Monitoring the effects of component structure and source on formulation stability and adjuvant activity of oil-in-water emulsions

doi:10.1016/j.colsurfb.2008.03.003

Colloids and Surfaces B: Biointerfaces

Volume 65, Issue 1, 1 August 2008, Pages 98-105

https://doi.org/10.1016/j.colsurfb.2008.03.003 Get rights and content

Abstract

Oil-in-water emulsions have shown promise as safe and effective adjuvant formulations for vaccines. In particular, formulations consisting of metabolizable oils such as shark-derived squalene and detergents such as egg phosphatidylcholine have been used to produce stable vaccine emulsion formulations. However, there is an emphasis in pharmaceutical regulatory bodies on using synthetic or plant-derived components from sustainable sources instead of animal-derived components. This study compares the physicochemical properties and biological efficacy of emulsions consisting of oil and detergent components from animal, plant, and synthetic sources. In particular, effects of component structure and source on emulsion stability and biological activity are examined. It is shown that oil-in-water emulsions using animal-derived components can be substituted with synthetic or plant-derived materials while still exhibiting satisfactory physicochemical and biological properties.

Introduction

A great deal of attention has been recently directed towards the development of new adjuvant formulations. These adjuvant systems are typically a combination of an immunostimulatory molecule and a delivery system such as alum salt particulates, emulsions, or liposomes. They are used in conjunction with vaccine antigens to potentiate immune responses (humoral and/or cell mediated) or to reduce the amount of antigen needed to elicit a given response [1], [2], [3]. Specific vaccine antigens may be more effective with one adjuvant system as compared to another [2]. Currently, alum is the sole FDA-approved adjuvant delivery system [4], [5]. Although alum-based adjuvants have exhibited an acceptable safety profile for many years, they have been known to induce local reactogenicity [6], and aluminum is a known neural toxin [7]. Therefore, a need exists to develop additional adjuvant systems that are both safe and effective.

Various adjuvant formulations other than alum are being researched, including aqueous formulations [4], [8], emulsions [9], [10], liposomes [11], [12] and other small particles [9], [13]. Stable emulsions (SE) are adjuvant delivery systems likely to garner FDA approval (approval in Europe has already been achieved for the emulsion formulation MF59) [9] and there are multiple ongoing clinical trials utilizing emulsion delivery systems [1], [9]. Typically, SE formulations are composed of metabolizable oil droplets, such as squalene, stabilized by one or a combination of natural and synthetic detergents in a buffered aqueous phase (oil-in-water or o/w) [14]. The converse is also an option, where water droplets are stabilized in an oil phase (water-in-oil or w/o). Squalene and phosphatidylcholine (PC), a natural detergent commonly used in SE formulations, are often from animal sources such as shark liver and chicken egg yolk, respectively. For regulatory and sustainability purposes it would be more desirable to utilize plant-derived or synthetic sources if SE is to become a widely used adjuvant delivery system. These plant- or synthetic-based systems must have stability and immunopotentiating profiles similar to traditional animal-derived emulsions while remaining cost effective.

This study aims to examine the stability of SE formulations consisting of oil and detergent components from animal, plant, and synthetic sources. Specifically, olive-derived squalene and the plant-derived Miglyol 810 oil were compared to shark-derived squalene. The contribution of egg yolk PC to emulsion stability as compared to soy-derived PC or synthetic PC was also investigated. Furthermore, the effects of the detergents polysorbate 80 (Tween 80^®) vs. Poloxamer 188 (Pluronic F68^®) on emulsion stability were evaluated. Finally, in vivo antibody responses were compared using the different emulsion components. Together, physicochemical and biological data from these emulsions indicate that plant-based formulations can be made that exhibit no significant reduction in adjuvanticity or stability compared to the animal-based formulations.

Section snippets

Materials

Shark liver squalene was purchased from Sigma–Aldrich (St. Louis, MO). Olive-derived squalene was purchased from Wilshire Technologies, Inc. (Carlsbad, CA) and NutriScience Innovations (Trumbull, CT). Miglyol 810, a capric/caprylic acid-based metabolizable oil, was obtained courtesy of Sasol Germany GmbH (Witten, Germany). Egg yolk PC, soy PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),

Results

Emulsion droplet size is an indicator of stability since it is thermodynamically more favorable for emulsions to minimize interfacial contact by decreasing droplet surface area/volume ratio until phase separation. After examination of visual appearance, emulsion particle size from each sample was analyzed periodically over at least 3 months using DLS. Within the scope of this study, an unstable emulsion was defined as visible evidence of phase separation of the oil and aqueous phases and/or oil

Oil source

There appears to be a slight improvement in the stability of shark-derived squalene emulsions compared to olive-derived squalene emulsions (Table 2, Table 3, Table 4). This discrepancy in stability may be due to differences in compound purity. Shark-derived squalene used in this study was at least 99% pure as stated by the manufacturer, whereas olive-derived squalene purity ranged from 85% to 97% depending on the manufacturer. Indeed, HPLC analysis revealed various unique peaks in the

Acknowledgements

This work was supported in part by grant #42387 from the Bill and Melinda Gates foundation and by a National Institutes of Health grant #AI25038. We thank Dr. Martin Friede for useful discussions.

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Monitoring the effects of component structure and source on formulation stability and adjuvant activity of oil-in-water emulsions

Abstract

Introduction

Section snippets

Materials

Results

Oil source

Acknowledgements

Vaccine

Neurosci. Biobehav. Rev.

Vaccine

J. Control. Rel.

Trends Parasitol.

Pharm. Sci. Tech. Today

Colloids Surf. B: Biointerf.

Eur. J. Pharm. Biopharm.

Adv. Colloid Interf. Sci.

Colloids Surf. B: Biointerf.

J. Control. Rel.

J. Control. Rel.

Trends Food. Sci. Technol.

Vaccine

Vaccine

Eur. J. Pharm. Sci.

J. Pharm. Sci.

Biophys. J.

J. Colloid Interf. Sci.

Molecular Biology of the Cell

Nat. Rev. Drug Discov.