Advances in manufacturing processes provide the ability for the high throughput production of liposomes containing a range of moieties, from small molecules to large biologicals (including proteins and nucleic acids for prophylactic and therapeutic applications). Whilst rapid quantification methods for small molecules are generally well established, the ability to rapidly quantify liposomal entrapment of proteins is limited. Indeed, most standard protein quantification techniques (including the BCA assay and Reverse phase-high performance liquid chromatography (RP-HPLC)) measure protein encapsulation indirectly, by measuring the amount of non-incorporated drug, and subtracting from the initial amount of protein added. However, this can give inaccurate and misrepresentative results. To address this, we have developed a range of methods to directly quantify protein entrapment within liposomes. The encapsulation efficiency within neutral, anionic and cationic liposome formulations was determined by three techniques; BCA assay, RP-HPLC and HPLC coupled to an evaporative light scattering detector, (HPLC-ELSD). All three methods are reliable for the quantification of protein, with linear responses and correlation coefficients of 0.99, and LOQ for all three methods being less than 10 µg/mL. Here within, we provide three methods for the rapid and robust quantification of protein loading within liposomal (and other bilayer) vesicle systems.

Formulation of inhalable delivery systems containing tuberculosis (TB) antigens to target the site of infection (lungs) have been considered for the development of subunit vaccines. Inert delivery systems such as poly (lactic-co-glycolic acid) (PLGA) are an interesting approach due to its approval for human use. However, PLGA suffers hydrolytic degradation when stored in a liquid environment for prolonged time. Therefore, in this study, nano- and microparticles composed of different PLGA copolymers (50:50, 75:25 and 85:15), sucrose (10% w/v) and L-leucine (1% w/v) encapsulating H56 TB vaccine candidate were produced as dried powders. In vitro studies in three macrophage cell lines (MH-S, RAW264.7 and THP-1) showed the ability of these cells to take up the formulated PLGA:H56 particles and process the antigen. An in vivo prime-pull immunisation approach consisting of priming with CAF01:H56 (2 × subcutaneous (s.c.) injection) followed by a mucosal boost with PLGA:H56 (intranasal (i.n.) administration) demonstrated the retention of the immunogenicity of the antigen encapsulated within the lyophilised PLGA delivery system, although no enhancing effect could be observed compared to the administration of antigen alone as a boost. The work here could provide the foundations for the scale independent manufacture of polymer delivery systems encapsulating antigens for inhalation/aerolisation to the lungs.