Host Cell Protein Detection & Mitigation
Recombinant therapeutics, including gene and cell therapies and vaccines are often contaminated with proteins from the host cell system from which they were produced. Called "host cell proteins" (HCP), these process-related contaminants can co-purify along with the therapeutic protein, and cause significant issues in terms of product safety and efficacy.
Haematologic Technologies offers Integrated HCP Services with a difference
HT offers a unique and comprehensive approach to biopharmaceutical industry to solve HCP challenges. Using both high resolution mass spectrometry (HRMS) and ELISA, HT provides phase-appropriate methods for HCP analysis of biotherapeutic products from early development through full cGMP release testing.
The best time to implement HCP analysis by HRMS is during process development: prevent HCP problems from happening vs. trying to go back and fix them after the process has been “locked down.” At HT, we have developed higher-throughput HRMS methods that can quickly and economically help companies purify their Product Protein.
Available services include:
- 1D/2D SDS-PAGE with gel band/spot excision and MS identification
- 1D/2D Western Blot for HCPs
- High Resolution Mass Spectrometry (HRMS) analysis for HCP identity and quantity in process intermediates and DS samples
- SEC-HPLC and collection of HMW impurities for HRMS analysis: this is often useful for determining which HCPs are “hitch-hikers”
- IEX-HPLC or RP-HPLC for impurity collection and HRMS identification
- Screen commercial HCP ELISA kits for appropriateness; validate commercial ELISA kits
- Develop, validate, and run ELISA for specific HCPs in a product
- Develop, validate, and run process-specific HCP ELISAs
- HRMS analyses to support ELISA development (characterization of the reference standard and HCP coverage)
- Data Independent Acquisition (DIA) HRMS methods
- LC-MRM targeted methods
- Identification of immunogenic HCPs in patients that have developed anti-HCP antibodies
Applicable drug substances
- Monoclonal antibodies
- Recombinant proteins: fusion proteins, chimeras, active enzymes, zymogens, etc.
- Gene and cell therapies
Why HCP testing? It’s All About Safety and Efficacy
Some HCP’s can reduce drug efficacy (e.g., glycosidases, proteases, lipases) or affect patient safety by triggering immunologic responses in patients.
High levels of HCPs can be detected by conventional methods such as SDS-PAGE (1D or 2D) or HPLC methods. Getting rid of those HCPs is somewhat straightforward. The challenge comes when one needs to get that final level of purity that is expected with today’s biopharmaceuticals. Much more sensitive methods are needed. However, none of the methods available today is a magic bullet – each has its own strengths and weaknesses, and so a combined approach is recommended.
The HCP ELISA is the workhorse and gold-standard in the biopharma world. Briefly, host cell proteins from a null cell line are injected into animals (goat, sheep, rabbit, etc.), and the antibodies generated against those HCPs are collected and purified and used in the ELISA. One of the main problems with this approach is that the HCP ELISA is immunologically weighted based on how those animals respond to the HCP antigens: some HCPs may generate lots of antibodies, but some may generate little or none. The ELISA result is therefore not strictly quantitative on an HCP-by-HCP basis, and the actual HCP profile of a sample is unknown. Despite this and other weaknesses, the HCP ELISA has several advantages: (1) high throughput, (2) the result is a simple single number that is conceptually easy (on the surface) to remember and use to track purification of the Product.
Mass Spectrometry (MS)
The power of MW is that it enables us to identify and quantify specific HCPs in samples. The proteins in a sample are digested into peptides, the peptides are separated by HPLC and injected into a mass spectrometer. MS analysis and the subsequent data processing enables one to identify and quantify what HCPs are in a sample. This level of detail is critical for rationally designing a purification process and insuring against being surprised by safety or efficacy problems during clinical trials.
Preventing HCP Problems During Process Development
The best way to avoid HCP problems is to purify them away as early as possible in the process. This is where MS analysis shines. Instead of basing process changes on an aggregate ELISA value or guessing at protein identities in gel bands or chromatograms, MS methods give the process development team the clarity and detail needed to rationally design the purification method based on HCP identity, MW, pI, and concentration. MS analysis enables the establishment of HCP clearance (on a protein-by-protein basis) at each step of the process. For example, suppose that as development and scale-up proceeds, the HCP ELISA value increases: is this increase caused by (a) more of the same HCPs in the product, or (b) new HCPs showing up in the product? The ELISA is incapable of determining the cause, whereas MS can provide the answer.