCP ELISA, more in-depth approaches for HCP characterization may ensure that risks to patients from HCPs are adequately assessed. Mass spectrometry methods provide rationale for targeted removal strategies through the provision of both qualitative and quantitative HCP information. A high pH, low pH, reversed-phase data independent 2D-LC-MS(E) proteomic platform was applied to determine HCP repertoires in the Protein A purified monoclonal antibody (mAb) samples as a function of culture harvest time, elution buffer used for DSP and also following inclusion of additional DSP steps. Critical quality attributes (CQAs) were examined for mAbs purified with different Protein A elution buffers to ensure that the selected buffers not only minimized the HCP profile but also exhibited no adverse effect on product quality. Results indicated that an arginine based Protein A elution buffer minimized the levels of HCPs identified and quantified in a purified mAb sample and also demonstrated no impact on product CQAs. It was also observed that mAbs harvested at later stages of cell culture contained higher concentrations of HCPs but that these were successfully removed by the addition of DSP steps complementary to Protein A purification. Taken together, our results showed how mass spectrometry based methods for HCP determination in conjunction with careful consideration of processing parameters such as harvest time, Protein A elution buffers, and subsequent DSP steps can reduce the HCP repertoire of therapeutic mAbs.

Proteomics in biomanufacturing control: Protein dynamics of CHO‐K1 cells and conditioned media during apoptosis and necrosis

“…For proteomics screening of cells and their conditioned media a high pH/low pH reversed phase 2D‐nano‐LC − MS E method was used which includes label‐free Hi3 quantitation and was previously introduced and validated for the determination of HCPs in purified mAb samples (Doneanu et al, ; Farrell et al, ; Silva et al, ; Silva, Gorenstein, Li, Vissers, & Geromanos, ). MS E is a multiplexed data independent acquisition mode.…”

“…LC‐MS based “omics”‐approaches are increasingly used to gain advanced understanding of biomanufacturing processes such as to study host cell proteins (HCPs), productivity during process scale‐up or cellular response to altered media composition (Farrell et al, ; Gao et al, ; Liu et al, ). The recent elucidation of the CHO cellular and secreted proteome further facilitates research in this area (Baycin‐Hizal et al, ; Kumar et al, ).…”

A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance

“…One DIA method, 19 MS E , has been coupled with two dimensional liquid chromatography (2D-LC) to identify and quantify low abundance HCPs, using the "Top 3 00 label-free approach (MS signal response for the three most intense tryptic peptides) for protein quantitation. 7,8,10,20,21 However, 2D-LC analysis may have reproducibility challenges and the nano-LC often used in the second dimension for improved sensitivity is not always robust. In addition, its 10-20 hours analysis time/sample makes it challenging to analyze multiple samples from in-process pools using the 2D-LC-MS/MS approach to provide real time support and quick response for addressing potential HCP concerns.…”

The growing role of two-dimensional LC in the biopharmaceutical industry

“…As part of an effort to help users understand the conditions under which 2D-LC provides more resolving power than 1D-LC separations for peptide analyses, the Heinisch group recently demonstrated that the peak capacity of 2D-LC separations is superior to that of 1D-LC separations for analysis times longer than about five minutes [18]. In the area of Host Cell Protein (HCP) analysis, Donenau and coworkers have demonstrated the use of heartcutting 2D-LC [19,20] and offline comprehensive 2D-LC [21] to detect trace-level peptide markers of HCPs. This is very important to quality assurance for biotherapeutic mAbs.…”