BEBPA Blog

Host cell proteins (HCPs) are a complex blend of various proteins that need to be depleted during the production of biotherapeutics. Any residual HCPs can pose diverse risks to quality, safety, and developability of a drug. There is manufacturing and clinical history indicating certain classes of HCPs which have been problematic, e.g. HCPs which,

1. Persist and become difficult to eliminate due to specific binding to the target molecule. Examples include Clusterin ¹ or PLBL2 ² binding to monoclonal antibodies.
2. Potentially cause adverse effects after drug administration due to their inherent bioactivity, such as cytokines like CCL2 (formerly MCP-1), which can exhibit effects even at very low concentrations ³.
3. Cause severe process / developability risks, as in case of proteases causing degradation of the active pharmaceutical ingredient (API) itself ⁴, or impacting stability via degradation of excipients like polysorbates used in drug product formulations, as seen with esterases ⁵.
4. Trigger immune responses against the HCP itself ⁶ or increase immune response against the API ⁷, posing a possible safety issue.

Standard HCP ELISA techniques (as described in USP 1132) may adequately detect these impurities; however, they only report a cumulative signal of the detected HCPs. Therefore it is deemed beneficial to apply orthogonal characterization in support of the ELISA e.g. using LC-MS based analytics (as described in USP 1132.1) also allowing for identification of residual HCPs. Hence, targeted analytics might become necessary when problematic HCPs are identified, to support process development, monitor depletion, and potentially even execute release analytics.

Analytical approaches for single HCPs include immunoassays such as single HCP ELISA, targeted LC-MS-based methods, as well as enzymatic assays to detect proteases or esterases. Each approach offers advantages and disadvantages including development timelines, sensitivity, throughput, and readiness for quality control (QC), etc.

• Single HCP ELISAs are considered well-established and economically beneficial. They are considered robust, deliver throughput, and are mostly straightforward in terms of development and validation. They are ready for quality control; however, they require the availability of antibodies and standards, which are often not easily accessible or need to be created first.
• Targeted LC-MS: techniques such as Multiple-Reaction Monitoring (MRM) are more and more established, deliver throughput, can be established relatively quickly by an expert. However, LC-MS instrumentation and expertise are not always easily at hand, especially not for QC purposes.
• Enzymatic assays: These not only monitor the impurity but also monitor the related activity. This is certainly beneficial as not every enzyme detected is truly active, and therefore the actual cause of any observed degradation. However, this technique is still relatively new for HCPs, and generic assay formats mostly lack the sensitivity to track trace amounts of specific enzymes.

Furthermore, specific sample preparation methods such as immunoaffinity based techniques, or the use of activity-based probes ⁸ are to be mentioned. These can be utilized to enrich certain HCPs increasing their detectability. Especially in the case of enzymes like esterases or proteases, minute trace amounts that are not detectable with common methods might still cause degradation of the API during long-term stability.

To reduce or prevent the risk for HCP related issues early on, more sophisticated phase appropriate development strategies for biotherapeutics are beneficial. These systematically incorporate advanced analytical characterization, e.g. like orthogonal LC-MS analytics at specific development checkpoints. This allows for detection of HCP related issues early on and can consequently enable process optimization, guided by more targeted analytics, to eliminate the respective HCPs before true issues arise, also reducing the need for their routine monitoring or release analytics later. Such strategies seek to achieve a balance between effort, time, and acceptable risk. This is a critical assessment to make as not all HCPs detected at trace amounts pose a clear risk, while others as listed above can clearly impact manufacturability, stability and patient safety. Within this context it is to be mentioned that assessing potential risks associated with HCPs can be complex and requires for a lot of expertise. When evaluating process risks, compliance related aspects, and most importantly safety risks cross-departmental considerations are often necessary. Also, these risk evaluations rarely yield straightforward conclusions, mostly due to the lack of information as well as the lack of appropriate tools especially for judging safety related aspects. Examples giving some guidance on risk assessments for HCP can be found in literature ^{9, 10}.

Topics such as development, implementation, and maintenance of both existing and novel HCP analytical methods, risk assessment and risk management for HCPs, strategies regarding analytics and process development, as well as approaches to reduce, deplete, or prevent problematic HCPs, are examined in depth during the annual BEBPA HCP workshop. This workshop facilitates comprehensive discussions, allowing a broad exchange of ideas and company perspectives, as well as providing insights into health authority expectations.