The innovation in gene therapy is poised to create a huge impact in the area of personalized medicine. Gene therapy holds promise for treating a wide range of diseases, from cancer and diabetes to rare genetic disorders, potentially revolutionize treatment modality making it an area of research of uttmost importance. Although some drugs have been discovered and approved, gene therapy has yet reach its full potential due to the complexity and regulatory procedures involved. This complexity also means that gene therapy can only be implemented in large bioproduction facilities.
Amongst different gene therapy materials production, viral vectors are a popular medium used to deliver genetic material for therapeutic application. Adeno-associated virus, or AAV, is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction, featuring high efficiency and excellent safety in human clinical trials. AAV-based products have been licensed to date in the US and EU for treatment of rare genetic diseases. Hurdles that have limited the pace of development of AAV-based gene therapies include those related to manufacturing capacity, quality control and deleterious immune responses that limit long-term expression.
As assay development scientist, I am very proud to be part of The Neuro-CERVO Alliance for Drug Discovery (NCADD) partnership between Affinite instruments and CERVO brain research center. The CERVO viral production platform, established more than 10 years ago, has been supplying researchers with research grade AAVs and other viral vectors. This project aims to develop new affordable quality control QC strategies to evaluate the potency and purity of AAV viral vectors throughout the course of the biomanufacturing process.
The AAV product is a function of vector and process design
During the production of AAV vectors, there are several quality control (QC) parameters that should be rigorously monitored to comply with clinical safety and efficacy. Two critical aspects for quality control are: viral potency and empty capsids impurities.
Viral potency determination is a critical characteristic that must be well established prior to use. Quantitative PCR techniques are the most frequent dose determining assay. Today at CERVO, droplet digital PCR technology is a de-facto. However, digital PCR is time consuming, expensive, and not easily integrable to the production process. This is where Affinité portable SPR can play an important role in terms of reducing time and cost, as well as integration into the quality control procedure. Using SPR technology, one of the key desired outcomes is to demonstrate the measure of total viral vector concentration using portable SPR.
Another key project objective is to determine empty/full capsid ratios. Empty viral capsids are a challenging and unique type of impurities for AAV vectors with the potential to induce toxicity and undesirable immune reactions, particularly if administered systematically in large doses. Another de facto technique today is the use of Transmission Electron Microscopy (TEM) for this analysis. Similarly to digital PCR, TEM is labour intensive and time consuming, as well requiring specialized trained personnel. Furthermore, TEM is normally performed towards the end of the production, detached from the continuous biomanufacturing process.
With the use of portable SPR, we hope to demonstrate the improvement of the manufacturing processes for pre-clinical and clinical AAV viral vectors with reliable and in-process AAV characterisation. In the coming months, a protocol will be developed for AAV titration and establishing a label-free method to measure the content ratio of a batch (full/total capsid) and providing inline monitoring of vector concentration during production that will significantly reduce costs, minimize work time and allow for disqualification of flawed patches early in the process.
Portable SPR a game changer for assay development and quality control
Coming from a medical background and having spent years in research laboratories particularly in the domains of immunology, virology, and infectious diseases research, I recall many innovative, promising research ideas that did not see the light due to financial and technical factors that resulted in time overrun frame or failed outcome. One such factor was due to the time consuming workflow of running an experiment like ELISA which can take hours (e.g. overnight incubation and 6-7 hours of work) to find out only towards the end that something went wrong, without any way to track the status during the run. Worse, there was no way to know what went run during an ELISA run and I often had to restart blindly, crossed my fingers and hoped for a better outcome. A portable SPR can improve such inefficient assay workflow by proving an orthogonal technique to ELISA to provide step-by-step debugging and real-time data.
In Figure 1 and Figure 2 below, a comparison between the AAV sample layout using ELISA and SPR shows that using ELISA requires more reagents and labeling, thus more expensive and time consuming in comparison to SPR which uses less reagents. And with SPR, the experimental results are in real time without the need for labeling.
Figure 1. Representation of AAV sample on SPR sensor chip
Figure 2. Representation of AAV sample on ELISA
Democratizing SPR can help to democratize gene therapy
Though promising, the road to democratize gene therapy can be a long winding road due to the need to have large biomanufacturing facility with expensive and slow quality control equipment. With the collaboration with CERVO, we hope to enable smaller labs and facilities also able to participate in the research of gene therapy using viral vectors. We are always looking for more partners to join this very exciting adventure. Contact us to learn how we are applying SRR protocols to set up a new measure of quality control at firstname.lastname@example.org