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22 BioPharm International eBook July 2021 www.biopharminternational.com timeline required to develop a prod- uct-specific immunoassay; the use of a commercially available generic HCP- detection enzyme-linked immuno- sorbent assay (ELISA) kit is common practice from early-stage through late- stage viral vector product development. This approach poses a significant risk; the generic HCP antigen used to gen- erate antibodies for a generic ELISA k it may not provide accurate HCP quantitation because the actual HCP antigen in a viral vector product will, de-facto, be different from that used to develop the ELISA kit. As shown in Figure 1, after com- paring the HCP content of adeno-as- sociated virus (AAV) drug substance samples measured using a generic kit-based ELISA, the actual content determined by quantitative l iquid chromatography (LC)-mass spectrom- etry (MS) showed consistently higher HCP values across many lots than were observed using ELISA, indicat- ing generic HCP k its may in some cases significantly underestimate HCP content by a wide margin. The impact of this underestima- tion is especially relevant to high-dose viral vector programs. The root cause of these f indings can be explained as insuff icient coverage by the anti- bodies used in generic ELISA kits, a s d e mon s t r ate d b y 2 D s o d iu m dodecyl-sulfate polyacr ylamide gel elect rophoresis (SDS-PAGE) a nd Western Blot ( Figure 2). Analysis of the samples by mass spectrometry shows that most of the HCP load is driven by four proteins, accounting for 67.5% of the entire HCP load. Presumably, those proteins are not well-covered by correspond- ing antibodies in the generic ELISA kit, resulting in poor recognition and signif icant under-estimation of the dominant HCP impurities ( Figure 3). HCP content is a critical qualit y attribute highly relevant to the safety of a gene therapy product. Therefore, the analytical methods used in process development and quality control should be suitable for their intended use by providing accurate results. In cases Biopharmaceutical Analysis Impurity Analysis Figure 1. Host cell protein content in adeno-associated virus drug substance. Generic enzyme-linked immunosorbent assay (ELISA) kit versus mass spectrometry (MS). Nano-liquid chromatography-MS-based, label-free quantitative proteomic analysis. ELISA kit underestimates HCP content in drug substance by an average factor of 33. ELISA MS 0 500 1000 1500 2000 2500 DS sample 1 DS sample 2 DS sample 3 DS sample 4 DS sample 5 DS sample 6 DS sample 7 ELISA 28 48 40 40 63 69 74 MS 1257 2058 1898 1394 960 1095 2464 ELISA MS Abundance ppm (ng/mg) Figure 2. Relative host cell protein (HCP) load in adeno-associated virus drug substance (DS). 4 of 24 HCPs present in DS account for 67.5% of the total HCP load in DS samples. 14.3 8.1 17.8 2.3 0.8 6.2 2.0 3.4 0.5 2.0 0.5 4.3 0.9 0.2 0.1 0.2 0.1 0.2 25.1 0.1 10.3 0.1 0.3 0.1 Relative HCP Load 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 HCP Protein Relative HCP Load 1 14.3 2 8.1 3 17.8 4 2.3 5 0.8 6 6.2 7 2.0 8 3.4 9 0.5 10 2.0 11 0.5 12 4.3 13 0.9 14 0.2 15 0.1 16 0.2 17 0.1 18 0.2 19 25.1 20 0.1 21 10.3 22 0.1 23 0.3 24 0.1 ALL FIGURES COURTESY OF THE AUTHORS.