Arguably, the biggest challenge in making biologics is flagging unwanted host-cell proteins (HCPs) that inevitably show up in the finished product. While nano in scale, these trace amounts can be highly immunogenic and can ultimately sink a potential biopharmaceutical. As a result, manufacturers must work hard to identify and control any impurities in the final product.
Loosely defined as any ingredients that are not part of the product, HCPs have traditionally been detected using commercial kits—off-the-shelf generic assays that are readily available and require no preparation. But the “black box effect” associated with this method can be hit or miss, causing some drug makers to turn to product-specific assays and mass spectrometry to quantify protein interlopers that can interfere with the drug.
This more time-consuming approach brings greater breadth and depth to HCP protein analysis, which is one reason why both the U.S. Pharmacopeial Convention, and its counterpart, the European Directorate for the Quality of Medicines and HealthCare, are writing new chapters that specifically address how drug developers deal with HCPs. Both groups are expected to release draft guidelines later this year.
Since this step-wise approach requires considerable expertise, here are five ways to improve host-cell protein assay performance using a customized approach.
Choose the right antigen. Each type of protein expression system has limitations that affect antigen selection, so it is important to select carefully. For instance, the majority of the prokaryotic expression systems keep the protein of interest within the organism (inclusion bodies), which lead typically to a whole-cell lysate (proteome) as the primary antigen, while yeast and mammalian expression cells secrete the product into the media making the secretome a suitable antigen for the 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.
Guard against unwanted immunogenic reactions. 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 foodborne antigens.
Qualify the antigen and antisera correctly. The ultimate goal of the assay should be to detect the greatest number of antigen species throughout the entire biologic development process. Currently the gold standard is 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. Ideally, the Western blot should show the same number of spots as the corresponding 2D total protein stain, but so far there is no acceptance criteria set in regulatory documents. Furthermore, a reduction of HCP assay performance down to a simple percentage number would be misleading and not reflect the complexity of this exercise.
Purify the antibodies carefully. Optimal performance of the HCP ELISA requires purification of the antibodies from a fraction of the crude serum drawn from inoculated animals. This is usually done by separating the biochemical components using affinity chromatography, which siphons off nonspecific antibodies that can make the HCP ELISA less specific or sensitive.
Consider mass spectrometry as a backup and orthogonal method. Mass spectrometry techniques can identify the “spots” that are not recognized using 2D gels and Western blot. These proteins are analyzed for highly immunogenic sequences, which are then used to make artificial peptides. The protein fragments are then 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. Significant improvements in mass spectrometry have allowed us to use the technique as a complementary method to immunoassays and gain more detailed intelligence on individual HCP patterns and species.