Bench to Bedside: Development of a cGMP Process for ORFV Viral Vector Based Vaccines

Abstract

The Orf virus (ORFV) has emerged as a promising platform for developing viral vector vaccines and immunotherapeutic approaches, owing to its high transgene capacity, potent immunomodulatory properties, the option for repeated booster vaccinations due to the low anti-vector immunity, and an overall favorable safety profile. However, until now there has been no systematically established, scalable process for reliably producing ORFV-based vaccines under GMP conditions. The aim of this work was therefore to develop an end-to-end manufacturing process for ORFV-based vaccines that ensures high yields, reproducibility, and GMP compliance. Using HEK293 cells in suspension culture, the upstream process was initially implemented in controlled bioreactors. By employing a systematic Design of Experiments (DoE) approach, critical process parameters - such as cell density at infection, MOI, pH, temperature, and dissolved oxygen - were precisely identified and optimized and successfully scaled up to a 50 L bioreactor. This enabled a more than one-hundredfold increase in virus titers (>10⁸ IU/mL) compared to the original Vero cell-based method. For the downstream process, a scalable clarification strategy was established, comprising nuclease treatment for targeted DNA reduction and a two-stage filtration with polypropylene-based depth filters. Subsequent chromatographic steps (ion exchange, hydrophobic, multimodal, and size-exclusion chromatography) achieved effective removal of host cell DNA and proteins at high yields. The purified virus batches met defined quality criteria for particle integrity, infectivity, and purity, thereby demonstrating suitability for clinical applications. To ensure final product stability, an extensive formulation screening was carried out. More than 30 excipients - among them disaccharides, recombinant albumin, and amino acids - were evaluated under various stress conditions. A formulation consisting of 1 % recombinant human serum albumin and 5 % sucrose in Tris buffer proved especially stable at 4 °C and under multiple freeze-thaw cycles. In addition, arginine-containing formulations enhanced stability at temperatures up to 37 °C, expanding the platform’s potential application range. Based on this developed manufacturing process, preclinical and clinical studies were conducted using multivalent ORFV-based SARS-CoV-2 vaccine candidates. The results demonstrate not only that the process development led to a robust production method but also that it lays the groundwork for future clinical and industrial applications of ORFV as a viral vector. Consequently, this dissertation lays a robust foundation for a scalable manufacturing platform that can be further leveraged for diverse ORFV-based vaccines and therapeutic applications.

Die Dissertation ist gesperrt bis zum 11. Juli 2027 !