Host cell proteins (HCPs) are residual protein impurities expressed along with the desired biologics product. Removing HCPs in the final drug substance (DS) to an acceptable level sometimes can be challenging because of the diverse nature of the HCPs and their potential affinity for the intended product, especially for non-monoclonal antibody (non-mAb) products due to the lack of affinity purification process. Failure to remove residual HCPs sufficiently during downstream processing can affect product quality, safety, and efficacy, or even induce adverse events in patients. Therefore, HCPs are considered an obligatory critical quality attribute (CQA) that should be minimized throughout the drug development and commercialization lifecycle.

The enzyme-linked immunosorbent assay (ELISA) is considered the industry standard for detection and quantitation of HCP due to many advantages, such as high sensitivity and selectivity, ease of use, high throughput, and versatility¹. However, ELISA cannot afford identification and quantitation information for individual HCPs. In addition, it may miss non-immunogenic HCPs. In the past decade, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as an orthogonal approach for HCP characterization². LC-MS/MS technology enables identification and quantitation of individual HCPs, which makes risk assessment of such HCPs possible to advance product and process understanding.

However, LC-MS/MS technology has its own challenges. A general LC-MS/MS method utilizes a bottom-up proteomics strategy, where both intended product and HCPs are digested together to yield peptides. All resulting peptides are then separated by LC and subsequently detected by a research grade mass spectrometer via both MS and MS/MS. Hence, this general workflow can potentially suffer from lower sensitivity and smaller dynamic range, especially considering what is required for characterizing HCPs.

To increase sensitivity and dynamic range of the LC-MS/MS technology, additional approaches were developed to either deplete the intended product and/or enrich the HCPs in the past several years. For example, a native digest protocol³ could be implemented to overcome the significant dynamic range requirement and afford capability to reliably detect residual HCPs at low ppm level. A slightly revised native digestion protocol has been reported to be applicable to AAV-based gene therapy product⁴. Furthermore, chromatography-based technologies can be applied to separate the intended product and HCPs, where fractions are collected and analyzed separately via LC-MS/MS for HCP analysis^5,6. Moreover, affinity-based technologies can be utilized to enrich certain types of HCPs, such as enzymes that could potentially induce polysorbate degradation⁷. Last but not least, different strategies can be implemented on LC-MS/MS instrumentation to gain better sensitivity in HCP detection, such as nanoflow setups, data-independent acquisition (DIA, as opposed to data-dependent acquisition DDA), and a targeted analysis method such as multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM)^8,9.

With the technological advancements mentioned above, more HCPs at low- or even sub-ppm level can be readily detected and quantified in an industrial laboratory. These technologies may not be able to support routine quality control (QC); however, they are beneficial to support product and process understanding under a non-GMP environment. This information can provide valuable insight on bioprocess development toward optimal HCP clearance, as well as formulation development toward better control of enzymatic activities that could cause change in stability or aggregation propensity of the intended biologics products.

On the other hand, analysts should be cautious about the capabilities of LC-MS/MS when it comes to reporting these HCPs at extremely low level. It will always be a case-by-case scenario, depending on not only the identity and level of such HCPs, but also other factors that are illustrated in previous publications^10,11. A patient-centric approach should always be considered as a priority during this cross-functional risk assessment activity. Risks might be different between an enzyme that causes degradation of the intended biological products and an HCP that elicits an immune response in patients, even when they are present at the same level in the final products. Risk assessment and management has been a hot topic at the BEBPA conferences via podium presentations and/or workshops in the past years, which are helpful to emphasize the importance of such topic to a broader audience. More importantly, cooperative discussions between industry peers and health authorities are prompted to enable better product and process understanding to ensure safety in patients.

Join us in Lake Bled, Slovenia for BEBPA’s 2025 Hybrid Host Cell Protein Conference May 28-30, 2025 to hear more talks on this topic and join the conversation. View the full agenda here. Register online before ticket prices increase!