We use a plant-based expression system to produce our immunotherapy-based biologics. Now we want to analyze the host cell proteins (HCPs) in our drug substance (DS). Unfortunately, since the expression system is quite unusual, commercial ELISAs are unavailable.
Can we perform HCP analysis without developing a process-specific enzyme-linked immunosorbent assay (ELISA)?
The short answer is yes; it is possible to measure and even quantify Host Cell Proteins from uncommon expression systems without developing a process-specific ELISA. One solution is to use liquid chromatography mass spectrometry (LC-MS).
Now, let’s go into more details.
The most common expression systems used to produce biologics are bacterial (e.g., E. coli) and mammalian (e.g., CHO and HEK) cell-based. However, other systems include plant-based, yeast, insect, viral, and cell-free systems.
Expanding host cell expression systems beyond E. coli, HEK, and CHO creates an increasing demand for new types of HCP-ELISAs. However, since these are not yet commercially available many companies are forced to develop platform- or process-specific ELISAs. We will get back to the pros and cons of this in a minute.
Fortunately, there are less time-consuming options available such as LC-MS.
Essential reasons for analyzing HCPs in biologics
But why measure Host Cell Proteins (HCPs) at all?
HCPs constitute a significant class of process-related impurities and are of particular concern in the drug manufacturing process. This type of impurity poses a risk factor for reduced drug efficacy, toxicity, and long-term immunogenicity in drug recipients. Therefore, it is essential to demonstrate process consistency and purity of the final DS by documenting and monitoring any HCPs during process development and manufacturing .
In addition, the identification of unwanted impurities guides manufacturers in adjusting purification steps and eliminating co-purifying impurities [1-3].
It is also a requirement from regulatory authorities.
Traditional methods for analyzing HCPs in biologics
The gold-standard method for HCP impurity analysis in biologics is ELISA. The advantages of ELISA include high sensitivity towards specific HCPs, simple and easy assay handling, short analysis time, and high sample capacity since you can analyze many samples simultaneously. Generic ELISAs are thus an adequate, semi-quantitative method to approximate the impurity level in the early processes [1, 4].
However, generic anti-HCP ELISAs can be scarcely available when using uncommon expression systems originating from, e.g., plants or insects. Such systems typically require the development of a platform assay to suit the specific cell line. Alternatively, a process-specific ELISA that covers all potential impurities in the biologic’s manufacturing process and final DS.
The process of developing a platform assay includes many optimization steps such as antibody production and characterization and ELISA development and validation. Unfortunately, several issues can arise in these steps: Low antibody sensitivity or affinity, cross-reactivity or unspecific binding, high background signals, etc.
Typically you also need to produce a null-cell line for a mock fermentation – again time-consuming and expensive.
A significant disadvantage of the platform and process-specific ELISA development is that it is time-consuming and may typically take at least a year. Also, it is not a one-time process: Once you use up the polyclonal antibody batches, new batches must be tested and validated. If these are unsuccessful, one would have to start again with antibody production [1-4].
Alternative to the development of a process-specific ELISA
As mentioned, there are other approaches available to analyze HCPs in your biologics. The LC-MS method works independently of antibodies. It is therefore not as vulnerable to purification or manufacturing process changes that may require new antibody production.
For LC-MS HCP impurity analysis, the drug sample is prepared for analysis by digesting proteins that include HCPs and drug substance proteins. Then follows the first sample preparation steps, where Intact protein standards are added. These standards act as quality controls and measures for the absolute quantification of unknown HCPs. After sample digestion, an ultra-sensitive LC-MS instrument analyzes the peptides, and a scientist searches the resulting data against databases of sequenced proteins to identify individual HCPs .
An advantage of the LC-MS approach is that it does not only provide the overall HCP impurity levels. The method also quantifies individual HCP impurity levels and gives detailed information about specific HCPs. E.g name, accession number, pI, and molecular weight. The data makes it more simple for manufacturers to optimize purification steps and improve the purity of the DS. LC-MS can therefore act as a quality tool to monitor batch-to-batch differences and control drug purity and efficacy .
To sum it up, the LC-MS HCP analysis gives a more detailed quantitative and qualitative result than any ELISA. Since LC-MS results usually are obtained within a few weeks, such an assay is notably less time-consuming than developing a platform or process-specific ELISA.
Where do I look for more information?
If you are interested in learning more about how to monitor HCPs throughout purification steps without developing a process-specific ELISA, these webinars are an excellent place to start:
- U.S. Pharmacopeia National Formulary, 2016; May 1, USP 39 Published General Chapter <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals. https://www.usp.org/sites/default/files/usp/document/our-work/biologics/USPNF810G-GC-1132-2017-01.pdf
- Bracewell D. et al., Biotechnol Bioeng 2015 Sep;112(9):1727-37, doi: 10.1002/bit.25628. The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control.
- Pilely K. et al., Anal. And Bioanal. Chem. 2021 Oct 1;1-12, doi: 10.1007/s00216-021-03648-2. Monitoring process-related impurities in biologics-host cell protein analysis.
- Zhu-Shimoni J et al., Biotechnol Bioeng 2014 Dec;111(12):2367-79. doi: 10.1002/bit.25327. Host cell protein testing by ELISAs and the use of orthogonal methods.