Thesis Raw Data: "Microfluidic manufacturing and development of a liposomal vaccine for Clostridium difficile"



Oral vaccination is a highly sought after route of administration due to significant advantages over other parenteral routes including high degrees of patient compliance as well as ease of administration. Furthermore, oral immunisation can generate both systemic and localised immune responses, making it an ideal for generating effective protection against many enteric pathogens such as the gram-positive spore forming bacterium Clostridium difficile (C. difficile). Despite these desirable attributes, design of an effective vaccine for oral administration remains a significant challenge. In order to reach the relevant sites for the induction of immunological responses, the vaccine must first traverse the hostile environment of the gastrointestinal tract. For vaccine subunit antigen acidic pH, degradative enzymes and bile salts can adversely impact upon structural stability, limiting the effectiveness of the immune response. In addition, the mucosal surface found throughout the gut can impair the transport of the vaccine antigen and limit the accessibility to the underlying antigen presenting cells. In order to circumvent these barriers, delivery systems can be employed. Lipid-based delivery systems are commonly used to deliver therapeutics via the oral route of administration, and included among these are liposomes. Liposomes generally exhibit low toxicity, enhance interactions with biological membranes and can be incredibly versatile in regards to what compounds they can incorporate (whether they are encapsulated within the aqueous core, within the lipid bilayer or surface associated). However, the manufacture of liposomes has been limited to batch scale production systems which has inherent risk associated. By employing microfluidics as a manufacturing platform, in conjunction with tangential flow filtration for purification, scalable production of a range of liposomal formulations is shown. Key production parameters were identified (lipid / antigen concentration and flow rate ratio) and formulations were selected based upon their physicochemical characteristics (such as size and charge) and protein loading efficiency. Selected formulations were screened for immunological effectiveness on THP-1 macrophage-like cells in regards to antigen processing ability and activation marker expression. Finally, a range of formulations were then administered orally to determine in vivo efficacy (systemic and localised antibody responses). Enhanced antibody responses (IgG) could be observed when antigen was administered encapsulated within liposomal formulation DSPC:Chol (alongside the potent adjuvant cholera toxin) when compared to free antigen alone, however poor localised IgA responses were observed for all liposomal formulations tested.

The work within this thesis presents a platform for the rapid and scalable manufacture, purification and at-line analysis of liposomal formulations incorporating protein, de-risking the translation of liposomal based protein products from bench to large scale production.
Date made available11 Mar 2021
PublisherUniversity of Strathclyde

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