Host Cell Protein Analysis
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 safety and efficacy problems.
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 Drug Substance or Drug Product samples
- SEC-HPLC and collection of HMW impurities for HRMS analysis: this is often useful for determining which HCPs are “hitch-hikers”
- Screen commercial HCP ELISA kits for appropriateness; confirm with HRMS as an orthogonal method
- Validate commercial HCP 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)
- LC-MRM targeted methods for specific HCPs
- 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 therapies
Why HCP testing? It’s All About Safety and Efficacy
HCP’s can reduce drug efficacy (e.g., glycosidases, proteases, lipases), trigger immunologic responses in patients, or the biological activity of an HCP may cause an adverse event.
For a review of recent HCP history in the clinic, see the following article co-authored by K. Van Cott, who runs Haemtech's HRMS HCP analysis group: Experience with host cell protein impurities in biopharmaceuticals
There is no such thing as the "perfect HCP detection method." Each has its own strengths and weaknesses, and so a combined approach is recommended. The core methods used today are HCP ELISA and mass spectrometry.
The HCP ELISA is the workhorse and gold-standard in biopharma. 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 production of 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.
High Resolution Mass Spectrometry (LC-MS/MS)
The power of mass spectrometry 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/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 develop a robust process where the clearance of HCPs has been validated for each column. Mass spectrometry gives the process development team the clarity and detail needed to rationally design the purification method based on HCP identity, MW, pI, and concentration. Mass spectrometry enables the establishment of HCP clearance (on a protein-by-protein basis) at each step of the process.
At Haemtech, we have developed highly efficient LC-MS/MS workflows that can provide answers quickly and economically. We have honed these methods over the last decade, and we do not sacrifice quality and the stringency of data analysis. Our specialty is working with companies to rapidly diagnose their HCP problems so they can be solved. The days of relying on gels, blots, and ELISAs are nearing their end. Contact us to see how we can help you quickly get over your HCP problems and start your clinical trials with a high quality product.
The Rich History of our HCP Projects
Many of our HCP projects at Haemtech are problem-solving projects. We specialize in working with startup companies who do not have the resources for HCP ELISA and mass spectrometry analysis. We also work with companies that are using a CDMO to develop the process, but the CDMO does not have the bandwidth to solve a particularly sticky HCP problem. We've also worked with companies with commercially licensed products and suddenly they have an HCP problem show up. In all these cases, we have seen over and over that complete reliance on traditional analytical biochemistry or even HCP ELISAs can be a dead-end. Only mass spectrometry can provide the insight needed to diagnose the problem, and then help solve the problem. Here are some example HCP projects we have worked on:
- In a commercially licensed product, the process-specific HCP ELISA signal started increasing with every new lot that was produced until finally there was an out-of-specification. What columns in the purification process were failing? Or was there a problem in the bioreactor? Was this ELISA signal increase caused by new HCPs showing up in the product? Or was the ELISA signal increase caused by higher levels of known HCPs in the product? The ELISA was incapable of answering these questions, whereas we used mass spectrometry to answer the question within two weeks. If mass spectrometry had been used earlier in the troubleshooting process, many lots and a lot of money could have been saved.
- An early-development product had highly inconsistent animal model results. We used mass spectrometry to identify a protease in the product that was responsible for stability problems. The purification group was able to focus their efforts on removing this protease early in the downstream process.
- An early-development glycoprotein had inconsistent glycosylation profile from batch to batch. We used mass spectrometry to identify glycosidases that were responsible for glycan degradation. The purification group was able to look up the pI and optimal pH of these enzymes and re-design the purification process to limit enzymatic activity and then remove these HCPs.
- A late-stage development product started having apparent proteolytic degradation problems, but it was inconsistent from one batch to the next. We analyzed process intermediate samples from multiple lots, filtered the HCP lists for proteases, and we were able to help the client identify which protease was responsible. In this case, it turned out to be a zymogen that sometimes got activated, and the mass spectrometry analysis was able to detect zymogen vs. active protease.
- Chimeric and highly engineered fusion proteins seem to have serious HCP hitch-hiker problems. We have extensive experience with numerous products like this, and we have seen a wide array of chaperones and other HCPs that tend to co-purify with these non-natural proteins.
- We have had several HCP projects where the endogenous version of the product protein was co-purified. In one case, commercial HCP ELISAs were virtually useless for tracking HCP levels in the products. In another case, the endogenous protein was present as an activated enzyme, and posed a potential safety risk to patients. In these cases, where there was >90% sequence identity between the recombinant Product vs. endogenous HCP, only mass spectrometry could tell the difference and quantitate the HCP.
- We have detected HCPs that are the endogenous target of a monoclonal antibody. In most cases, the HCP has been at very low levels and was not a safety concern, but if that endogenous HCP were a cytokine, it would be a different story.
- Our HCP projects have involved bacterial, yeast, and mammalian expression systems. We have worked with cell-free systems.
- We also have extensive experience doing contaminant protein analysis of plasma-derived therapeutics.
- Our "infamous" HCP project was one in which a significant percentage of patients developed antibodies to HCPs during the clinical trial. The trial was halted, and the regulatory agency demanded that immunogenic HCPs be identified. Haemtech has a rich history in plasma protein purification, and we rapidly isolated patient antibodies, and used mass spectrometry to identify the immunogenic HCPs. This knowledge was then used to re-design the purification process, and mass spectrometry was used to confirm these immunogenic HCPs were removed to below detectable limits. The product went on to be commercially licensed.
For more information, please contact us.