David Chimento (Rockland Immunochemicals, Inc., Limerick, PA 19468, LinkedIn), about host cell protein assay development
What is a Host Cell Protein immunoassay and what is it used to monitor?
A HCP immunoassay is a specialized assay that is against all of the proteins of the cell line used to manufacture the therapeutic biopharmaceutical. It is a very complicated assay where there is an antibody reagent generated against all of the proteins or as many of the proteins as can be generated against and it is used to monitor in-process consistency in the purification of that molecule. So, what will happen is during the bioprocessing with multiple different steps samples are be taken and this anti-host cell protein immunoassay will be used to measure the level of total HCPs present and this is used to set specifications for that bioprocess.
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What kind of reporter systems support HCP assays? What reporter systems are most often used?
HCP assays are not limited by reporter systems and frequently most of the common immunoassay systems that are used. So we can start with the basics which can be either a directly conjugated anti-host cell protein reporter with an enzyme such as horseradish peroxidase or alkaline phosphatase. Or it can also be conjugated to biotin and then using a streptavidin HRP or alkaline phosphatase (“alk phospho”) reporter molecule. These are the common basic assays that are used.
It can also be engineered to support some of the higher throughput platform systems, whereby the specific reporters for those platforms can just as easily be conjugated to the host cell protein antibodies, and a high throughput system can very easily be engineered from that.
What are some of the critical performance parameters that need to be investigated for an HCP assay?
Some of the critical parameters for development of a HCP assay are common to other assays. In this case the matrix effects of the different buffer systems is important. This assay is going to see samples that come from very far upstream in the bioprocessing workflow all the way to final product. During its duty in that workflow it is going to see samples that are going to be at very acidic pH, it will see samples that may be in high buffers, and it is going to see samples which have final formulations and the components of final formulation.
So the assay must be able to perform across a quiet diverse set of matrices that the sample will be containing. Another key feature of this particular type of assay for host cell proteins is the dilutional linearity.
Because the nature or the complexity of the samples with so many proteins with a very wide dynamic range of concentrations of those proteins, it can be challenging to get an assay that functions with good dilutional linearity across all of the different steps of the bioprocessing workflow. So that is something that really has to be robustly worked out in order to have a well functionally assay.
What is the right assay calibration standard?
The assay calibration standard should be the same one typically that used to generate the HCP antibody. So typically what ought to be done is a very large amount of that material should be treated as a gold standard material, and then aliquots of that can be used to generate the HCP antibody and then the other fractions can be put in reserve. And that should be used as the calibration standard for the HCP assay by which the host cell protein levels are determined.
Are there any other recommendations to produce a good reagent and assay?
The way to do that has to begin with the end in mind. Start with thinking about the manufacturing platform that your drug product will be produced from.
In case of therapeutic monoclonals, that is typically a CHO cell line, consider whether or not you have a single program or if you have multiple different programs or a platform based assay by which you need to consider how much material you are going to need to support your products for more than a decade. Those are kind of the critical parameters that you are thinking about depending on what your angle is.
Backing away from that you have to start looking at what are the specifics or your platform regarding how your cell line is being used, how it is being grown, where you are going to take your upstream sample from that is representative of your process.
If you consider all those things and make your best informed choices about that, it is very likely that you will be able to produce a good reagent in sufficient quantity such that it can produce a robust HCP immunoassay to support all of your programs for the timeline of the life of those products.