Despite the advent of technologies like next-generation sequencing, quantitative PCR (qPCR) remains a critical pillar of biopharmaceutical development at every stage. From biomarker discovery to candidate selection, scale up to sterile fill and finish, this highly sensitive and reliable tool enables qualitative and quantitative genomic analysis, with applications including gene expression, genotyping, and microbial detection.
Regulatory authorities recommend qPCR for verification and validation, quality assurance, and quality control—for example, the monitoring of process impurities picked up or passed down during biomanufacturing—across a wide variety of therapeutic modalities and specializations. Here we explore a few select examples of how the sensitivity and specificity of qPCR can be applied to biopharma use cases.
qPCR can support the drug development process from discovery to cGMP manufacturing
Given its ease of use and reliability, the benefits of qPCR are appreciated across the development pipelines of nearly every, if not every, biopharma therapeutic modality.
Take the development and production of proteins such as monoclonal antibodies and traditional vaccines. Plasmid constructs contain not only the sequence coding for the protein of interest, but also the promoter that drives its expression. The protein may be “humanized” to minimize the possibility that it is recognized by the immune system as foreign. Codon use is optimized for expression in the host species. qPCR can verify/confirm those plasmid sequences.
Researchers need to confirm the transgene copy number in the cell lines being considered as investigational candidates. They also need to establish the levels of transcription from these constructs, assayed by reverse-transcription qPCR. By tracking these numbers over time, qPCR can also be used to determine the stability of cell lines.
The manufacture of cell and gene therapy products—including CAR-T cells and viral vectors—has a similar need for assessing genetic constructs and their in vitro expression. It is additionally important to know the degree to which these cells or viral particles have been taken up by the patient, therapeutic effectiveness, and persistence over time. qPCR is a robust and sensitive tool ideally suited for these clinical assessments, as it is for establishing identity and freedom from adventitious agents for cell banking.
Biopharmaceuticals, by definition, are produced in living organisms, including yeast, bacteria, insect, and mammalian cells. Once the biologic is harvested and purified it is essential and required by regulatory agencies to confirm that host DNA does not contaminate the final product. Manufacturers must monitor the process each step along the way, documenting there has been no carry-over, and qPCR has become a gold-standard technology to achieve this goal.
Similarly, the presence of virus or viral particles must be guarded against during biopharmaceutical manufacture. To ensure viral contamination is detected and contained as early as possible, steps such as selective absorption, size exclusion (nanofiltration), and mechanical and chemical inactivation are used at various points in the manufacturing process. Viral targets are routinely detected using qPCR to assure that these measures are effective. In virus clearance validation, a virus can be spiked into a preparation prior to the clearance step, then the output can be tested using qPCR, to document successful removal of the contaminant.
For application in oncology, qPCR provides a fast, computationally simple, and sensitive way to provide genotypic and expression screening post-discovery. For example, qPCR can be used to identify specific genetic mutations in resected tumors that may help to develop companion diagnostics. Transcriptomic profiling in cultured cells may help determine the effect of a drug on gene regulation. And microRNA profiles in plasma or serum liquid biopsies may be leveraged as diagnostic biomarkers.
While the technology to perform qPCR is available to a large percentage of laboratories, a thorough understanding of best practices seems not to be as ubiquitous. Despite publication of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines in 2009, the drum beat of pleas in presentations, articles, and blog posts indicate continued calls for standardization, consistency, and accuracy.
These calls include, but are certainly not limited to, best practices in: sample preparation, including extraction of nucleic acids from a given matrix and its dilution to minimize interference with enzymatic reactions; controls and replicates, including determining how many and what type are necessary for statistical power; design and use of sequence-specific primers and probes; standard curves, quantification, and choosing appropriate normalization targets; and data analysis, including thresholding.
Done right, qPCR provides for an inexpensive, sensitive, tried-and-true means to quickly and reliably test for the presence of, and to quantify, nucleic acids at every stage biologic manufacture.
With the Applied Biosystems' QualTrak™ Biopharma offering, Thermo Fisher Scientific has carefully curated a qPCR ecosystem to help simplify drug filings for biologics manufacturers. For more information about tailored workflows, products manufactured under GMP principles, and 21 CFR 820-compliant instruments and software, explore QualTrak Real-Time PCR Solutions for Biopharma.