Host cell proteins (HCPs) are impurities derived from the host organism used in the production of genetically engineered biopharmaceuticals. Their removal is crucial for the safety and efficacy of the final product, as they can provoke unwanted immune responses and/or have undesired biological/enzymatic activities [1-4]. The sandwich enzyme-linked immunosorbent assay (ELISA) remains the most widely used method for HCP quantification throughout biopharmaceuticals product development lifecycle due to its high sensitivity, throughput, and selectivity in complex sample matrices. HCP ELISA typically employs polyclonal antibodies to capture and detect a heterogeneous mixture of HCPs that may co-purify with the therapeutic protein. These antibodies are often purified from antisera generated from animal immunization using either specific or platform null cell antigens that represent HCPs co-produced with the product during upstream manufacturing process [5-8]. Commercially available generic HCP ELISA kits are also widely used to monitor HCP clearance during early phase development of biopharmaceuticals but are mostly replaced by upstream platform-specific or process-specific assays during late phase development to mitigate HCP detection risk, as often required by regulatory agencies [5, 6].

Regardless the assay of choice, regulatory requirements and best practices mandate that anti-HCP antisera (or purified polyclonal antibodies) used in the ELISA be qualified for their ability to detect process-specific HCP impurities through coverage assessment. Traditionally, this involves two dimensional (2D) SDS-PAGE gels and Western blots analysis to determine the estimated percentage of HCP impurities recognized by the antisera, commonly known as 2D coverage assessment [9, 10]. In addition to traditional methods, affinity-based techniques such as immunoprecipitation (IP), antibody affinity extraction (AAE), or direct ELISA enrichment, combined with advanced analytical methods like liquid chromatography-tandem mass spectrometry (LC-MS/MS) or 2D differential in-gel electrophoresis (2D-DIGE), are increasingly used to evaluate HCP coverage [11-14]. These methods involve immobilizing ELISA antibodies to a solid matrix, binding HCPs, and analyzing the bound HCPs in comparison with the loading HCPs through either 2D-electrophoresis (2D-E) or proteomics LC-MS/MS.

While either approach can provide a scientifically sound assessment of HCP coverage, simplifying HCP coverage based on isoelectric point (pI) and molecular weight (MW) distribution in 2D and solely relying on the percentage values calculated from either the number of protein spots from 2D SDS-PAGE coupled with Western blots detection, or the number of HCP identifications (IDs) from LC-MS/MS before and after affinity enrichment often do not reveal the full picture of coverage, and sometimes can generate misleading information on antibody reagents quality. For example, an HCP ELISA with a broad pI and MW coverage in 2D with reactivity more evenly spread-out would be good in detecting trace levels of residual HCPs in DS but could fall short in detecting certain hitch-hiking HCPs or other significantly over-expressed HCPs, due to antigen excess [2, 15]. In contrast, a process-specific HCP ELISA with antibody reagents that heavily target the prominent HCPs may turn out to be less effective than platform HCP ELISA with a broad 2D coverage if those prominent HCPs are easy-to-remove during downstream purification process and not present in DS[16]. Therefore, a thorough evaluation of coverage data in three dimensions (3D) is critical to better understand the HCP detection risk associated with ELISA antibody reagents. The added 3^rd dimension is mostly focused on the understanding of relative abundance and binding affinity differences of individual HCP to the polyclonal antibodies and can be evaluated through one or more of the following angles:

• The relative abundance differences of HCP spots detected in 2D-DIGE or HCP IDs detected by proteomic LC-MS/MS when comparing the reference standard used in the ELISA assay to process-specific HCPs expressed along with the therapeutic protein. Generally, the more similar the relative abundance levels of individual HCPs between the HCP standard used in the assay and the co-expressed HCPs from a specific manufacturing process, the better quantitation accuracy can be achieved on total HCPs. The similarly of HCP profile in HCP standard and manufacturing process-specific HCPs can also be examined through the dilution curve shape (i.e., parallelism) in ELISA.
• Immunoaffinity of reactive HCP spots on 2D Western blots or relative abundance changes of individual HCPs through affinity enrichment under native conditions. This will provide information on the estimated weight of each detected HCP in the total signals generated and help understand the depth of antibody reagents coverage for individual HCPs.
• ELISA sample dilutional linearity and other performance characteristics such as the accuracy of HCP standard spike recovery. A good spike recovery and sample dilutional linearity often indicate that the antibody reagents have good coverage in 3D. Both antibody excess (prozone) or antigen excess (post-zone) could lead to sample dilutional non-linearity issues impacting assay performance.
• HCP downstream process clearance patterns and residual HCP compositions (ID and relative abundance) in the drug substance. ELISA antibody reagents don’t need to have 100% coverage to detect all HCPs since many of HCPs with similar biochemical properties are removed from downstream purifications in a similar way. However, HCP ELISA measurement will be more accurate when the residual HCP profile in a test sample matches the HCP profile in the HCP standard, although this is rarely the case. Generally, HCP ELISA with antibody reagents demonstrating a majority coverage in 2D fit for its intended purpose of monitoring HCP clearance through downstream purification processes and measuring the residual levels in DS for controlling manufacturing consistency but the quantitation results will vary depending on the antibody reagents used in the ELISA.

To learn more on why a holistic evaluation of HCP coverage in 3D is crucial and how the 3D coverage evaluation can be done with latest technologies, please join us in Lake Bled, Slovenia for BEBPA’s 2025 Hybrid Host Cell Protein Conference May 28-30, 2025.