Ana Sofia Moreira

The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 10¹¹ total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.

Chronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP’s therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG’s anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1β, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.

Biopharmaceuticals are currently becoming one of the fastest growing segments of the global pharmaceutical industry, being used in practically all branches of medicine from disease treatment to prevention. Virus-like particles (VLP) hold tremendous potential as a vaccine candidate due to their anticipated immunogenicity and safety profile when compared to inactivated or live attenuated viral vaccines. Nevertheless, there are several challenges yet to be solved in the development and manufacturing of these products, which ultimately can increase time to market. Suchlike virus-based products, the development of a platform approach is often hindered due to diversity and inherent variability of physicochemical properties of the product. In the present work, a flow-through chromatographic purification strategy for hepatitis C VLP expressed using the baculovirus-insect cell expression system was developed. The impact of operational parameters, such as residence time and ionic strength were studied using scaled-down models and their influence on the purification performance was described. The flow-through strategy herein reported made use of radial-flow chromatography columns packed with an anion exchanger and was compared with a bind and elute approach using the same chromatography media. Overall, by selecting the optimal operational setpoints, we were able to achieve higher VLP recoveries in the flow-through process (66% versus 37%) with higher removal of DNA, baculovirus and host-cell protein (92%, 99% and 50% respectively).