In an ideal world, how should the process of identifying and assessing your biologics for host cell proteins work? In the old days, it would have involved less sensitive approaches like chromatographic methods and total protein stains. But these techniques have given way to more customized approaches that optimize how the enzyme-linked immunosorbent assay (ELISA) uses antibodies to detect antigens and the threshold immunoligand assay (ILA) measures proteins. The four main assay performance drivers are choice of antigen, immunization, qualification of antigen and antisera, and purification of the polyclonal antibodies from the crude serum.
Choice of antigen. The selection of an appropriate antigen is crucial for the development of any HCP assay. But there are limitations that typically occur with each type of protein expression system. For instance, in a prokaryotic expression system, where DNA transcription and translation occur simultaneously—you need to start with a whole cell lysate as an antigen for immunization. Other things to watch out for are hormones, serum and other media additives that can trigger non-HCP specific antibodies—imagine a lot of white noise in the background of the immunoassay. Growing cells in serum-free media or thoroughly washing the cells or bacteria can eliminate unwanted antigens.
Immunization. This is the most critical, time-consuming and unpredictable part of developing an exquisitely sensitive assay for HCPs. It becomes necessary, therefore, to maintain good biological karma. Using specific pathogen free (SPF) animals that are housed in controlled environments and that adhere to a defined nutrition plan help to reduce unwanted immunogenic reactions against food-borne antigens. Typically, two animal species (four rabbits and two goats) provide blood samples three times over three months in sufficient enough quantities to supply hundreds if not thousands of ELISA plates.
Qualification of antigen and antisera. The ultimate goal of the assay should be to detect the greatest number of antigen species throughout the entire biologic development process. One way to maximize those chances is to by using high-resolution two-dimensional (2D) electrophoresis gels stained with silver to visualize the HCPs and then comparing the results to the standard Western blot (see image below). 2D gels analyze complex mixtures of proteins by separating and identifying antigens in two dimensions—the first on the basis of the net electrical charge and then by protein mass. Ideally, if your silver stain detects 600 spots in the 2D gel you would hope to see 600 spots in the Western blot because that would mean the animals were reacting to all the antigens, but antibody coverage of about 80% is actually considered very good.
Purification of the antibodies. A fraction of the crude antisera drawn from the animals is then processed further by affinity purification and labeling to yield an even smaller quantity of anti-HCP antibody. Optimal performance of the HCP ELISA requires purification of the antibodies from crude serum. This is usually done by separating the biochemical components using affinity chromatography, which siphons off non-specific antibodies and other essential stuff that can make the HCP ELISA less specific or sensitive.
A new additional tool – HCP-GAPex
The spots not recognized using 2D gels and Western blot can be cut out and sent to organizations like Protagen Protein Services that identify proteins using mass spectrometry. These proteins are analyzed for highly immunogenic sequences which be used to make artificial peptides that can be re-injected into animals to induce specific antibodies that match the original peptide sequence. In the field of HCPs, this is a fairly new approach.
Once the antibodies are accounted for and the protein quantitation completed, the assay can be validated using a number of parameters such as accuracy specificity, recovery and precision of the HCP test. For instance, testing the specificity of the assay with product protein and a HCP mixture should find no signal above the detection limit.
Customizing assays from the earliest stages of drug development to the finish line is, obviously, a time-consuming process. And not every laboratory has the expertise or budget to do it. But in this Age of Big Proteins, it might be fruitful to view assay development as a race between the tortoise and the hare. Developing clear-cut standards for how we develop and validate these assays may take more time but it could ultimately help developers get their products across the finish line first.
You can find the first part of my blog series here. It highlights impending guidelines on HCPs in the US and across the pond.