Developing a Sterile and Scalable Polishing Step for a Large Live Virus Purification

Live attenuated virus (LAV) vaccines offer the benefit of stimulating humoral and cellular immune responses, which is recognized as an advantage over many subunit vaccines. However, process development of LAV vaccines presents many unique challenges due to their size, molecular complexity, and structural heterogeneity. Downstream purification is further limited by a number of factors, including low binding capacity for many viruses on traditional chromatography resins, constraints on operating conditions (e.g. pH, use of detergents), and the requirement of closed-system aseptic processes for those viruses that are too large for terminal sterile filtration. This work examines several purification methods as a polishing step for a candidate LAV vaccine requiring significant purification as well as aseptic processing. A variety of purification approaches were evaluated including membrane supports, newly designed bead-based media for viruses and large biomolecules, and non-chromatographic methods such as selective precipitation and centrifugation. Polishing steps were evaluated based not only on usual performance criteria such as purification potential and yield but also on their ease to be performed aseptically and their potential for scale up.