By Mark White, Ph.D.
Cell and gene therapies, once a research pipe dream, are now healthcare realities. As of July 2021, 15 approved cell and gene therapies are being administered to patients1 and over 400 more are undergoing clinical trials in the United States.2 These powerful treatments can correct genetic mutations, attack cancer cells, and more.
Cell and gene therapies are often lumped together in biopharma industry discussions because they both involve gene editing. However, scientists may be doing themselves a disservice by failing to distinguish between these technologies when considering unique manufacturing needs.
Gene therapies: Detecting contaminants and confirming titer
For gene therapies, scientists’ primary concerns revolve around the viral vectors used to deliver healthy gene copies to patients. Thus, therapeutic doses need to contain enough viruses — and that needs to be all they contain.
Adeno-associated viruses (AAVs) are among the most popular vectors for gene therapies due to their versatility and low immunogenicity. However, since recombinant AAVs are produced using living host cells instead of in perfectly controlled in vitro conditions, some variability is involved in their manufacturing. Furthermore, manufactured vectors must be concentrated multiple times to obtain sufficiently high titers for effective treatment. Therefore, to ensure patients receive the correct dose every time a gene therapy is administered, it is essential to determine the viral titer in every batch precisely.
Since gene therapies are delivered directly into patients’ systems, the cultures in which these therapies are produced must be kept completely free of impurities. However, they’re prone to multiple contaminants such as empty capsids, immunogenic protein impurities, host cell DNA fragments, and potentially harmful organisms like mycoplasma. Some contaminants can decrease the efficacy of the therapy by artificially lowering the de facto dosage, while others can be actively harmful. For example, host cell DNA fragments can potentially integrate into the patient genome and become oncogenic. Host cell proteins can trigger dangerous immune responses. Mycoplasma and other bacteria can put patients at risk of dangerous respiratory illness or other infections. Unfortunately, many of these contaminants are notoriously difficult to detect because of their small size or low concentration. Developers need to make sure they have sensitive, efficient tools on hand to test batches at every step and make sure all the contents are accounted-for.
Cell therapies: characterizing cells and their behavior
While concerns like avoiding contamination still apply for cell therapies, manufacturers’ primary concerns in this field revolve around creating effectively transformed cells and closely observing their behavior once introduced back into patients’ bodies.
Cell therapy developers generally transfect patients’ cells with therapeutic transgenes, such as the CAR gene intended to prime the immune system against cancer. Each cell must contain the correct number of transgenes to strike a balance between efficacy and safety — a cell with too few transgenes won’t perform its intended function. Still, a cell with too many might create dangerous side effects. With ideal transgene copy numbers as low as 1-4 copies per cell, only the most sensitive nucleic acid detection assays can ensure that cells fall in the right range.
Even after successfully developing a cell therapy, scientists and physicians cannot perfectly predict how the live cells will behave inside a patient once the therapy is delivered. Therefore, they need tools to monitor patients’ blood samples to see how long the cell therapy persists at significant concentrations. Ideally, these tools should be not only accurate but also cost-effective to make serial monitoring practical.
Solving challenges with sensitive assays
To address these diverse manufacturing challenges, many cell and gene therapy manufacturers are transitioning from qPCR to Droplet Digital PCR (ddPCR) technology for detecting contaminants, determining dosages, monitoring cell persistence, and more.
ddPCR assays offer higher precision and sensitivity; furthermore, since this technology does not rely on standard curves, it offers absolute quantification and reduces chances for measurement errors. By delivering an absolute count of proteins or nucleic acids in a therapeutic sample, ddPCR assays can increase scientists’ confidence in their standard dosage determinations and subsequent quality control measurements.
Cell and gene therapies are game-changing tools in the fight against cancer, rare disease, and other threats to human health. Leveraging ddPCR technology to fine-tune these therapies is the best way to ensure we realize that potential. Bio-Rad experts are intimately familiar with the myriad different analytes biomanufacturers may need to measure, so we’ve made it a company priority to help developers find the ready-made assays they need or design fully-functional custom assays in just minutes.
Explore Bio-Rad’s Droplet Digital PCR Assay Design Engine to find assays for every step of cell or gene therapy manufacturing.
1Center for Biologics Evaluation and Research. Approved cellular and gene therapy products. Retrieved July 01, 2021, from https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products
2 U.S. National Library of Medicine Search Engine. ClinicalTrials.gov. Retrieved July 01, 2021, from https://clinicaltrials.gov/ct2/results?term=%E2%80%9Cgene%2Btherapy