New test methods can provide improved quality and efficiency, but they must be validated to demonstrate equivalency.
The biopharmaceutical industry is advancing at an unprecedented rate as new cells and viruses are being used to generate biological therapeutics in greater yields with shorter production cycles and more efficacious properties, according to Audrey Chang, executive director of global services at BioReliance.
“New cell substrates (e.g., transformed cell lines, insect cells), new product types (e.g., gene and cell therapy, tissue engineering), new production process, and the discovery/detection of new contaminants have contributed to an elevated complexity of product testing to evaluate safety and efficacy,” Chang explains. Newer techniques for lot-release testing can offer numerous advantages in this situation, but there are challenges to assuring equivalence to standard methods.
New test methods can improve quality and promote efficiency, according to Niall Dinwoodie, global coordinator of analytical testing in the Biologics Testing Solutions business of Charles River Laboratories. “New methods or improved versions of traditional methods allow quality control laboratories to provide a greater level of information about the finished product, whether through improved detection limits, discriminating power, or quantification,” he observes. “In addition, newer techniques tend to be more automated, allowing faster sample throughput in larger numbers.”
The use of newer analytical techniques can also help mitigate risks, according to Chang. “The cost and safety impact of contamination has led the industry to embrace quality-by-design principles with respect to building quality into a product and process. As a result, biopharmaceutical companies are starting off with knowledge of the risks involved and how best to mitigate those risks, which includes the use of the most effective analytical methods,” she says.
The adoption of alternative methods into a regulatory program can be based on several factors, such as the desire to reduce/eliminate the need for animal testing, shorten the turnaround times for testing, and/or provide a more comprehensive safety profile through either increased sensitivity or specificity, according to Chang. Some alternative test methods are in fact improvements over existing techniques rather than true alternatives and rely on the same properties of the molecules to perform the analyses, according to Dinwoodie. He points to capillary electrophoretic methods as substitutes for gels to obtain greater resolution of closely related proteins within the product and provide more robust quantification of these components. In addition, he suggests the use of ultra high-pressure liquid chromatography (UHPLC) methods instead of high-performance liquid chromatography (HPLC) to reduce run times and increase sample throughput as an example. New technological methods for rapid detection, identification, and quantification of adventitious agents have also emerged, according to Chang. “Many of these techniques are molecular-based, such as polymerase chain reaction (PCR) for mycoplasma, next-generation sequencing for virus screening, and digital PCR for residual DNA quantification,” she comments. Charles River is also seeing growing interest in rapid microbiology methods for sterility testing of autologous or allogeneic cell therapies where cells need to be infused into patients before traditional sterility results would be available, according to Dinwoodie. He also believes that PCR coupled with mass spectroscopy (Plex-ID) and massively parallel sequencing may provide advantages over current techniques for detecting adventitious agents in cell substrates and biological products. “New techniques for determining sub-visible particulates that enable the detection and evaluation of particles of much smaller sizes are also providing conformational data that goes beyond traditional light obscuration methods,” adds Dinwoodie.
To prove equivalence statistically, a large data set is needed. “Generating a data set to show equivalence requires testing multiple samples that cover all potential degradation routes and impurity profiles. Therefore, in order to show equivalence of a new method, testing must be conducted on a bank of retain samples from historical production runs and new stress and stability samples,” Dinwoodie states.
Beyond this issue, companies often face the challenge of correlating the conclusions generated from the old and new methods when there are apparent differences between the data sets, according to Chang. “A good example is the use of PCR, where the presence of the nucleic acid is used as a substitute to monitor the presence of live/infectious organisms. Frequently, the term ‘false positive’ is used when a positive result is seen with a PCR assay and a negative result is obtained with the corresponding cell culture assay (e.g., mycoplasma PCR and mycoplasma cell culture assays). In actuality, the PCR result was not a false positive; the presence of the DNA produced the PCR positive result as the assay was intended.”
The alternative method may also identify new or increased levels of known impurities, according to Dinwoodie. “If batches used for safety and clinical assessments are still available for testing and can be shown to contain similar levels, the impact of these impurities can be more readily assessed.” In addition, in the case of quantitative assays, he notes that the alternative methods may be concordant (i.e., show similar dose-response curves) but not truly equivalent. “Understanding the relationship between data sets derived from the two assays is critical, particularly in the case of potency assays,” Dinwoodie stresses.
Another key challenge for Chang is correlating the limit of detection (LOD) of the two assays. “Again, using the mycoplasma example, the PCR LOD is reported as genomic copies (GC), whereas the cell-based method is reported in colony-forming units (CFUs). The lack of an industry standard mycoplasma stock can make it difficult to compare results because the GC/CFU ratio can vary depending on the species, storage, handling, and processing parameters,” she explains.
Plan and document
“The best way to show equivalence is through statistical comparison of the results for the same samples tested under standard and alternative methods; the greater the range of samples, the more convincing the statistical argument,” asserts Dinwoodie. He adds that it is important to recognize that the process will be a long one, and that alternative methods should be introduced to stability programs when there is typically sufficient sample available and they can be run alongside the current method to provide an initial assessment of equivalence.
If an alternative method has been used from the start of product development, it is possible to justify using that method rather than the standard approach, according to Dinwoodie, but ultimately it is necessary to provide data to show that the method is fit for purpose. “In general, though, traditional techniques are commonly used in the early stages of product development and newer, more sensitive, technologies are introduced as the product moves through clinical trials; this approach gives time to run the stability studies and gather data on multiple batches before the final submission,” he notes.
The key message from Chang is the need to have well-executed validation strategies. “Good science requires well-planned, well-executed, well-documented assays that generate meaningful interpretation of the results. The overall validation approach needs to be risk-based, with the level of validation required for each step established. The goal is to define the risk and validation strategies employed, as well as outline the steps in the validation lifecycle. If this approach is adopted, industry/regulators will be encouraged to move away from old techniques and embrace the next generation of novel platforms that are far more sensitive and rapid,”
Pitfalls to avoid
It is crucial to include degraded or historical production samples in the equivalence test when changing a method, according to Dinwoodie. In addition, if the alternative is to be used from the start of the development program, the validation of the method must be considered. Chang adds that it is important to assess an alternative assay’s readiness for use in a release program, which requires planning and accounts for performing the validation and documentation of the information. “Companies need to ensure that resources are dedicated to a validation quality program to ensure that the performance characteristics of the alternative method meet the requirement for the intended application. In addition, for all methods, but particularly for new/alternative methods, plans need to be in place to monitor assay performance over time,” she states.
Finally, Dinwoodie recommends that regulatory authorities be consulted whether considering an alternative for an existing method used for an approved product or when developing the specification for a new product at the investigational new drug stage.
About the Author
Cynthia A. Challener is a contributing editor to BioPharm International.