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


Association of CpG ODN with Emulsion Formulations

The association of CpG with the oil (squalene) phase is thought to be weak because of the unfavorable energetics of hydrophobic interactions. The partitioning of oligonucleotides is dominated by strong solvation in the aqueous phase, though surface or interfacial effects may be occurring through mediation of surfactants. However, even weak association with particles may have an effect on biological activity. Because of the small difference in density of the two phases, it is necessary to increase the density gradient by adding a high MW sucrose polymer such as Ficoll PM400 (GE Healthcare). Ficoll can be dissolved in water to create a solution that has a significantly higher density than water because of its high molecular weight and excellent water solubility. This allows separation of the phases in an emulsion using a standard microcentrifuge. Alternatively, an ultracentrifuge may be used to separate the phases without adding the sucrose polymer.3


Table 2. Association of CpG ODN with oil-in-water emulsion
To determine the extent of CpG ODN associated with the oil particulates in the SE formulation, a 30% (w/v) solution of Ficoll in water was mixed at a 1:1 ratio with the SE formulation (10% oil v/v and 1.25 mg/mL CpG ODN) in a microcentrifuge tube and vortexed briefly. The sample was centrifuged for 30 min at 16,000g. After centrifugation, the sample was carefully removed and the tube bottom carefully punctured with a 27g half-inch needle to extract an aliquot of the lower aqueous phase. The absorbance at 260 nm was then measured and compared to a CpG ODN standard. The difference in absorption represents the amount of CpG ODN associated with the oil phase (Table 2). In this example, all of the CpG ODN was in the aqueous phase, as was the case for a CpG-MF59 formulation that underwent ultracentrifugation.3 This probably is because these emulsions use nonionic surfactants or neutral phospholipids as emulsifiers. Emulsions made with charged emulsifiers show higher CpG ODN association with the oil phase and somewhat better immune activity; however, strong oil association is not necessary for synergistic adjuvant effects demonstrated with neutral emulsion-CpG ODN formulations.3

Because CpG ODNs are efficiently ionized at physiological pH, a change in electrophoretic mobility (zeta potential) of the emulsion can be observed if the CpG ODN molecules associate preferentially into the interface between the oil and water phases. Differences in the composition and local chemical potentials at the interface can adjust to balance the electrostatic–hydrophobic interactions that shift as the activity of CpG grows with increasing solution concentration. To measure the change in emulsion interfacial charge caused by CpG ODN interactions, a titration of zeta potential versus CpG ODN concentration was performed. The emulsion used here was the MF59-like emulsion described above. The emulsion was diluted 10-fold in water to permit light beam transmission of the scattering solution, as well as lowering the ionic strength to decrease the conductivity and increase the Debye length, permitting a more accurate measurement.


Figure 4. Zeta potential measurement of the titration of CpG 1826 in an MF59-like emulsion. Error bars represent triplicate measurements from a single experiment.
The diluted emulsion was titrated against 4.0 mg/mL CpG ODN and changes in emulsion surface charge and curvature were measured by measuring both zeta potential and particle size as described above. In this example, the effects were subtle, with a gradual downward slope in zeta potential from 0 to ~0.1 mg/mL CpG ODN, a gradual upward slope from ~0.1 to 0.25 mg/mL (Figure 4), and an abrupt shift of ~2 mV at ~0.25 mg/mL CpG ODN, possibly caused by a reorganization of the molecules in the interfacial Stern layer to accommodate the changes in the aqueous phase as solvation of the added CpG becomes more extensive. At higher CpG ODN concentrations up to 0.9 mg/mL, the zeta potential is relatively constant.

Conclusion

Appropriate formulation of CpG ODN can significantly improve vaccine adjuvant activity. Rational design of CpG ODN formulations is enabled by thorough characterization of fundamental formulation parameters. In addition, changes in vaccine excipient composition should be monitored to determine effects on CpG ODN formulation. In this work, simple but informative physicochemical analytical techniques have been effectively used to determine the extent of CpG ODN association with particulate formulations, namely aluminum hydroxide and oil-in-water emulsion formulations.


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