SUPERVISOR: Nico LINGG

PROJECT ASSIGNED TO: Tomas MESURADO

Bionanoparticles (BNPs) are an emerging class of biopharmaceutical products. Bionanoparticles include viruses, virus-like particles (VLP) and extracellular vesicles (EV). VLPs have many advantages over other types of BNPs because they have precise and repeating structures and large carrying capacities. This type of nanomaterial can be produced in a variety of systems, including mammals, plants, insects, and bacteria. In particular, enveloped VLPs (eVLPs), which are encapsulated in a lipid bilayer from the cell in which they are expressed during the assembly and budding process, are gaining popularity in preventive medicine such as vaccines due to their high immunogenicity. Their surface composition is strongly dependent on the viral encoded proteins, the replication cycle and the host cell type in which they are produced, which in most cases increases the complexity and heterogeneity of the sample to be analysed. Moreover, determination of VLP concentration can be complicated because of the protein/nucleic acid content of the particles.

In addition, eVLPs tend to be more sensitive to the external environment than non-enveloped VLPs, which consist only of a protein capsid. Any change in conditions such as temperature, shear force and the purification process used can affect the integrity and stability of these particles and reduce their efficacy. Contaminants and impurities, including cell debris, host cell proteins, DNA and lipids, make the purification process a major challenge. The purity of the final product is a CQA that can affect the antigenicity of the eVLP. Analytical data is required to ensure that vaccine batches are equivalent in terms of potency, purity and physicochemical integrity in accordance with Good Manufacturing Practice (GMP).

As BNP based therapies continue to evolve, advances in analytical tools will be essential not only to determine particle concentration and calculate yield after each step, but also to evaluate process performance and gain more information from the sample characteristics. Current analytical methods typically require high laboratory workloads, long analysis times, low sample throughput and high variability of results. The lack of rapid at-line analytical methods to accurately characterize and quantify bionanoparticles remains a challenge and a problem for the development of viral therapies. Due to their overlap in size, buoyant density and similarity in membrane composition particle separation and discrimination from process and product related impurities have been a major challenge to accurately describe the quality and quantity of bionanoparticles.

The aim of this PhD project is to establish and optimize a downstream purification process based on non-woven fibers applicable to various bionanoparticles of interest, as no robust approach has yet fully resolved the issues of batch-to-batch variation, low yield and low purity. In parallel, HPLC combined with chromatography or asymmetric flow field fractionation (A4F) as separation techniques coupled to physical detectors such as UV, MALS and DLS will be used for particle separation and characterization.