By Ben Butkus
LONDON – Representatives from big pharma weighed in on the increasing use of quantitative PCR in areas such as companion diagnostic development and vaccine analytical testing, and provided common pitfalls and tips for success in applying the technique at a qPCR industry meeting held here this week.
In the keynote presentation at Oxford Global's qPCR, Next-Generation Sequencing, and Analytical Genomics Congress, Yiu-Lian Fong, director of diagnostic development for Novartis Molecular Diagnostics, shared with attendees strategies for analytical validation of PCR-based in vitro diagnostic tests, with a particular focus on companion diagnostics.
Meantime, in another presentation, Azeem Ansari, deputy director of analytical research and development for Sanofi Pasteur, discussed with conference-goers how qPCR has in recent years become the go-to method for vaccine analytical testing, an essential component of the vaccine-development process.
First up was Novartis' Fong, who outlined the opportunities that exist for pharma companies to develop and validate biomarkers for use in diagnostics, prognostics, or theranostics, and cited market analysis that predicts 38 percent annual growth in the use of biomarkers in clinical trials through 2015.
However, the current reality is that companion diagnostic development "often does not start until the later stages of drug development" — around the time of phase II and III clinical trials, Fong said. Due to low drug-development success rates, "it has made no sense to invest the time and money into companion diagnostic development" earlier in the process for drugs that stand a better chance of failing than succeeding, she added.
This strategy has its drawbacks, however, because for those drugs where a companion diagnostic is deemed necessary, the development and approval of the test in the later phases of clinical development can hold up the drug's approval, "which is what we have seen in the last few years," Fong said.
For example, the European Medicines Agency initially rejected Amgen's marketing application for the colorectal cancer drug Vectibix in May 2007. The company then resubmitted its application with data showing that patients with certain mutations in the KRAS gene would not respond to the drug. In September 2007, EMEA recommended that Vectibix be marketed in non-KRAS-mutated patients.
As such, Fong said that regulatory requirements for companion diagnostic development need to be considered early on in the overall drug development process, and outlined a score of challenges that exist in doing so.
For example, one of the biggest challenges facing test developers is that tests used in drug trials might not be readily transferred into IVD tests, Fong said. This could be a result of different assays, procedures, instruments, and reagents being used at different sites in a multi-site clinical trial; or that the assay technology is not "robust" enough and does not meet GMP or CLIA standards.
Further, the analytical performance of molecular tests used in drug trials may not have been fully vetted. For instance, Fong said that common problems include a lack of validation for analytes and other substances found in a specimen that might interfere with the molecular testing system; and a lack of validation of software, controls, and test stability.
Fong also pointed out that clinical samples used to develop a molecular test may not have been collected or stored in an appropriate form for IVD development and validation. Examples of this include Qiagen's PaxGene kit for purifying RNA from whole blood versus unadulterated whole blood samples; isolated genomic DNA versus whole-blood or tissues for DNA-based tests; and fresh frozen tissue compared with formalin-fixed, paraffin-embedded tissue.
To help address many of these issues, Fong suggested that test developers first identify the "intended use" of the test, which will help define variables such as the target analyte, specimen type, indications for use, and target population.
Further, developers must conduct a rigorous analytical validation of a molecular test by identifying the key parameters that affect its performance; review and follow guidelines set forth by regulatory authorities; and minimize differences between assay design and assay procedures.
Lastly, Fong stressed the importance of working with life science tool providers who have FDA-approved platforms and instrumentation for IVD development from the very start.
The upshot is that companion diagnostic developers need to consider validation of the "total test system," and not just the PCR assay, Fong said.
In his presentation, Sanofi Pasteur's Ansari stressed the increasing importance of analytical testing in vaccine development, and the increasing role of qPCR in such testing.
The role of an analytical lab like the one Ansari oversees is to use analytical testing methods to determine a vaccine's potency, or "whether it's going to work when injected;" purity; and, perhaps most importantly, safety, Ansari said.
As such, qPCR is "fast becoming the method of choice for vaccine analytical laboratories," replacing analytical testing using cell culture, immunology, or other biochemical methods.
For instance, for potency testing, laboratories have traditionally used a viral plaque assay in cell culture, which can take anywhere from two to 14 days to perform to completion, and often relies on human visual analysis. As such, this method is low-throughput, labor-intensive, subjective, and variable. "Give an analyst the same plate within a two-hour period and it will have different plaque results," Ansari said.
A PCR-based infectivity test, on the other hand, skirts many of these issues because it can measure viral genomes instead of whole virus replication, Ansari added. He said that Sanofi Pasteur's labs typically run qPCR-based viral infectivity tests on a Roche LightCycler 480 platform.
Another important issue in vaccine analytical testing is identifying adventitious pathogenic organisms that might be introduced to a formulation during manufacturing. Ansari said there are "hundreds of potential contaminating agents," and although there are other tests available – for instance, virus replication testing in eggs, rodents, or cells from simians or humans – they usually take too long and are unable to detect many key contaminating viruses.
"These days the method of choice is qPCR because vaccine manufacturing units have become very good at getting rid of most" but not all potential contaminants, Ansari said, meaning that a highly sensitive test is needed to detect remaining contaminants. "qPCR is the only method sensitive enough to do this," he added.
Lastly, Ansari noted that qPCR is essential for "method validation," or determining gene copy numbers to ensure vaccine development process consistency.
Although many vaccine developers have begun to realize how qPCR methods can improve their analytical testing, "it is incumbent upon analytical laboratories … to influence the FDA and other regulatory agencies to apply state-of-the-art technologies, including PCR, to make safer vaccines," Ansari said, especially considering that the agencies themselves are "sometimes slow to adopt these methods because the field is so heavily regulated that once something works, the FDA is slow to change."
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