Host cell proteins (HCPs) are impurities present in all therapeutics derived from biological sources, and as a critical quality attribute (CQA), they must be characterized in detail and controlled through the manufacturing process and in the final drug product.
The individual protein contaminants that make up the HCP profile may vary significantly among individual biologic products including cell and gene therapies and expression systems, even for very closely related molecules. As such, the HCP profile must be analyzed to identify even low levels of potentially concerning species in a biologic drug.
To ensure consistency of product manufacture and shelf-life stability, and to minimize potential adverse clinical reactions, significant attention must be paid to identifying HCPs that remain in a biologic product following purification. Once identified, HCPs are often monitored and the levels controlled by process development decisions, to consistently yield a safe and efficacious product.
While ELISA methods have historically been the main approach to detecting total HCP content, LC-MS based HCP detection methods have increasingly become an expected orthogonal standard in successful biologic development.
As a modern approach with advanced instrumentation, HCP profiling by LC-MS provides comprehensive identification and relative quantitation of these impurities down to low single-digit ppm. LC-MS analysis of HCPs has quickly developed into a set of approaches that can be performed to profile the diverse set of species and target quantitation of specific components. HCP characterization can be conducted at an early stage to assess the profile quickly and then maybe applied with greater rigor at a later stage to support BLA/regulatory filings.
The most basic method framework typically involves subjecting the sample to enzymatic digestion, followed by LC separation of the resulting peptide fragments with high-resolution MS detection. The MS data is then analyzed to enable identification and quantitation of HCP impurities. A comparison of HCP profiles from multiple lots can be used to assess process consistency and differences in profiles from samples generated using different process parameters to provide information that guides decision making in process development.
To completely identify the HCPs in any given test article, a combination of two MS data acquisition settings (methods) may be required. Data-dependent MS acquisition targets known peptides for identification from a pre-existing library is used because it is fast, robust, and sensitive, which is well-matched for monitoring impurities found at low levels. Data independent acquisition (DIA) is used to identify untargeted HCPs, and the results of DIA can be verified and provide quantitation using parallel reaction monitoring (PRM).
To obtain accurate information using LC-MS profiling of HCPs to guide decision making depends on a few key aspects. We take a rigorous approach to ensure high-quality results for all projects through:
HCP profiling is applied to a wide range of biologic products, from antibodies to enzymes to gene/cell therapies, which are manufactured using diverse cell types. There are substantial differences in the possible HCP impurities derived from different host cell sources, and we have developed complete peptide libraries for common expression systems, including CHO, HEK, and E.coli. We have also worked with our clients on unique host cell systems to develop customized, corresponding libraries.