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June 19, 2020

Better coverage assays for better HCP ELISAs: Quantifying the benefits

By Article first published in Genetic Engineering and Biotechnology News


Biotechnology has turned cells into machines capable of making biological medicines that are more effective and better targeted than traditional drugs.

But manufacturing in a living system is difficult. In addition to the desired molecule, the cell lines used in production also make their own proteins, which they need to grow, divide, and survive.

This is a problem because these “host cell proteins” (HCPs) can affect the product. For example, enzymatically active HCPs can degrade therapeutic proteins, thereby lowering yields. More importantly some HCPs can pass through processing steps and end up in the finished product. This is a major challenge because they are associated with increased risk of immunogenicity [1].

As a result, drug firms must make efforts to first quantify and then remove HCPs from processes in order to minimize their presence in finished products.

Enzyme-linked immunosorbent assays (ELISAs) are used to quantify HCPs. However, even the best assay cannot recognize all cellular proteins. So firms must assess ELISA “coverage” before determining what downstream removal steps are needed.

This article will look at innovations in HCP ELISA coverage assays that have the potential to streamline testing and accelerate process development.

HCP quantification

“HCPs are regarded as a process-related impurity in the production of biologics,” says Joe Hirano, Cytiva. “While significant efforts are made to remove them, biologics contain a small amount of HCPs in the final product.”

Hirano notes that a reason for this difficulty is, “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

The ImageQuant 800 biomolecular imager helps save time in Western blot imaging

Fig 1. Understanding host cell protein populations is an important part of process development and plays a vital role in optimizing downstream purification

At present, ELISAs are the gold standard for industrial HCP quantification. The concept is straightforward. Researchers gather proteins from the unmodified cell line and raise a population of polyclonal antibodies against them in an animal host. These antibodies are used to create an HCP ELISA that can bind and quantify the HCPs present in culture fluid and in-process samples. Process developers then use the ELISA to measure HCP levels to check the efficacy of the purification steps in removing the HCPs.

This approach is widely used in industry. Philip Ridley-Smith, Cobra Biologics, tells us, “We currently use an ELISA method for the measurement of residual E. coli host cell proteins in purified plasmid DNA bulk.”

This is echoed by James Graham, D.Phil., Technical Director, Analytical R&D, at Lonza Pharma & Biotech, who says, “ELISA is the current industry standard for release testing of manufacturing batches, as described in US Pharmacopeia Chapter 1132.”

Other methods like liquid chromatography–mass spectrometry (LCMS) have the potential to be used to analyze harvested cell-culture fluid (HCCF) and provide quantitative information on HCPs.

However, says Anne-Sophie Bres, Cytiva, “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 to every host cell protein with which they are immunized. Enzyme-linked immunosorbent assays made using these antibodies do not recognize all HCPs. This is referred to as the assay’s coverage

Fig 2. Animals do not produce antibodies to every host cell protein with which they are immunized. Enzyme-linked immunosorbent assays made using these antibodies do not recognize all HCPs. This is referred to as the assay’s coverage

Despite being the gold standard, HCP ELISAs have limitations. No assay can detect every HCP made by a cell line because of the way its antibodies are produced.

ELISA antibodies are made by injecting animals with HCPs extracted from a cell line. The animal responds by producing antibodies that bind foreign proteins, enabling the immune system to remove them. It is these antibodies that form the basis of the assay.

HCP ELISAs do not have antibodies for all HCPs because immunized animals do not mount an equal immune response to every single protein. Put simply, some HCPs are more immunogenic than others.

At most the animals generate antibodies to 70-80% of the proteins, which means the resulting ELISA will miss up to 30% of the HCPs made by the cell. This is referred to as the ELISA’s “coverage.”

Coverage is a major preoccupation for industry. Dr. Graham says, “Lonza has implemented a number of different HCP ELISA test methods over the years as technology has evolved, with the latest version reaching over 80% coverage against proteins from culture supernatant or cell extract.

“More complete protein coverage is useful, as it reduces the risk that changes could occur within a protein sub-population that cannot be detected by the ELISA method,” he points out.

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

Most commonly, scientists assess HCP ELISA coverage using 2D gel electrophoresis combined with chemiluminescence Western blot.

In this approach, scientists run a sample on duplicate gels, transfer one gel to a membrane using the ELISA anti-HCP antibodies in a Western blot, and apply a stain to the other gel. To determine ELISA HCP coverage, scientists overlay the images and identify spots detected by the stain that have a corresponding spot on the Western blot.

However, while 2D provides an effective assessment of ELISA coverage, the approach is time consuming and technically challenging. Gel-to-gel and blot-to-blot variation can be considerable. Also, transferring HCPs to blots is a difficult procedure, as is aligning the images for comparative purposes. All of these issues can reduce confidence in the determination of ELISA coverage.

A new technique — 2D DIBE™, or differential in blot electrophoresis — has the potential to eliminate the challenges associated with traditional approaches to coverage assessment.

In DIBE, HCP from the null cell line is pre-labeled with a fluorescent dye. The sample is separated in a gel which is then transferred to a membrane where it is exposed to anti-HCP antibody, i.e., the HCP ELISA antibody, to carry out the Western blot. The Western blot signals are detected with another fluorescent dye which has a different fluorescence wavelength from the total HCP labeling. Once the Western blot is complete, the fluorescent signals from total HCPs and Western blot signals are detected by a multi-channel fluorescent scanner.

Coverage can be determined visually with dedicated 2D image analysis software that enables the user to identify labeled proteins bound to a labeled antibody. This approach reduces the number of gels required, minimizes variations, and makes interpretation of the results more straightforward.

Other limitations

Limited ELISA coverage is not the only challenge biomanufacturers face. For example, regulators insist that biomanufacturers monitor HCP levels throughout the drug life cycle. As a result, manufacturers need to maintain supplies of ELISA antibodies for as long as the drug is being made; sometimes up to two decades.

Production costs can be significant for firms that make their own antibodies, and getting approval from regulators can take a long time. Consequently, some companies employ “commercial” antibodies designed for the cell line they are using. However, this strategy has associated risk. Anything that affects supply, e.g., changes at the manufacturer, will affect the ability to conduct consistent assays.

Issues associated with dilution linearity are another challenge unique to 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 consequence, 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 potential to determine HCP ELISA coverage more rapidly and with better accuracy is beneficial for the biopharmaceutical sector, particularly given the time and effort required 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.”