CIM TECHNOLOGY: ENABELING ECONOMIC VACCINE PURIFICATION

Vaccines are diverse and complex biological molecules, complexes and particles. Different production technologies are used for vaccines manufacturing and every production process require somekind of vaccine purification. Purification of vaccines is technically challenging and was traditionally inefficient and partially neglected due to different economical and technical reasons. Density gradient ultracentrifugation, introduced in1960s is still a major purification step for many vaccines present on the market. As a complementary techniques, cross flow filtration and chromatography has been used. Conventional chromatography supports designed for protein purification have relatively small pore sizes with restricted access for large molecules and viruses. In addition mass transfer in resins is based on diffusion and as such is not optimal for large molecules. An alternative to conventional resins are monoliths characterized by large flow through channels and convective mass transfer which results in a high and flow-independent dynamic binding capacity and resolution. Together with high low rates these properties enable increased productivity which makes monoliths attractive chromatographic supports for viruses and plasmid DNA purification. The presentation will focus on development and optimization of a purification processes for influenza viruses, adenoviruses, bacteriophages, and plasmid DNA. The characteristics of plasmid DNA and viruses are peculiar obstacles for chromatography and inherent limitations will be presented. Chromatography methods with an emphasis on ion exchange and hydrophobic interaction monolithic chromatography will be discussed and impact of monolith chromatography on process economic will be presented

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification

HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation