Host Cell Protein Detection and Analysis

Biological medicines have transformed the pharmaceutical industry since their introduction¹ and are due to comprise about half of all Top 100 prescription drug sales by 2024.² Most biologics are manufactured within plant or animal cells,³ with Chinese Hamster Ovary (CHO) cells, for example, being among the most common hosts for producing therapeutic proteins.⁴

Unfortunately, as well as producing the therapeutic protein, organisms also often excrete other proteins. These may be necessary for growth or survival of the cell, and are usually only produced in tiny quantities (e.g., nanograms per milligrams of the recombinant protein). Nonetheless, these host cell proteins (HCPs) act as contaminants to the drug and can cause harmful immune responses in patients.⁵

Techniques for HCP detection

Three common techniques are used for HCP detection and analysis: enzyme-linked immunosorbent assay (ELISA),⁶ western blot, and liquid-chromatography-mass spectrometry (LC/MS).

Some benefits and limitations of these techniques are listed in the table below.

Table. Benefits and Limitations of the Three Most Common HCP Detection Techniques

Western blot

Western blot is a common laboratory method that can be used to detect residual HCP among a mixture of other proteins. For this technique, the protein samples are mixed with a detergent that unfolds them and gives them a negative charge. They can then be separated according to size using gel electrophoresis and transferred from the gel onto a blotting membrane (hence, the name western blot). The blotting membrane now carries the proteins in size-sorted bands and can be incubated with an antibody that detects the HCP and, after that, with a reporter enzyme that produces color or light.⁹

This process is, unsurprisingly, time-consuming, according to Maria Pulina, Ph.D., Product Manager at Enzo Life Sciences. “With western blot, you can only use a few samples,” she says. “[It’s useful] if you don’t need high sensitivity or throughput.” Her colleague, Deborah Kim Holzapfel, Ph.D., Senior Commercialization Manager and Head of the Product Management Team at Enzo, adds that western blot is popular in academia as it’s cheap to carry out, and there isn’t a high emphasis on detection speed.

Enzyme-linked immunosorbent assay (ELISA)

ELISA is the most common detection method for HCPs, according to Pulina. At its most basic, ELISA involves adding a sample to a multi-well plate on which capture antibodies have been immobilized. If the HCP of interest is in the sample, it will bind to the antibodies, and can be detected using a second antibody with a color-change marker.^6,10

Enzo and several other companies supply ELISA kits for detecting HCPs, which Holzapfel explains are very simple to use. “It’s a fairly basic technique, it’s cheap and you can [screen] quite a few samples at once,” adds Kevin Gamber, Ph.D., Vice President Marketing at Canopy Biosciences, which also offers ELISA kits.

Canopy and Enzo offer off-the-shelf kits for different cell types, including E.coli, HEK293, and Chinese Hamster Ovary (CHO) cells. These standard kits are useful for early-stage drug development using established cell lines, according to Kerstin Pohl, Senior Global Marketing Manager for Gene Therapy & Nucleic Acid at SCIEX. As Gamber explains, they are simple to run because most people are familiar with ELISA and the only additional equipment needed is a plate reader.

However, for late-stage product testing or development, many customers require process-specific ELISAs. These are often developed by the same companies that offer standard kits. However, they can take several months to develop, Pohl explains.

Liquid chromatography-mass spectrometry (LC/MS)

Liquid-chromatography/mass-spectrometry (LC-MS) uses a chromatography column to separate protein analytes in solution by their size or other physical properties. The liquid is evaporated, and the analytes ionized, and then drawn into the mass spectrometer where they are subjected to electromagnetic fields. Through this process, the ions can be mapped based on their amount and physical structure—allowing specific proteins to be identified.¹¹

LC/MC is highly costly, as you need both to own a mass spectrometer and to have it regularly serviced, according to Holzapfel. It’s also more complex to operate than a plate reader, putting it beyond the use of many small biotechnology companies.

But LC/MS has several advantages over ELISA/western blot. The main benefit is being able to identify and quantify hundreds of thousands of different HCPs—rather than simply detecting an overall amount, says Pohl. This allows companies to understand which exact proteins are present. Using this information, they can adjust their processes to more efficiently remove HCPs based on their physical properties, she says, without needing to develop a protein-specific ELISA.

LC/MS can be performed using more than one type of chromatography column, to improve sensitivity and specificity by unpacking a complex mixture of proteins, says Steven Martin, Ph.D, Vice President of Research at Waters. He suggests using LC/MS for late drug discovery or early-stage development to identify what HCPs may be present, before moving to chromatography-based tools during manufacturing.

Due to the complexity of LC/MS systems, Martin says they’ve introduced a simpler LC/MS system that is suitable for a wide range of users. They believe this will reduce the barrier to entry when using LC/MS systems, allowing them to compete with ELISA on ease of use.

References

1. Oxtoby, K. (2019) How biologics have changed the rules for the pharmaceutical industry. Chemistry World 

2. Unlisted author (2019) EvaluatePharma® World Preview 2019, Outlook to 2024, 12th edition, June 

3. Unlisted author (accessed 2023) How do Drugs and Biologics Differ? Biotechnology Innovation Organization 

4. Li, F. et al (2010) Cell culture processes for monoclonal antibody production. MAbs, vol. 2 (5), pp.466-77

5. Wang, X. et al (2009) Host Cell Proteins in Biologics Development: Identification, Quantitification and Risk Assessment. Biotechnology and Bioengineering, vol. 103 (3), pp. 446-58 

6. Unlisted author (accessed 2023) ELISA principles and types. Abcam 

7. Jiang, W. et al (2020) A protocol for rapid western-blot: shorten the time to 1-3 hours V.2. Protocols.io 

8. Ryding, S (accessed 2023) ELISA versus Mass Spectrometry for the Detection of HCPs. News-Medical 

9. Unlisted author (accessed 2023) Western blot. Scitable by Nature Education 

10. Unlisted author (accessed 2023) ELISA testing. Biobest, 

11. Kailasam, S. (2021) LC-MS – What Is LC-MS, LC-MS Analysis and LC-MS/MS. Technology Networks: Analysis & Separations. 

12. Steve Martin at Waters (see body text)