Host cell protein quantification by fourier transform mid infrared spectroscopy (FT‐MIR)

Abstract

Process development in up- and downstream processing requires enhanced, non-time-consuming, and non-expensive monitoring techniques to track product purity, for example, the level of endotoxins, viral particles, and host cell proteins (HCPs). Currently, HCP amounts are measured by laborious and expensive HCP-enzyme-linked immunosorbent assay (ELISA) assays best suited for measuring HCP amounts in the low concentration regime. The measurement of higher HCP amounts using this method requires dilution steps, adding dilution errors to the measurement. In this work we evaluated the suitability of attenuated total reflection spectroscopy for HCP quantification in process development, using clarified cell culture fluid from monoclonal antibody producing Chinese hamster ovary-cells after treatment with different polyelectrolytes for semi-selective clarification. Forty undiluted samples were chosen for multivariate data analysis in the middle infrared range and predicted HCP-values were in good agreement with results obtained by an ELISA-assay, suggesting the suitability of this new method for HCP-quantification. As this method is able to quantify HCP titers ranging from approximately at least 20,000-200,000 ng mL(-1), it is suitable especially for monitoring of process development steps with higher HCP concentrations, omitting dilution errors associated with ELISA assays.

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

“…Potential risks associated with specific HPCs may include immunogenicity, adjuvant activity, proteolytic activity, and direct biological activity of potent molecules (e.g., cytokines); examples of all of these are documented in the literature. [1][2][3][4][5] Currently, immunoassays are almost universally used to monitor total residual HCP levels in the industry (the use of Fourier transform mid infrared spectroscopy has also recently been reported 6 ), but whether specific, critical HCPs should be individually measured is an open question. 7,8 Monoclonal antibodies (mAbs) constitute a substantial portion of the biopharmaceutical industry's current biotherapeutics portfolio, and downstream purification of mAbs has evolved into platform processes that are highly similar across the pharmaceutical industry.…”

At-line mid infrared spectroscopy for monitoring downstream processing unit operations

“…The sensitivity of the model may also depend on the covered concentration range. The here presented models cover a relatively small concentration range between 700 to approximately 2000 ng ml −1 of HCPs, whereas previously reported models cover concentration ranges factor > 40 larger and exhibit limits of quantification of around 2000 ng mL −1 and higher [16]. Another parameter to be monitored was the amount of mAb aggr .…”

“…protein A chromatography which typically yields a purity of >98%, with residual HCP levels between hundreds and several thousands of ppm [22]. Additional better-suited applications include upcoming alternative means of protein purification such as precipitation, resulting in elevated HCP profiles [16,17]. It may also be useful for determining HCP levels in samples derived from chromatography runs in case the expected HCP levels are higher and meet the required amounts for IR-based quantification.…”

“…HCP levels in samples derived from Martillac were too low to be monitored by FT-MIR, thus model design for these samples was omitted. Yet, for samples from Darmstadt, three models could be obtained, based on previously published wavenumber ranges [16] as well as additional wavenumber ranges chosen after visual inspection for correlations between peak intensities within the IR spectra and HCP levels determined by ELISA. Model A was solely based on the following wavenumber ranges: 1557-1545, 1514-1505, 1424-1417, Despite using different mathematical pre-treatment steps ( Table 2), these models showed similar accuracies and were able to determine HCP levels in samples, covering a range between 700-3000 ng mL −1 .…”

“…Additionally, Capito et al showed the principal suitability of this technique to selectively quantify and monitor mAb amounts, host cell protein (HCP) levels and amount of aggregated antibodies (mAb aggr ) [16][17][18]. However, research studies have so far been limited to less common protein purification systems, using either semi-selective precipitation with copolymers to obtain varying amounts of the respective HCPs and mAbs, or spiking defined amounts of mAb aggr to non-aggregated mAb.…”