Monitoring process-related impurities in biologics-host cell protein analysis

Abstract

During biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC-MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC-MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC-MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC-MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.

Keywords: ELISA coverage analysis; ELISA-MS; Host cell protein analysis; Liquid chromatography mass spectrometry; Process-related impurities.

Fig. 1

Objectives of HCP assays for monitoring HCPs in biologics. The assay should measure the majority of proteins in the early process, i.e., most of the HCPs that could potentially end up in the final product as well as the small amount of residual HCPs in the purified drug substance [8, 12]. In case of a process failure, e.g., a leaking chromatography column, the HCP assay should be able to detect the changed HCP profile. In case of process change, the chosen HCP assay should be capable of measuring any new HCPs

Fig. 2

Selecting an HCP monitoring method. (a) Commercial ELISA kits developed from generic strains are widely applied in early drug development stages. Commercial ELISA kits may be used for licensed pharmaceuticals on the market if the assay has been validated with process-specific performance. (b) If no commercial ELISA kit with sufficient HCP coverage is available, it is recommended to develop a product-specific ELISA kit or to use a platform ELISA kit. During the production of the product-specific HCP antibodies, it is important to select and characterize the antigen used for immunization and to consider the species immunized with HCP antigens. (c) Whether using a commercial, platform, or product-specific HCP-ELISA, it is necessary to do a fit-for-purpose validation to the actual process and product, including choosing an HCP standard representative for the samples, checking for quantitative dilutional linearity, and determining sensitivity and the limit of quantification (LOQ) for the drug substance samples, and to characterize the critical reagents: the HCP antigen and the HCP-specific antibodies. The HCP antibody’s ability to cover the HCPs in the process must be evaluated by an HCP coverage analysis. The most common coverage methods are 2D-PAGE and Western blotting; immunoaffinity–chromatography(IAC) and 2D-PAGE (2D-DIGE); and Immunocapture followed by LC–MS. (d) If no HCP-ELISA kit shows sufficient coverage of HCPs, LC–MS is recommended as an orthogonal method to HCP analysis by ELISA. HCP analysis by LC–MS provides identification and quantification of the individual HCPs, as well as the total HCP amount. When using LC–MS for HCP analysis, it is important to do a fit-for-purpose validation of the assay reproducibility, as the analysis is composed of multiple steps and a complex analysis workflow, as well as to qualify the robustness and sensitivity of the chromatography and MS system. This also includes characterization of the critical reagents such as qualification of standard proteins for quantification, i.e., intact proteins spiked-in, in known amounts into each sample

Fig. 3

HCP clearance during the downstream manufacturing process involving six purification steps. The process HCP data are shown for three PPQ batches, PPQ1–3. a The total number of HCPs throughout the process. b and c Clearance of two major HCPs, showing their amount in parts per million (nanograms (ng) HCP per milligram (mg) total protein). Note the difference in the scales