HCPs are process-related impurities that may co-purify with biopharmaceutical drug product. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, difficult to purify and/or degrade both product molecules and excipients. Why should the biopharmaceutical industry worry about these high-risk host cell proteins? What approach could be taken to deal with these high-risk HCPs?

Intravitreal injection of therapeutic antibodies into the eye has proven to be a remarkable success for treatment of diseases such as neovascular age-related macular degeneration and diabetic macular edema. Here we will discuss one program where initial large animal studies with a monoclonal antibody fragment identified severe inflammatory reactions that correlated with high levels of host cell protein in the preparation. In later stages of manufacturing development, host cell protein was reduced by an order of magnitude; this was accompanied by an elimination of the inflammatory reaction and advancement of the protein into the clinic.

What do we do when we have two differing total HCP results by two different HCP ELISAs? We present a case study where we characterized the specific HCPs and the HCP ELISA reagents to select an HCP ELISA that best ensures patient safety. Multiple complementary methods were used during the characterization: HCP clearance from in-process pools and dilution linearity analysis by ELISAs, antibody coverage by 2D SDS-PAGE/Western Blot and LC-MS/MS, immunoaffinity enrichment followed by LC-MS/MS, and a suite of other analytical methods. Based on the lessons learned in this case study, we recommend applying a diverse HCP characterization toolbox to select the HCP ELISA that is sensitive to the changes in the total HCP levels in the lifecycle of the product, thereby ensuring patient safety.

Host cell proteins are process related impurities that need continuous monitoring for clearance through process purification steps. Choosing a suitable ELISA assay to monitor HCP levels coupled with a control strategy are key components of establishing long term assay support for product release. This presentation will focus on evaluation strategies to assess the suitability of commercially available ELISA assays. Approaches towards bridging between commercial kits will also be explored.

CHO host-cell protein (HCP) levels in mAb process streams are substantially reduced by Protein A chromatography and are extremely low in the final drug substance, but the number of HCPs involved at each step may be surprisingly high. This presentation will compare HCP removal patterns in several mAb processes, which reveal important similarities but also distinctive differences. Further investigations to explain these patterns are discussed as well.

Sandwich immunoassays are typically used to measure HCP levels.  Readouts in these assays are dependent on the anti-HCP antibodies.  Since HCPs with low immunogenicity may not be detectable in these immunoassays, additional orthogonal methods are needed.  A case study will be presented where a DS had no detectable results in our platform HCP immunoassay, yet LC-MS showed ~100 ppm levels of HCP X.  We investigate why our HCP assay does not detect HCP X and describe our mitigation strategy for HCP X. 

Residual host cell proteins (HCPs) in biotherapeutic products can pose a risk to patients and product quality and therefore must be monitored and controlled.  Mass spectrometry has become an increasingly common approach to identify and quantitate HCPs.  Here we present an update on USP’s efforts to develop documentary standards outlining best practices for HCP analysis and physical standards to support identification and quantitation of high risk and high abundance HCPs.

Host cell proteins (HCPs) are monitored to ensure product purity, stability, efficacy, and safety. Using untargeted mass spectrometry (MS) on samples from throughout the purification process, high HCP sequence coverage has been obtained. This allows an increased depth in understanding for many HCPs, including processing and purification of processed forms. The processing of a variety of HCPs will be discussed, along with the potential impacts these proteins might have on product quality.

HCP ELISAs are typically used during the purification process to ensure removal of HCPs and to demonstrate process consistency and final drug substance purity. Unfortunately, the ELISA does not inform which HCPs are present or to which HCPs the assay reacts.  Orthogonal Antibody Affinity Extraction and Mass Spectrometry method provides enhanced coverage analysis and identifies HCPs in the Drug Substance.

Upstream and downstream process development during early phases of clinical trials will likely require process improvements.  To avoid costly GLP toxicology studies and support comparability studies due to process changes, we have used quantitative proteomics to identify and quantify host cell protein impurities from microbial expression systems.  This strategy, as part of a comparability protocol, has facilitated the clinical development of a recombinant malaria vaccine from phase 1 to phase 2 without a new toxicology study.

Monitoring of host cell proteins (HCPs) is a crucial step during the development of biosimilars to prove product stability and consistency to the reference product. ELISA is the standard method for HCP detection. However, there is currently a growing need to provide robust and sensitive LCMS analysis of HCPs as complimentary method. Here, a case study is shown demonstrating the supportive role of LCMS for HCP analysis during biosimilar development.

To improve the sensitivity and reliability for HCP detection, we developed a high pH-low pH two-dimensional reversed phase LC-MS/MS approach in conjunction with offline fraction concatenation.  For the first time, we demonstrate an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for HCP detection. The method performance has high impact on pharmaceutical company practices in using advanced LC-MS/MS technology to ensure product quality and patient safety.