ddPCR in GMP product testing quantifies without calibration curve & determines absolute DNA concentration

Weihong Wang, Manager, WeihongWang@eurofinsUS.com; Jon Kauffman, Ph.D., Vice President, BioPharma Biologics, JonKauffman@eurofinsUS.com

In recent years, the number of biopharmaceutical products incorporating nucleic acids has been steadily increasing in the drug development pipeline. Some of these have made it to the market in a very visible manner such as COVID-19 vaccines that deliver messenger RNA to encode for spike protein, and gene therapies for the unmet needs of those with rare genetic disorders. Characterisation of the purity, efficacy and safety of these products is highly dependent on a technique known as Polymerase Chain Reaction (PCR). A relatively new technique for the detection and quantification of nucleic acid is digital droplet Polymerase Chain Reaction (ddPCR). In this technique, the PCR mix containing the test sample is partitioned into a large number of water-oil emulsion droplets, and PCR amplification of the target DNA sequence occurs in each individual droplet. Following PCR amplification, each droplet is assessed to determine the fraction of positive droplets in the sample. These data are analysed using Poisson statistics to determine the target DNA template concentration in the original sample.

Compared to quantitative real-time PCR (qPCR), which has become a standard methodology in most molecular biology laboratories, ddPCR has several advantages, especially in a GMP QC testing environment. First and foremost, samples can be quantified without the need for a calibration curve. In qPCR methods, a calibration curve is typically prepared from a DNA reference standard, and used to interpolate sample results. Therefore, the quality and concentration assignment of this reference standard can greatly influence the accuracy and even the validity of the reported sample results. ddPCR however, determines absolute DNA concentration through the power of statistics, thanks to the creation of tens of thousands of droplets that allow for the generation of large numbers of data points from each sample. This is particularly useful when a well characterised reference standard truly representative of the test sample is not possible, such as in the case of viral vector genome copy determination. Another advantage of ddPCR is that it is generally considered less susceptible than qPCR to PCR inhibitors that may be present in samples. This feature is especially important in the context of residuals testing, where assay sensitivity is critical. The better tolerance of ddPCR to inhibitors allows for the testing of samples without extraction, therefore greatly reducing the necessary volume of the drug substance and/or drug product allotted for testing. In the case of viral vector product testing, this can result in better preservation of products that are often produced in much smaller batch sizes compared to traditional biologics.

With the advantages discussed above, ddPCR has gained rapid momentum in QC testing laboratories. Eurofins in Lancaster, PA, has installed and validated the BioRad QX-200 ddPCR system within its molecular biology laboratories. The team has successfully performed method development, transfer, and validation projects, with a majority of them supporting viral gene therapy products. In addition to customised methods for individual clients, we are also developing generic ddPCR methods targeting consensus sequences within various viral vector backbones to support testing, including viral genome and infectious titer determination. Our in-house method validation, in conjunction with a product-specific qualification, will allow for the quick implementation of GMP testing of many sample types. For more information, visit: www.eurofinsus.com/bpt