Analytical characterization of cell and gene therapy (CGT) products remains a challenging, costly, and time-consuming task. Additionally, the relatively high sample volume typically required for analytical testing of such products is often considered a challenge by itself. To overcome those issues, new technologies can be used, and we would like to present a proof-of-concept work demonstrating application of multidimensional chromatography for characterization of Adenovirus type 5 (Ad5)-based product. In our case study, we focused on Ad5 quantitation by AEX-HPLC and identity by RP-HPLC described in the literature [Blanche et al., Gene Ther., 7 (2000) 1055-1062 and Lehmberg et al., J. Chromatogr. B, 732 (1999) 411-423]. In the first step, we were able to significantly reduce the sample run time by using modern chromatographic columns and optimizing gradients, while maintaining selectivity and overall quality of the individual assays. In the second step, we combined two analytical assays into one functional 2D LC assay allowing for Ad5 quantitation and confirmation of its identity.
The resulting 2D LC method includes first-dimension AEX separation, which generates the data that can be used for quantitation of Ad5 viral particles using the area-under-the-curve approach and an appropriate calibration curve, followed by automated transfer of the material representing the Ad5 peak from the first to the second dimension, and second-dimension RP separation, which generates the characteristic pattern of Ad5 capsid proteins that can be used for verifying identity of Ad5.
The approach presented here not only allowed us to increase productivity, but also to limit the sample volume needed for the analysis. We envision that by applying the concept presented in this proof-of-concept work, it will be possible to shorten the time necessary to perform analytical testing of CGT products and reduce the required volume of samples needed to complete the analysis.