Benefits in DD and Development
“The technology is ideal for monitoring cell surface and intracellular events mediated by target-binding protein and cell-based drugs,” pointed out David Gordon, senior manager immunoanalysis at Aptuit. “FC allows you to simultaneously measure multiple analytes, demonstrate mode of drug action at the cellular level, and carry out cell subtype, counting, and distribution analyses.
“The technique generates semiquantitative or quantitative endpoints and can be used for applications ranging from simple binding assays to more sophisticated functional transcription response assays that evaluate linked biochemical processes in heterogeneous populations of primary cells.” The potential uses of FC thus range from drug discovery and early development mechanistic/efficacy assays to immunotoxicology, bioanalysis (PD markers), immunogenicity, and biopharmaceutical production, according to Gordon.
Despite the obvious utility of FC in a nonclinical setting, manufacturers also still view the hospital and academic markets as their bread and butter, Dr. Jepras said. “Innovation in instrumentation, reagents, and software is, therefore, still largely targeted at these markets.”
One of the major issues facing FC in drug discovery and development is the “lack of clearly defined regulatory guidance, both for assay development and with respect to validated software and informatics,” pointed out Garry Takle, Ph.D., vp of operations at WuXiAppTec.
“Flow is one of the most widely used methods for cell analysis and monitoring and really needs to be brought out of the academic and clinical labs and into the GLP/GMP arena. The meeting served to highlight how many companies are already using flow extensively, and the real need to start formalizing what the general expectations are for the use of FC and its associated data-management tools in a regulated environment.”
Scientists at AstraZeneca are spearheading the drive to bring the lack of guidance to the regulators’ attention. Dr. Coldwell and colleagues Jo Cunliffe, Ph.D., and Nicola Derbyshire, Ph.D., senior scientists, clinical pathology, are working to draft a paper that will hopefully promote further discussion within the industry and the regulatory bodies.
“Because there are few standardized procedures or methods for FC, and as the flow-derived data can be subjective, initial attempts to draft validation procedures will only be able to suggest minimum requirements,” Dr. Derbyshire suggested.
While industry is only too willing to help develop the foundations of a flow-cytometry validation package as a starting point for the regulatory authorities, the task won’t be easy, according to Mark Wing Ph.D., group director, clinical and translational sciences at Huntingdon Life Sciences. “Over the last five or six years, increasing numbers of our clients have been asking us to use FC in support of preclinical and clinical development programs, particularly in biomarker applications.
“About 20 percent of our biomarker services now use FC methods, but nobody has really come to grips with how you would go about developing a validation framework for FC assays.” The problem is compounded further, he pointed out, because there are no guidelines for biomarker assay development, whatever the method used.
“Whereas the FDA BMV guidelines clearly set out how you go about validating a method for PK analysis, biomarker assays present a greater challenge and typically follow the fit-for-purpose approach to validation. The absence of suitable reference material makes the assessment of accuracy for most FC assays challenging.
“For a PK method, negative samples would be spiked with purified drug, to assess the method’s ability to recover that drug. There aren’t readily available purified populations of cells that would allow you to assess the accuracy of a flow-based assay, however. That said, truly quantitative PK analyses are possible by FC when the reference material is available.”
With accuracy evaluation problematic for biomarker assays, assessing precision is key to in-house validation of an FC assay, Dr. Wing continued. “You, at least, want to demonstrate that your assay performs the same each time, regardless of how often it is carried out. It may be inaccurate, but as long as it is consistently inaccurate, that may be sufficient to derive meaningful data.”
Assessing precision and reproducibility brings its own problems, he admitted. “The analytes, i.e., cells, aren’t particularly stable. Whereas with immunoassay or mass-spectrometry techniques, your analytes tend to be stable and can be frozen for repeated analyses over a number of days if necessary, cells don’t freeze well. The use of stabilized cell preparations as surrogates for the real analyte may well be the best way to get around this.”
Despite these issues, a number of companies are successfully using FC in an increasing range of applications. “We have successfully used FC to support GMP quality control of biopharmaceuticals, to assess drug potency in different species, and to measure biomarkers in preclinical and clinical trials as part of submissions to the regulatory authorities,” Dr Wing said.
Indeed, as long as you can adequately demonstrate what the technology can achieve by showing evidence and defining boundaries, there should be no real hindrance to submitting FC-derived data as part of a regulatory package, suggested Gordon. Aptuit has been using FC technology for the last couple of years, primarily for preclinical work in areas such as species specificity.
“When evaluating the effects of a drug in a nonhuman species, it is vital to ensure that your animal is relevant and reflects the activity of the drug in humans,” he explained. “Recent European guidelines on strategies to identify and mitigate risks for first-in-man studies highlighted the need to demonstrate the relevance of an animal model. With increasing numbers of protein-based therapeutics such as antibodies coming into development, FC technology has come into its own as a method of choice for examining whether the extent of binding of a protein-based candidate to human and animal cells is similar.”