The use of capillary electrophoresis in Gene Therapy

Abstract

Background & Aim Capillary Electrophoresis (CE) has undergone a huge resurgence over the last 15 years as the pharmaceutical industry moved from traditional small molecule pharmaceuticals into more complex biologicals and protein and peptide-based biopharmaceuticals. Today there is a drive to look for more target approaches to treat rare diseases and one of the areas which is seeing a rapid increase in funding is the development of gene therapy-based products. Gene therapy products often use a viral vector (capsid) as a carrier to transfer DNA/RNA to cells and these products provide an additional level of complexity over antibodies so profiling and analysing such products has thrown up new challenges for analytical chemists. In this work we will present how CE has been used to profile and analytical test new Gene Therapy products. The methods takes advantage of the traditional approaches of CE used in monoclonal antibody testing and adapts them to analysing the building blocks of the gene therapy product as well as the final product itself. Methods, Results & Conclusion The methods used are based on CE-SDS protein assays or capillary isoelectric profiling methods (cIEF) using commercially available capillaries and consumables. In the analysis of viral proteins which make up the adeno associated virus (AAV) vectors a new protein derivatisation step has now been developed to fluorescently tag the proteins in a simple 2 step process to enhance the sensitivity and detect viral capsid proteins. The sensitivity improvement is required due to the lower protein concentrations found in gene therapy products. The CE-SDS method developed using this new flourescent tag has allowed AAV viral proteins to be detected at a viral particle concentration of 1.1 × 10 10 GC/ml and is very reproducible. In a new application using cIEF we will show how CE is now able to profile empty vs full AAV gene therapy products and is capabale of being applied to several different serotypes. In conclusion CE can now be used in the analysis of gene therapy products and can not only profile the proteins making up the viral vectors but can also be used to profile the AAV final products to provide information on empty versus full particles.