Optimizing manufacturing and composition of a TLR4 nanosuspension: physicochemical stability and vaccine adjuvant activity.

BACKGROUND: Nanosuspensions are an important class of delivery system for vaccine adjuvants and drugs. Previously, we developed a nanosuspension consisting of the synthetic TLR4 ligand glucopyranosyl lipid adjuvant (GLA) and dipalmitoyl phosphatidylcholine (DPPC). This nanosuspension is a clinical vaccine adjuvant known as GLA-AF. We examined the effects of DPPC supplier, buffer composition, and manufacturing process on GLA-AF physicochemical and biological activity characteristics. RESULTS: DPPC from different suppliers had minimal influence on physicochemical and biological effects. In general, buffered compositions resulted in less particle size stability compared to unbuffered GLA-AF. Microfluidization resulted in rapid particle size reduction after only a few passes, and 20,000 or 30,000 psi processing pressures were more effective at reducing particle size and recovering the active component than 10,000 psi. Sonicated and microfluidized batches maintained good particle size and chemical stability over 6 months, without significantly altering in vitro or in vivo bioactivity of GLA-AF when combined with a recombinant malaria vaccine antigen. CONCLUSIONS: Microfluidization, compared to water bath sonication, may be an effective manufacturing process to improve the scalability and reproducibility of GLA-AF as it advances further in the clinical development pathway. Various sources of DPPC are suitable to manufacture GLA-AF, but buffered compositions of GLA-AF do not appear to offer stability advantages over the unbuffered composition.

Cryogenic transmission electron microscopy of recombinant tuberculosis vaccine antigen with anionic liposomes reveals formation of flattened liposomes.

Development of lipid-based adjuvant formulations to enhance the immunogenicity of recombinant vaccine antigens is a focus of modern vaccine research. Characterizing interactions between vaccine antigens and formulation excipients is important for establishing compatibility between the different components and optimizing vaccine stability and potency. Cryogenic transmission electron microscopy (TEM) is a highly informative analytical technique that may elucidate various aspects of protein- and lipid-based structures, including morphology, size, shape, and phase structure, while avoiding artifacts associated with staining-based TEM. In this work, cryogenic TEM is employed to characterize a recombinant tuberculosis vaccine antigen, an anionic liposome formulation, and antigen-liposome interactions. By performing three-dimensional tomographic reconstruction analysis, the formation of a population of protein-containing flattened liposomes, not present in the control samples, was detected. It is shown that cryogenic TEM provides unique information regarding antigen-liposome interactions not detectable by light-scattering-based methods. Employing a suite of complementary analytical techniques is important to fully characterize interactions between vaccine components.

In vitro evaluation of TLR4 agonist activity: formulation effects.

Effective in vitro evaluation of vaccine adjuvants would allow higher throughput screening compared to in vivo studies. However, vaccine adjuvants comprise a wide range of structures and formulations ranging from soluble TLR agonists to complex lipid-based formulations. The effects of formulation parameters on in vitro bioactivity assays and the correlations with in vivo adjuvant activity is not well understood. In the present work, we employ the Limulus amebocyte lysate assay and a human macrophage cellular cytokine production assay to demonstrate the differences in in vitro bioactivity of four distinct formulations of the synthetic TLR4 agonist GLA: an aqueous nanosuspension (GLA-AF), an oil-in-water emulsion (GLA-SE), a liposome (GLA-LS), and an alum-adsorbed formulation (GLA-Alum). Furthermore, we demonstrate the importance of the localization of GLA on in vitro potency. By comparing to previous published reports on the in vivo bioactivity of these GLA-containing formulations, we conclude that the most potent activators of the in vitro systems may not be the most potent in vivo adjuvant formulations. Furthermore, we discuss the formulation considerations which should be taken into account when interpreting data from in vitro adjuvant activity assays.