(T1030-08-52) Characterization of Host Cell Protein Impurity Coverage by a Process Specific Assay Kit for Its Suitability for Use during Late Phase Manufacturing of an Investigational Therapeutic Fc-Fusion Protein

Purpose: Host-cell proteins (HCP)are process-related impurities that need to be adequately removed during biologics manufacturing to minimize their potential impact to product quality, efficacy, and patient safety. Sandwich enzyme-linked immunosorbent assay (ELISA) is commonly used across product development lifecycle to monitor HCP downstream process clearance and residual HCP content in drug substance during (DS) release testing. It is a general regulatory expectation that process-specific HCP ELISA needs to replace generic ELISA method in late phase development to mitigate HCP detection risk. In this study, we aimed to characterize HCP coverage differences between process specific ELISA and commercial kit to evaluate the former’s suitability to replace the latter for residual HCP testing during late phase and commercial manufacturing of an investigational therapeutic Fc-fusion protein (TFFP).

Methods: The process-specific ELISA assay antibody’s coverage to the HCPs that potentially co-purify with the TFFP was compared side-by-side with the commercial kit antibody’s coverage by resolving the Cye fluorescent dye labeled mock HCPs obtained from a null cell culture on two-dimensional difference gel electrophoresis (2D-DIGE) gel and Western blotted using each antibody as primary antibody. The specificity and sensitivity of process specific ELISA in comparison with the commercial kit was also evaluated by the performance of each ELISA method in detecting HCPs in the process intermediates and DS of representative lots of TFFP during method validation and bridging. The downstream clearance of HCPs was also visually examined by 2D-DIGE. Additionally, proteomic LC-MS/MS method was used to identify individual HCPs in each process intermediate and assess the process clearance of HCPs during downstream processing to gain a holistic understanding of HCP expression profile and downstream clearance during the production of TFFP using commercial process.

Results: Process-specific anti-HCP antibody demonstrated broader coverage than the commercial kit antibody on 2D Western blots and the superiority of HCP coverage by the former was more substantial for proteins in the low molecular weight (LMW) and the basic region (high pI) of 2D Western blot images (Fig. 1). The process-specific assay’s broader HCP coverage was also confirmed by the improved sample linearity and increased detection sensitivity when bridging the two assays using TFFP process intermediates and DS (Fig. 2). Orthogonal LC-MS/MS characterization further confirmed the effective clearance of HCPs during TFFP downstream processing, with an average of 904 (n=3) HCPs being identified in the production HCCF and after affinity purification and two polishing chromatography steps only 1 HCP was consistently detectable in the DS with an estimated method detection sensitivity at 10 ppm (Fig. 3).

Conclusion: While both the process-specific ELISA and commercial kit methods have demonstrated its suitability in monitoring HCP downstream process clearance, the broader coverage and increased detection sensitivity and specificity from process-specific ELISA makes it a superior assay in mitigating HCP detection gap risk for commercial manufacturing of TFFP. The orthogonal characterization by LC-MS/MS and 2D-DIGE showing robust HCP downstream process clearance provided deeper understanding on the HCP clearance pattern and further confirmed the process specific assay’s suitability for HCP detection and measurement due to its high sensitivity and high throughput.

Figure 1. Side-by-side comparison of HCP coverage between a process specific assay anti-HCP antibody and commercial kit anti-HCP antibody by 2D-Western blots and 2D-DIGE.

Figure 2. Side-by-side comparison of HCP downstream process clearance as measured by commercial kit assay (red curve) and by process specific HCP ELISA (blue curve) showing increased detection sensitivity in all process intermediates and DS samples from 3 representative late phase manufacturing lots. AFFINITY PP = Affinity purification product; POLISHING 1P = polishing chromatography column 1 product; POLISHING 2P = polishing chromatography column 2 product; DS = drug substance.

Figure 3. TFFP HCP downstream process clearance as indicated by the decrease in the # of HCPs identified along the downstream purification process in each process intermediate by proteomics LC-MS/MS. AFFINITY PP = Affinity purification product; POLISHING 1P = polishing chromatography column 1 product; POLISHING 2P = polishing chromatography column 2 product; DS = drug substance.