Biotechnology has turned cells into machines capable of making biological medicines that are more effective and better targeted than traditional drugs.
But manufacturing biopharmaceuticals in a living system is difficult. As well as 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 impact 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, which 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 to minimize their presence in finished products.
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,” according to Joe Hirano, GE Healthcare, who says “while significant efforts to remove them are made, biologics contain a small amount of HCPs in the final product.”
A reason for this difficulty, notes Hirano, is that “quantitative measurement has been a challenge and so far, we do not have a specific and sensitive enough method 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
At present ELISAs, enzyme-linked immunosorbent assays, 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 able to bind and quantify the HCPs present in culture fluid and in-process samples. Process developers then use the ELISA measure the HCP levels to check how the purification process is effective to remove the HCPs.
The approach is widely used in industry. Philip Ridley-Smith, Cobra Biologics, tells GEN “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, GE Healthcare “Mass spectrometry requires a highly skilled operator and access to expensive equipment” adding “at present the process is too time consuming and costly to be practical.”
Assay challenges
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 their removal by the immune system. It is these antibodies that form the basis of the assay.
HCP ELISAs do not have antibodies to 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 ELISAs ‘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 it requires a unique anti-HCP polyclonal antibody.”
“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, 2D gel combined with chemiluminescence Western blot is used to assess HCP ELISA coverage.
In this approach, a sample is run on duplicate gels one of which is transferred to a membrane using the ELISA anti HCP antibodies in a Western blot. A stain is applied to the other gel. ELISA HCP coverage is determined by overlaying the images and identifying the spots detected by the stain that have a corresponding spot on the Western blot.
However, while 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, as is aligning the images for comparative purposes. All of these issues can reduce confidence in the determination of the ELISA coverage.
A new technique -2D DIBETM, or differential in blot electrophoresis, has potential to eliminate the challenges are associated with traditional approaches to coverage assessment.
In DIBETM, total HCPs from the null cell line is pre-labelled 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 different fluorescence wavelength from the total HCP labelling. Once the Western blot is completed, the fluorescent signals from total HCPs and western blot signals are detected by a multi-channel fluorescent scanner.
Coverage can be determined visually with a help from a dedicated 2D image analysis software, by identifying the labelled proteins that are bound by a labelled antibody. The approach reduces the number of gels required, minimizes variations and makes interpretation of the results more straightforward.
Limited ELISA coverage is not the only issue biomanufacturers face. For example, regulators insist HCP levels are monitored 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 as long as two decades,
For firms that make their own antibodies, production costs can be significant and getting approval from regulators may take a long time As a consequence some companies employ “commercial” antibodies designed for the cell line they are using. However, this strategy has associated risk. Anything that impacts supply, e.g., changes at the manufacturer, will impact the ability to conduct consistent assays.
Coverage assays
The potential to determine HCP ELISA coverage more rapidly and with better accuracy is positive for the biopharmaceutical sector says Hirano, particularly given the time and effort required for current approaches.
“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
