The absolute quantitation of HCP assays is exceedingly difficult for several reasons. First of all, there is no recognized reference preparation of HCP against which we can calibrate our assays. While ELISA is inherently a quantitative method when applied to a single analyte, ELISAs that attempt to measure simultaneously all of the hundreds of potential HCP impurities in the same well using a single reporter/detector system are at best semi-quantitative assays. Many arbitrary choices and assumptions are used when preparing anti-HCP antibodies and later the choice of material to use when preparing “standards”.
How to obtain the HCP material for immunogen is the first choice that must be made.
- Do we use a null cell line or a mock transfected cell line or one with the actual product plasmid and what differences in HCP might we see from each?
- At what point in the purification process do we obtain the HCPs for the immunogen or standards?
In almost every case the array and relative concentrations of HCPs in final product are different from the HCP in the ELISA standards.
- How does one assign a total HCP concentration to an indeterminant mixture of many proteins with different molecular weights?
- What effect will the different affinities of antibodies to various HCPs as well as the different concentrations of those antibodies have on the ability of the assay to be quantitative?
- How do we know that we have antibody to all the HCPs present in a given sample type?
All of these questions and arbitrary choices mean HCP assays will at best be semi-quantitative methods capable of making only relative estimates of HCP concentrations from one sample to the next. We estimate the potential error in reporting an HCP level in a sample that has a different array of HCPs from the standards to be as high as 4-fold even if you have a very good broadly reactive antibody. The commonly expressed theoretical concern is that HCP assays might under-estimate results due to incomplete coverage of all HCPs. In reality, the quantitative errors in HCP assays due to the arbitrary choices and fundamental limitations of the method as discussed above can result in either over-estimation or under-estimation and tend to do so to about the same degree. In our experience, a well generated and affinity purified antibody will react to more than 70% of individual HCPs as demonstrated by traditional 2D Western blot correlated to silver stain. What is more important than the number of individual proteins with antibody reactivity, is the total mass of those proteins that are detected. In our experience and as you might expect from theoretical considerations, the proteins in highest concentration have the best chance of generating an antibody. As such, an antibody with about 70% reactivity to an individual HCP will react with those proteins that account for more than 95% of the total mass of HCP. Given this level of uncertainty in the absolute HCP concentration, the most important criteria by which we can judge any HCP assay is on other objective parameters by which any analytical method should be qualified or validated. Those criteria include specificity and accuracy as demonstrated by sample dilution linearity and spike recovery experiments, precision, robustness, and sensitivity. Provided the assay has sensitivity to detect at least a relative portion of the HCP in your final drug substance and the assay also meets the other objective analytical parameter specifications, such an assay is a valuable analytical method capable of demonstrating process control and reporting relative levels of HCP contamination from lot to lot as a release test.
Due to the impracticality of obtaining real final product HCPs that co-purify with product down to the final step, most of our HCP assays will utilize a source of HCPs from very upstream in the purification process. Those HCPs typically do not come from the actual product cell line but rather a null cell or mock transfected cell line. Once we have decided on the source material for the kit standards we first perform some partial purifications to remove non-HCP materials such as growth media additives, buffer salts and extraction reagents. Our initial approach to calibrate this processed material is to simply perform a BCA total protein assay using bovine serum albumin (BSA) as an arbitrary standard. When standards made with the HCP material calibrated by BCA give reasonable “stoichiometric agreement” with the amounts of antibody used in the ELISA then we feel the calibration by BCA is good enough. What is meant by “stoichiometric agreement” is that we know how much antibody is used in the ELISA. It is the quantity of antibody that actually dictates the analytical range and dose response curve of the ELISA. If we assume an average molecular weight for the total HCPs, then we can reasonably estimate HCP concentrations across the valid analytical range of the assay. When the BCA assay concentrations do not reasonably approximate the ELISA stoichiometry, we process the HCP material further. Such processing involves various purification steps to remove components registering in the BCA assay but that are in fact not HCP or at least not immunoreactive HCPs. This processing might also involve affinity purification against the anti-HCP antibody. The purification stops as soon as the BCA concentration gives a realistic stoichiometric agreement in the ELISA.