Characterization of Aluminum Hydroxide Gel and Oil-in-Water Emulsion Formulations Containing CpG ODNs - Adjuvant activity can be greatly improved by appropriate formulation of cytosine


Characterization of Aluminum Hydroxide Gel and Oil-in-Water Emulsion Formulations Containing CpG ODNs
Adjuvant activity can be greatly improved by appropriate formulation of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs).

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CpG ODNs. CpG oligodeoxynucleotides (ODNs) were obtained from Avecia Biotechnology and Coley Pharmaceutical Group. Several different sequences are available and efficacy varies between animal models depending on the sequence. The ODNs described here are a modified CpG ODN similar to 10103 (the order of two sequential base pairs were switched from CpG ODN 10103): TCG TCG TTT TTC GGT GCT TTT, CpG 2395: TCG TCG TTT TCG GCG CGC GCC G, and CpG ODN 1826: TCC ATG ACG TTC CTG ACG TT.8,9 CpG ODN (similar to 10103) from Avecia is supplied as a dry powder and stored at –20 C. CpG 1826 was purified with an ethanol precipitation step and reconstituted in 10 mM pH7 Tris with 1 mM EDTA. CpG ODN absorbance values were measured using a Beckman Coulter DU 640i or a Hitachi 3900H UV-Vis spectrophotometer.

Aluminum. Alhydrogel "85" was manufactured by Brenntag Biosector. Alhydrogel "85" consists of aluminum hydroxide gel containing small aggregates (~1 to 10 mm) of high surface area and positive charge.10 X-ray diffraction and infrared spectroscopy data indicate that the structure of aluminum hydroxide adjuvant resembles boehmite with the chemical formula AlO(OH), instead of the implied formula of Al(OH)3.10,11 The aluminum content in Alhydrogel "85" is 1% or 10 mg/mL.12 Alhydrogel is stored at room temperature and settles with time, so vigorous shaking is required before use. Aluminum hydroxide zeta potential measurements were measured using a Zetasizer Nano-ZS and an MPT-2 multi-purpose titrator from Malvern Instruments.

Oil-in-water emulsion. An oil-in-water stable emulsion (SE) was manufactured at the Infectious Disease Research Institute. Squalene (Sigma-Aldrich), purified from shark liver, was used for the oil source and was present at 10% (v/v) in the final product. High purity (99%) phosphatidylcholine (Avanti Polar Lipids) from chicken egg yolks, was used as a natural emulsifier and was present at 1.92% (w/v) in the final product. Poloxamer 188 (Pluronic F68 from BASF), a synthetic triblock copolymer nonionic surfactant, was used as a coemulsifier at a final concentration of 0.09% (w/v). To create an isotonic formulation, glycerol (Sigma-Aldrich) was included in the SE at 1.8% (v/v). Alpha-tocopherol (Spectrum Chemical) was used as an antioxidant and present at 0.05% (w/v). A 25 mM ammonium phosphate buffer was used to maintain the aqueous phase at pH 5.1 0.05.

The oil phase, consisting of the squalene, phosphatidylcholine (PC), and alpha-tocopherol, was prepared by sonication in a heated (~50 C) water bath until the PC was fully dissolved (typically 1–2 h). The aqueous phase was prepared by combining deionized water with glycerol, Pluronic F68, and ammonium phosphate buffer. The buffered aqueous phase was added at 90% (v/v) to the oil phase and then mixed with a Silverson heavy-duty laboratory mixer emulsifier (3/4 inch tubular square hole high shear screen attachment) at ~8,000–10,000 rpm for several minutes. The mixture was subjected to high-pressure homogenization using the Microfluidics M-110EH-30 for 12 passes at ~207 MPa (~30,000 psi).

Particle diameter was ~100–110 nm as measured by dynamic light scattering using the Malvern Instruments Zetasizer Nano-S. Visually, the SE had the appearance of homogenized milk. The SE formulation was stored in rubber-stoppered glass vials at 2–8 C and demonstrated good stability (minimal change in particle size and no visual phase separation) for at least 1 year. The aqueous phase separation procedure described in the text was carried out using Ficoll PM400 obtained from GE Healthcare. In addition to the manufacture of SE, an MF59-like emulsion was manufactured using similar procedures to produce an oil-in-water formulation containing 5% v/v squalene and 0.5% v/v each of Tween 80 and Span 85 as surfactants; the water in this case was unbuffered.

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