Abstract
Subunit vaccines require particulate adjuvants to induce the desired immune responses. Pre-clinical manufacturing methods of adjuvants are often batch dependent, which complicates scale-up for large-scale good manufacturing practice (GMP) production. The cationic liposomal adjuvant CAF09b, composed of dioctadecyldimethylammonium bromide (DDA), monomycoloyl glycerol analogue 1 (MMG) and polyinosinic:polycytidylic acid [poly(I:C)], is currently being clinically evaluated in therapeutic cancer vaccines. Microfluidics is a promising new method for large-scale manufacturing of particle-based medicals, which is scalable from laboratory to GMP production, and a protocol for production of CAF09b by this method was therefore validated. The influence of the manufacture parameters [Ethanol] (20–40% v/v), [Lipid] (DDA and MMG, 6–12 mg/mL) and dimethyl sulfoxide [DMSO] (0–10% v/v) on the resulting particle size, colloidal stability and adsorption of poly(I:C) was evaluated in a design-of-experiments study. [Ethanol] and [DMSO] affected the resulting particle sizes, while [Lipid] and [DMSO] affected the colloidal stability. In all samples, poly(I:C) was encapsulated within the liposomes. At [Ethanol] 30% v/v, most formulations were stable at 21 days of manufacture with particle sizes <100 nm. An in vivo comparison in mice of the immunogenicity to the cervical cancer peptide antigen HPV-16 E7 adjuvanted with CAF09b prepared by lipid film rehydration or microfluidics showed no difference between the formulations, indicating adjuvant activity is intact. Thus, it is possible to prepare suitable formulations of CAF09b by microfluidics.
Highlights
The liposomal vaccine adjuvant CAF09b (Statens Serum Institut, Copenhagen, Denmark) is capable of inducing robust cytotoxic T-lymphocyte (CTL) responses [1], and is currently in first-in-man phase I clinical trials in a therapeutic vaccine combined with the peptide-based tumor associated antigen BCL-Xl against prostate cancer (NCT03412786) and in a neoepitope-based peptide cancer vaccine (NCT03715985)
Manufacture of CAF09b by the high shear mixing method is a several step process performed in batches involving the formation of a dry lipid film, a rehydration step to prepare the liposomes and a subsequent step for addition of poly(I:C) [1]
The total flow rate (TFR), on the other hand, was found not to affect the physicochemical characteristics of the liposomes. This is in accordance with a study applying microfluidics to formulate a variety of liposomes composed of different lipids, which showed that the flow rate ratio (FRR) was the main factor affecting the particle size, whereas TFR mainly had an impact on the speed of manufacture [21]
Summary
The liposomal vaccine adjuvant CAF09b (Statens Serum Institut, Copenhagen, Denmark) is capable of inducing robust cytotoxic T-lymphocyte (CTL) responses [1], and is currently in first-in-man phase I clinical trials in a therapeutic vaccine combined with the peptide-based tumor associated antigen BCL-Xl against prostate cancer (NCT03412786) and in a neoepitope-based peptide cancer vaccine (NCT03715985). Sonication or vortexing, which are methods often used to produce liposomes, the mechanism of action for high shear mixing is to break apart preformed large vesicles to form small-sized liposomes by using shear forces [4,5]. With this method, a lipid film is formed by evaporating organic solvent from dissolved lipids, forming sheets of stacked lipid bilayers deposited on the surface of the preparation vial [5]. Reduction of liposome particle sizes is regulated by varying the energy input, i.e., the applied shear force in high shear mixing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
