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

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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).


BioPharm International Supplements


Results and Discussion

CpG-aluminum hydroxide adsorption isotherm. Because of the negatively charged phosphate groups that make up the CpG ODN backbone, these molecules are electrostatically attracted to the positively charged Alhydrogel. Besides electrostatic considerations, other mechanisms, including phosphate ligand exchange and hydrophobic interactions, also can attract adjuvants or antigens to the aluminum adjuvant.10


Figure 1. Adsorption isotherm of a modified CpG ODN (similar to CpG 10103) on aluminum hydroxide (Alhydrogel "85") as measured by centrifugal separation and absorbance at 260 nm.
The following illustrates two complementary procedures for determining the extent of CpG ODN adsorption to Alhydrogel "85". These techniques also are applicable to antigen adsorption studies.10 Aliquots of 100 mL Alhydrogel "85" (10 mg/mL aluminum) were pipetted into each of 10 microcentrifuge tubes. One hundred mg of CpG ODN (1 mg/mL) were added to the first microcentrifuge tube, 200 mg to the second tube, etc., with the last tube containing 1 mg of CpG ODN. Each tube contained the same total solution volume (1.1 mL). After briefly vortexing, the samples were incubated for 1 h at room temperature to allow for binding of CpG ODN to Alhydrogel "85". The samples were then centrifuged for 9 min at 16,000g to separate Alhydrogel "85" from aqueous solution (Alhydrogel "85" settles). The absorbance of supernatant at 260 nm was measured in each sample. The non-adsorbed concentration of CpG ODN using a standard curve was determined and subtracted from the original CpG ODN concentration to calculate adsorbed CpG ODN. Adsorbed CpG ODN versus original CpG ODN concentration in solution is plotted in Figure 1.


Figure 2. Adsorption isotherm of a modified CpG ODN (similar to CpG 10103) on aluminum hydroxide as measured by zeta potential titration. Error bars represent duplicate measurements at each concentration in three separate experiments.
A CpG ODN-aluminum hydroxide adsorption isotherm also can be constructed by measuring the zeta potential of aluminum hydroxide particles during CpG ODN titration. Increased negative charge on the particles reflected in lower zeta potential represents more bound CpG ODN, whereas a plateau in zeta potential represents maximum CpG ODN adsorption. To measure the zeta potential of CpG ODN molecules adsorbed to Alhydrogel "85", a stock solution of aluminum hydroxide was prepared at 1 mg/mL aluminum and CpG ODN at 4 mg/mL. The titrator was configured to inject 0.05 mg/mL aliquots of CpG ODN and collect duplicate measurements of zeta potential after each injection. Of course, if a titrator is not available, the zeta potential measurements can be carried out by preparing separate solutions, as was done for the UV absorption experiment above. The titration was continued until the final CpG concentration was 1.25 mg/mL, corresponding to 1.8 mg CpG ODN/mg aluminum. Zeta potential versus CpG ODN/aluminum w/w is plotted in Figure 2. Notice that the agreement between both adsorption isotherms is quite good (Figures 1 and 2).


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