Development of a continuous downstream process for microbial produced virus-like particle based Group A Streptococcus vaccine

Abstract

The current pandemic outlines the need for efficient and mass-produced vaccines to fight infectious diseases. Group A Streptococcus is a human pathogen causing hundreds of millions of infections every year and leading to over 500,000 deaths annually. Unfortunately, there is still no efficient vaccine developed today. A novel class of vaccines that can overcome some of the limitations of traditional vaccines are virus-like particles (VLPs) that can induce strong and long-lasting immune responses. However, caused by lengthy and complicated production routes they are also among the most expensive. Microbially-expressed modular virus-like particles with inserted foreign antigens are a promising platform technology for rapid and low-cost vaccines but their current purification methods lack scale-ability and process performance. A main burden during processing is the formation of soluble aggregates and poor performance during chromatographic purification. Processing of viral capsomeres usually requires hard-to-scale unit operations and enzymatic treatment to remove DNA. The first half of my project, therefore, investigated the cause of the aggregate formation and investigated possible purification pathways. It was found that a main source of soluble aggregates during processing is DNA-capsomere aggregates, caused by the strong DNA affinity of viral capsomeres. This aggregation does not only lead to poor product quality during viral assembly but is also responsible for poor chromatographic performance as the large-size aggregates cannot enter the pores of the chromatographic resin and hinder the efficient removal of DNA. It was furthermore shown, that these DNA-capsomere interactions can be controlled by adjusting the ionic strength of the buffer system and high salt buffers can suppress DNA-capsomere interactions. Controlling DNA-capsomere interactions did not only lead to better product quality and improved DNA removal, but it also enables efficient chromatographic processing at elevated salt concentrations using salt-tolerant mixed-mode ion exchange resins. The novel approach of processing viral capsomeres at elevated salt concentrations, suppressing DNA-capsomere interactions, with novel salt-tolerant mixes mode ion exchange resins led to the development of several integrated purification pathways, that were optimized using high-throughput binding studies. The optimized purification pathways were comparatively evaluated using a range of analytical techniques. After optimization, the preferred integrated purification pathway was capable of producing highly pure virus-like particles without any of the common hard-to-scale unit operations found in other described methods and showed outstanding process performance. The finding of these two studies are not only relevant for my specific virus-like particle but are also of high interest if other viral proteins are processed as the underlying DNA binding sites are conserved within many viruses. Furthermore, the finding can explain many challenges occurring during the processing of virus-like particles and viral proteins, that are described in the literature. The second part of my studies focuses on the transition of the batch process to a continuous one. Continuous bioprocessing gains significant attention within the biopharmaceutical industry in recent years, as it promises faster, cheaper, and more consistent production. However, unlike other industries, continuous biomanufacturing is still in its infancy. Research in this field mostly concentrates on antibody production and VLPs are not addressed yet. This project, therefore, aims to widen the research field of continuous biomanufacturing to virus-like particles, and an automated, continuous production route was developed. Based on the findings of the first part of my studies the first fully integrated and continuous purification pathway for virus-like particle vaccines was developed. The process showed a robust behaviour over a wide range of inlet stream concentrations and produced Group A Streptococcus virus-like particle vaccines of constantly high quality over the examined period. A novel key concept is the integration of a flow-through step with multi-modal cation exchanger in a periodic counter-current chromatography (PCC) set-up, that allows seamless integration of the two-unit operations without buffer adjustment in between. The process also shows the possibility to remove the reducing agent DTT during column elution using chromatography, eradicating an additional buffer exchange unit operation before in-line viral assembly. A main challenge to solve was the development of a new control strategy for continuous periodic counter-current chromatography subject to unstable/fluctuating inlet stream concentrations. Current control strategies fail if the inlet stream concentration does not remain constant during column loading. The developed approach was modelled in-silico, using mechanistic models, and enables a more robust controlling of PCC processes than common approaches. Apart from describing the first continuous purification pathway for virus-like particle vaccines, the described process might lay the fundamentals for continuous production for a wide range of biopharmaceuticals. The process can be easily adapted to other biopharmaceuticals as the process can be changed using different chromatographic resins and is not constructed around a specific affinity chromatographic step. This project outlines the development of a continuous manufacturing process for microbially expressed VLP vaccines against Group A Streptococcus from scratch. It shows exemplarily the development and optimization of a batch process and the subsequent transition to a continuous and integrated process and therefore can serve as a blueprint for the development of continuous processes for biopharmaceuticals other than VLPs.