Literature
Question:
We use a plant-based expression system to produce our immunotherapy-based biologics. Unfortunately, since the expression system is quite unusual, commercial ELISAs are unavailable.
We want to analyze the host cell proteins (HCPs) in our drug substance (DS). Can we perform HCP analysis without developing a process-specific enzyme-linked immunosorbent assay (ELISA)?
Answer:
The short answer is yes; measuring and even quantifying Host Cell Proteins from unique expression systems is possible 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 particularly concerning in the drug manufacturing. This 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 [1].
In addition, identifying 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 unique 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 covers all potential impurities in the biologic manufacturing process and final DS.
Developing a platform assay includes many optimization steps such as antibody production and characterization and ELISA development and validation. Unfortunately, several issues can arise during 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 must start with antibody production again [1-4].
Alternative to the development of a process-specific ELISA
As mentioned, other approaches are 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.
The drug sample is prepared for analysis by digesting HCPs and drug substance proteins for LC-MS HCP impurity analysis. Then follows the first sample preparation steps, with adding intact protein standards. 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 [3].
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 simpler for manufacturers to optimize purification steps and improve the purity of the DS. LC-MS can act as a quality tool to monitor batch-to-batch differences and control drug purity and efficacy [3].
To sum 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:
References
[1] 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
[2] 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.
[3] 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.
[4] 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.
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