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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Adsorption Effects of Excipients, Other Adjuvants, or Antigen

Table 1. Adsorbtion of CpG ODN to aluminum hydroxide gel
The excipients present in some antigen or adjuvant stock solutions can alter the binding behavior of the CpG-Alhydrogel complex. One must be especially cautious of ligand exchange with phosphate-containing solutions. Before the effects of the antigen on the adsorption of CpG and vice versa can be monitored, the effects of the excipients present in the antigen preparation must be evaluated. To determine excipient effects on CpG-Alhydrogel adsorption behavior, the binding capacity protocol from above was repeated using a CpG ODN solution containing the excipients from a lyophilized antigen preparation instead of water. Thus, for a 100 mL in vivo injection consisting of 10 mg CpG and 100 mg aluminum, a sample phosphate buffer containing EDTA and sucrose was mixed with the adjuvant and aluminum. The sample was centrifuged as described above and the absorbance of supernatant was measured at 260 nm. For this example, it was found that the adsorption of CpG ODN to Alhydrogel was not diminished in the presence of the excipient mixture (Table 1). It should be noted that order of addition of each component and time to bind also may affect adsorption behavior. The effects on CpG-Alhydrogel adsorption of several different buffers and excipients has been discussed elsewhere.13

A similar procedure can be followed to determine the effects of other adjuvants or antigens on CpG-Alhydrogel adsorption. However, detection using spectrophotometry becomes more complicated since other adjuvants and especially antigens can interfere with the 260 nm absorbance signal. In these cases, other detection methods may be desirable. Supernatant CpG ODN can be detected by SDS-PAGE/laser densitometry, in addition to spectrophotometry.13 Size exclusion HPLC can detect CpG ODN present in the supernatant after centrifugation.

Figure 3. HPLC detection of CpG 2395 in TE buffer
The mobile phase for size exclusion HPLC methods often is the same as the sample buffer, making these methods simple to perform. Other HPLC methods for ODNs or DNA, including reversed phase and ion exchange, also have been demonstrated.14,15 In addition, a dissociation step may be necessary before HPLC analysis to separate CpG ODN bound to protein or other materials.14 In this case, because the CpG ODN was stored in TE buffer, a mobile phase was prepared consisting of 10 mM Tris buffer at pH 8. The flow rate was set to 0.5 mL/min (TSKgel G3000PWXL column), with an injection volume of 20 μL, and the UV-Vis absorbance detector set to 260 nm. To develop the detection range for this method, samples of CpG 2395 were prepared at 1, 0.1, and 0.01 mg/mL, with a blank of TE buffer. Detection of CpG ODN was possible at a concentration of 0.01 mg/mL (Figure 3).

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