The current COVID-19 global pandemic has highlighted the importance of streamlining the time and cost of bioanalytical testing to support therapeutic development for viral infections such as coronavirus and influenza. To achieve this, however, requires that we apply scientific ingenuity. In a recent webinar, my colleague Jessica Weaver and I and walked through an example of how BioAgilytix translated a diagnostic virus neutralization method into a quantitative cell-based assay for the determination of relative potency of an anti-influenza antibody. This study, conducted with our sponsor Visterra, confirmed the ability to translate a diagnostic readout into therapeutic potency within cGMP regulatory constraints, and highlighted its utility over time-consuming in vivo testing for potency estimation.
Below, we recap the main highlights and key questions covered in the live Q&A session of the webinar:
How can cell-based systems expedite potency testing of viral neutralizing antibodies?
A range of modalities are being used to develop biologics for viral neutralization, ranging from vaccines to engineered neutralizing proteins, anti-serum, polyclonal antibodies, and monoclonal antibodies. While much of their specific development needs differ, they all require potency assays which have been traditionally performed in vivo. However, animal testing is both expensive and time-intensive, and a push by the industry and global regulatory agencies to address the ‘3Rs’ – replace, reduce, and refine processes to minimize the use of animal testing – has driven a transition to in vitro cell-based assays. Cell-based assays help to reduce the cost and expedite the process of potency testing. They can also be used not only for protein-related modalities but also for gene therapies that ultimately lead to expression of these proteins or similar in their modes of action.
How can a diagnostic assay be adapted to determine relative potency?
A viral neutralization test requires three main components: infectable (host) cells, a neutralizing agent (such as antibodies in a serum), and a source of the virus. The antibodies in the serum or neutralization agent in the host cells can vary, as can the virus. The combination of the virus with the neutralizing agent, followed by incubation with the cells, results in a viral titer, which can be measured and used to determine immunogenicity.
For our study, we wanted to demonstrate proof of concept that a mirconeutralization method for detection of viral immune response could be transformed into a GMP method for relative potency of a neutralizing agent. Specifically we chose the World Health Organization (WHO)’s diagnostic assay for H1N1 as a platform to work from. In this assay, the heat inactivated sera are incubated with H1N1 virus and then further incubated with MDCK cells. MDCK cells are often chosen for these types of studies since they have a long history with influenza; they were essentially designed and optimized with the intent for vaccine use. An ELISA evaluation is then performed against a nucleoprotein, which results in a readout of the viral titer.
To adapt this general concept to a viral neutralization assay for relative potency, we made one small change: the addition of a reference standard. The same process still occurs and a viral titer is determined, however, the reference standard is now also part of this determination, measuring the neutralizing activity or relative potency of the therapeutic under investigation. In our specific example, we replaced antibodies in a serum with the monoclonal antibody VIS410, with a final readout being a series of titration curves which allow relative comparison between reference standard and the samples of interest to determine a relative potency. This is where the power and capabilities of this method come into play. While our study was specific to a monoclonal antibody, it has broad applicability across many modalities.
What assay parameters were evaluated during development?
For the assay, there were several important factors to consider, among them cell characterization to understand the long-term support of the assay, activity measurements of the VIS410 neutralizing agent, defining the dilution series, and plate compatibility. To start, we performed experiments to determine median tissue culture infectious dose for the TCID50 at various cell densities to verify the virus titer. We also took a look at the activity of the VSI410 to determine if dose-response could be obtained and, if so, to find the dilution series needed to reflect the optimal reference curve. We focused on drug dilutions of the EC50 and the distribution of points on the curve to determine the optimal drug dilution series. Other parameters examined during the development included timing of dosing of cells, assay plate type, and preparation and incubation of the substrate.
I invite you to watch the full webinar for a detailed description of how the assay was optimized for these critical attributes and then qualified and validated to current standards for linearity of response, precision, intermediate precision, accuracy, robustness, and specificity. I also want to thank all of the attendees who joined us for the live session and asked many thoughtful questions during the Q&A portion of the event. We’ve captured a few of the asks and our responses below.
What are the critical attributes for this assay in order to achieve consistent relative potencies?
Critical attributes are well characterized cells and viruses. They must be matched to meet the mode of action. Also, having a robust system that produces robust curves and analytical data is necessary to arrive at the relative potency via the comparison of the curves and the responses. It is also important for the neutralizing agent to have well characterized materials and a consistent process for characterization.
How do you determine the similarity between curves?
We looked at this in different formats, in both constrained and unconstrained curve bits. For the constrained curve, we looked at the F test and determined if that met parallelism. For the unconstrained curve, we looked at equivalency ratios between the upper and lower asymptotes as well as slope ratios. We also looked at the difference in upper and lower asymptotes for the similarity between the curves.
How was the reference standard characterized?
We took the reference standard and ran multiple experiments at 100% NDC as well to characterize the upper asymptotes, the slope, and the lower asymptotes. We set ranges around what we typically see for that and the C parameter, and constrained those before looking at other samples. However, there were other biophysical tests performed—biocollaborators—that we don’t have details on that this time.
What was the percent CV of the accuracy test?
The overall percent CV across the NDCs for the accuracy test was 18%.
It was mentioned that one passage after F/T was optimal—how much did it improve the assay?
Greatly. If you solved the cells and then pledged them at the first passage, the assay would not meet the control step for the cell control as well as the virus control for the assay. So, the assay would not work unless you had at least one passage. After that passage, it was continued to culture, harvesting, and plating and the assay could then actually meet the control standards for the plate.
What are the key considerations to apply to other virus neutralizing agents, systems & modalities?
The same critical attributes for this specific assay are applicable to other neutralizing agents and modalities. The key is to have a good cell line: one that can be robustly maintained, well characterized, and ideally, one that is commercially available. It’s also important to have a good source of the virus of interest which is consistent, robust, and can also be well characterized and maintained throughout the duration of the study and all phases of development.
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Thank you again to those that submitted your questions and to everyone who joined us for the live session. Jessica and I welcome you to reach out with any additional inquiries or to discuss your anti-viral therapeutic development program and how BioAgilytix can support your bioanalytical needs.
Download the On-Demand Webinar
To access the full recording of the BioAgilytix live webinar and Q&A session, click here.
