In 2017, the U.S. FDA approved the first gene therapy after decades of hope, setbacks, and finally success. Several others have since been approved and dozens more are in pivotal trials, cementing this targeted approach as a viable treatment option for a range of genetic disorders. These therapies can potentially be a one-time treatment, furthering expanding their potential.
Gene therapies work by providing a functional gene that can replace or compensate for a dysfunctional one. These can be delivered to the patient in a variety of ways. One such method is a non-replicative viral particle called adeno-associated virus (AAV).
Because of its newness, there are many challenges that must be overcome to ensure this type of therapy is safe, effective, and scalable. Based on our work in this area, we provide some key considerations how to navigate the field as a gene therapy developer.
Adeno-associated viruses (AAVs) are often used to deliver the DNA encoding the functional gene, drawing on their inherent ability to infect cells. A common challenge developers face is making sure the viral capsid is completely full. During production, some capsids may be empty or partially empty. This can cause several complications. For one, these partially full capsids can compete with their full and functional counterparts. This means a higher dose is needed to give the patient the desired therapeutic effect. The ineffective capsids can also trigger adverse complications, including life-threatening immunotoxicities.
There are several ways to measure the empty vs. full capsid ratio. During downstream processing, this type of impurity can be decreased with analytical ultracentrifugation or chromatography. While analytical ultracentrifugation is a standard in the field, high-pressure liquid chromatography (HPLC) with full/empty analytical columns is emerging as a superior option because it requires less of the AAV sample to run. With minimal sample input, HPLC provides highly robust quantification when separating full, partial and empty capsids.
Peptide mapping is used during process development and quality control to characterize the protein sequence of viral capsid proteins and to ensure that critical post-translational modifications are present on the AAV drug substance.
Liquid chromatography with tandem mass spectrometry is the used to conduct peptide mapping. This method provides a map of the primary protein sequence and post-translational modifications necessary for the AAV particle to function properly within the cell. It can also be used to identify any relevant changes that occur during capsid production, including any increased impurity levels associated with the manufacturing strategy. Scaling up production is inevitably complex, but with liquid chromatography with tandem mass spectrometry, developers can confidently manufacture a consistent product from batch to batch.
A safe and effective gene therapy must also be free of a variety of impurities that can arise during the development process. Two common residual impurities are polyethylenimine (PEI), which is used during AAV purification, and iodixanol, which is used to generate density gradient for the purification of the viral particles. HPLC is a good fit for the detection of both of these residual impurities. Conversely, Benzonase, which is used to digest superfluous DNA or RNA during the bioprocess, is best detected with ELISA or selective ion monitoring mass spectrometry.
Another important component is ensuring that the vector is pure and that there are no bioprocess intermediates, host cell protein contaminants or degraded/aggregated parts of the vector present. Typically, researchers use SDS-PAGE to test for vector purity. However, capillary electrophoresis and HPLC are superior methods that offers high precision, robustness and sensitivity.
The Future of Gene Therapy
For gene therapies to become a mainstream treatment option, developers and manufacturers need to work together to improve efficiency and decrease costs. Enhancements made at the process development and quality testing stage will decrease the production costs associated with what is now a highly expensive therapy.
For this level of efficiency to be achieved, there will need to be more process standardization. Finding the most effective way to measure things such as strength, potency, and particle-to-infectivity ratio of vectors will help researchers more effectively compare the therapy across different labs and studies.
Industry guidance is also constantly evolving. Just this past January, the FDA released seven new guidances. However, some developers are pushing the field forward and view these as the minimum standard. As the industry progresses and more is learned about gene therapy, we can expect these industry guidelines to progress with it.
There is great, life-changing potential that will soon be realized through gene therapies. By better understanding some of the common analytical challenges and working together as an industry to advance process efficiency, more patients will be able to benefit from these inspirational treatments.
Khanh provides clients with unparalleled expertise and tactical knowledge of protein biochemistry and molecular biology. Her experience extends from R&D to analytical method development, validation, implementation, method transfer, and optimization of test methods for the cGMP setting per USP and ICH guidelines. This understanding of the entire process helps guide successful and productive collaborations across different laboratories, sites, and functions. Khanh is also adept at protein expression and purification from E. coli and mammalian cells, in vitro potency assays, protein/DNA/peptide binding studies, ELISAs and other immunological methods, analytical chromatography, forced degradation studies, product quality investigations, and manufacturing investigations. Her technical background provides the foundation for effective authorship of analytical sections in BLAs and MAAs, as well as responses to requests and questions from the FDA, EMA, and PMDA.