ELISAs are the standard for HCP quantification, but not all assays are created equal. Better coverage assays help to minimize HCP and keep development on track. Learn how recent innovations in HCP ELISAs can help you improve testing and purification.
Article first published in Genetic Engineering and Biotechnology News
Modern biotechnology can use cells to make medicines that are more effective and better targeted than traditional drugs.
But working with living cells comes with challenges. The cells produce the desired drug, but they also continue to make the proteins and other molecules they need to survive.
Some of these byproducts cause problems down the line. For example, host cell protein (HCPs) can degrade therapeutic protein and lower yields. And if they end up in the finished product, they can increase the risk of immunogenicity [1].
It’s up to the manufacturer to identify and remove HCP to minimize their concentration in the final product.
You can use enzyme-linked immunosorbent assays (ELISAs) to quantify HCP, but even the best assay cannot recognize all cellular proteins. So manufacturers need to assess coverage and use this to determine what removal steps are needed downstream.
In this article, we will discuss recent innovations in HCP ELISA coverage assays that can help streamline testing and expedite development.
HCP quantification
“HCPs are regarded as a process-related impurity in the production of biologics,” says Joe Hirano, PhD, Program Manager, Imaging & Western Blotting, Cytiva. “While significant efforts are made to remove them, biologics contain a small amount of HCPs in the final product.”
Joe says there is a reason for this challenge: “Quantitative measurement has been a challenge and so far, we do not have a method specific and sensitive enough to detect all traces of HCPs in drug products, in order to develop better removal methods, we need better measurement methods that are more specific and more sensitive.”
Measuring methods
Understanding host cell protein populations is an important part of process development and plays a vital role in optimizing downstream purification.
ELISAs are today’s standard for industrial HCP quantification. Researchers gather proteins from the unmodified cell line and raise a population of polyclonal antibodies against them in an animal host. Using these antibodies, researchers create an ELISA that can bind and quantify the HCP in the sample. Then, process developers use the same ELISA to measure HCP levels and check how well each purification step removed HCP.
Other methods like liquid chromatography–mass spectrometry (LCMS) could potentially be used to analyze harvested cell-culture fluid (HCCF) and quantify HCP.
However, Anne-Sophie Brès, Global Product Manager, Impurity Testing, Cytiva says: “Mass spectrometry requires a highly skilled operator and access to expensive equipment. At present, the process is too time-consuming and costly to be practical.”
Assay challenges
Animals do not produce antibodies for every host cell protein, so enzyme-linked immunosorbent assays made using these antibodies do not recognize all HCP. This discrepancy is referred to as the assay’s coverage.
While they are today’s standard, HCP ELISAs have limitations. No assay can detect every HCP made by a cell line. The reason for this is because of the way its antibodies are produced.
Researchers make ELISA antibodies by injecting animals with HCPs extracted from a cell line. In response, the animal produces antibodies that bind foreign proteins and enable their immune system to remove them. The resulting antibodies form the basis of the assay. But immunized animals do not mount an equal immune response to every single protein – so no ELISA has antibodies for all HCP. In other words, some HCPs are more immunogenic than others. At most, animals will generate antibodies to 70%-80% of the proteins – which means the resulting ELISA can miss almost a third of the cell’s HCP. This discrepancy is referred to as the assay’s coverage. For manufacturers, coverage is a major concern.
This view is shared by Hirano: “Each drug molecule comes into late clinical Phase II or III, when all upstream and downstream bioprocesses have been fixed for production. In a sense, anti-HCP ELISA antibody and the biologics production process is a ‘catch-22’ situation. To optimize and finalize the biologics production processes, we need a very good anti-HCP ELISA antibody. But to generate a good anti-HCP ELISA antibody, the biologics production processes must be finalized.”
Determining coverage
To determine HCP ELISA coverage, scientists often use 2D gel electrophoresis combined with chemiluminescence Western blot.
For this approach, scientists run a single sample on duplicate gels. They apply a stain to one gel, and transfer the other gel to a membrane using the ELISA anti-HCP antibodies in a Western blot. Then they overlay the images and identify spots on the Western blot that were also detected by the stain. This method can effectively assess ELISA coverage, but it is also time-consuming and technically challenging. Transferring HCPs to blots and aligning the images are both difficult procedures, and gel-to-gel and blot-to-blot variation can affect results. Combined, these issues can reduce a manufacturer’s confidence when determining ELISA coverage.
A new technique — differential in-blot electrophoresis, or 2D DIBE™ — can potentially eliminate the challenges of traditional approaches to assessing coverage. In DIBE, scientists use a fluorescent dye to pre-label HCP from the null cell line. They separate the sample in a gel and transfer it to a membrane before exposing it to the HCP ELISA antibody for the Western blot. Using another fluorescent dye with a different wavelength, the scientist detects signals from the Western blot. After, they use a multichannel fluorescent scanner to detect the Western blot signals and fluorescent signals from total HCP.
Additionally, scientists can determine coverage visually. Dedicated 2D image analysis software enables users to identify protein bound to a labeled antibody. This approach requires fewer gels, reduces variation, and makes it more straightforward to interpret results.
Other limitations
In addition to limited ELISA coverage, manufacturers face other challenges. For example, manufacturers must monitor HCP levels throughout the drug development cycle to meet regulatory requirements. That means manufacturers need a steady supply of the ELISA antibodies they used for as long as the drug is produced – sometimes up to twenty years.
For organizations that make their own antibodies, production costs can be high, and the regulatory approval process can be lengthy. Some manufacturers choose to use commercial antibodies for this reason, but that can come with risks. Outsourcing antibodies opens the door to manufacturer changes that affect supply down the line, and, as a consequence, the ability to conduct assays.
Dilution linearity and its associated issues present another unique challenge for HCP ELISAs. Hirano explains, “As the HCP ELISA antibodies are a mixture of antibodies, some antibodies may be insufficient to detect the total volume of those detected HCPs in the sample. As a result, the diluted sample will not show any reduced read-out. It just continues to give the same read-out despite the sample dilution.”
Coverage assays
The biopharmaceutical sector would benefit from being able to determine HCP ELISA coverage more accurately and rapidly – especially considering the effort and time needed for current approaches.
“Being able to determine ELISA coverage more quickly and effectively will ultimately allow biopharmaceutical companies to focus their resources more effectively,” says Hirano. “The most important advantage of having better ELISAs would be to shorten the development phase and time to commercialization. This helps to deliver effective and safe drugs quicker to those patients who need them.”
References
[1] https://www.ncbi.nlm.nih.gov/pubmed/27739010
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- Article – Enhanced host cell protein analysis in biologics manufacturing
- Blog post - Why, why, why… ELISA? A look at the benchmark HCP assay
- Blog post - How well are your HCP ELISAs covered?
- Webinar - Challenging host cell protein assays for improved risk mitigation
TRADEMARK NOTE:
DIBE is a trademark of General Electric Company