Automated, Quantitative Capillary Western Blots to Analyze Host Cell Proteins in COVID-19 Vaccine Produced in Vero Cell Line

Abstract

Background/objectives: Host cell protein (HCP) content is a major attribute for biological and vaccine products that must be extensively characterized prior to product licensure. Enzyme Linked Immunosorbent Assay (ELISA) and Mass Spectrometry (MS) are conventional methods for quantitative host cell protein analysis in biologic and vaccine products. Both techniques are usually very tedious, labor-intensive, and challenging to transfer to other laboratories. In addition, the ELISA methodology requires 2D SDS PAGE and 2D western blot antibody reagent validation to establish reagent coverage. This reagent coverage provides a rather weak link that is currently accepted, as the western blot is run under denaturing conditions and the ELISA is run under native conditions. Simple Western™ is a relatively new, automated, capillary western blot-based technology that allows for the separation, blotting, and detection of proteins. But, unlike traditional western blots, Simple Western™ is quantitative, allowing for the quantification of HCP content in biologic and vaccine samples. Antibody reagent validation is much more straightforward, as the reagent coverage can be directly linked between the 2D methodology and Simple Western™, as they are both run under denatured and reduced conditions.

Methods: Herein we describe the development of a capillary western blot method to quantify the HCP content in samples generated using a Vero cell line for the production of an investigational live virus vaccine candidate (V590) for Coronavirus Disease-2019 (COVID-19). The HCP content in COVID-19 vaccine samples was evaluated using three methods: the new capillary western, the gold standard ELISA, and SDS-PAGE.

Results/conclusions: Strong agreement was observed in the HCP content data between the capillary western and SDS PAGE methods, whereas the ELISA HCP data were outliers, suggesting that the capillary western is generating HCP concentrations closer to the true concentration. This is the first report of using capillary western technology in analyzing HCP in vaccine samples.

Keywords: COVID-19; Simple WesternTM; V590; capillary western; host cell protein (HCP).

Figure 1

Example electropherogram traces and their peak area integration showing the broad MW range of HCP present in Vero cell in the (A) Vero HCP reference standard material at 25 μg/mL and (B) V590 COVID-19 UFP sample.

Figure 2

(A) Electropherograms of serial dilution of reference standards (purple: 3.20 μg/mL, Green: 6.25 μg/mL, Blue: 12.5 μg/mL, Red: 25.0 μg/mL and Black: 50.0 μg/mL); (B) the HCP standard curve from 3.20–50.0 μg/mL with r² > 0.990.

Figure 3

Comparison between HCP level and S protein production during (A) viral growth in bioreactor from 0 to 3 dpi; (B) HCP concentration level during V590 COVID-19 purification process; and (C) the percentage of HCP level clearance from HVF to DS as compared to the S protein production.

Figure 4

HCP electropherograms of each V590 COVID-19 purification step from HVF to DS showing the HCP MW distribution profiles across the purification process. The HCP electropherogram profiles during viral growth in bioreactor are similar to HVF.

Figure 5

The SDS PAGE of V590 COVID-19 purification samples showing the rVSV purity increases from HVF to UFP (see Table 1). The DS was not included in % purity calculation since DS contains rHSA.

Figure 6

HCP correlations among three different assays: (A) SDS PAGE versus SW (R² = 0.92); (B) ELISA versus SW (R² = 0.60); and (C) SDS PAGE versus ELISA (R² = 0.36).

Figure 7

SDS PAGE profile for visual comparison between UFP sample and HCP reference standard (RS), loaded according to the measured HCP concentrations by SDS PAGE, SW, and ELISA for two different samples: lot 4 and lot 8 (from Table A1).