Formulation Studies of an Adsorbed Conjugate Vaccine - How a thorough formulation study ensured the development of a stable and effective vaccine. - BioPharm International

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Formulation Studies of an Adsorbed Conjugate Vaccine
How a thorough formulation study ensured the development of a stable and effective vaccine.


BioPharm International Supplements



Table 3. Summary of the time points for the long-term stability studies at 2–8 C (data shown in Figure 2)
The stability data for PBS and saline formulations are presented in Figure 2. The time points for each study are summarized in Table 3. The specifications for saline and PBS formulations are presented in Table 4. The saline formulation provided stable adsorption with some drift in pH. The PBS formulation provided a stable pH, but, the percent adsorption dropped initially and stayed low. The vaccine remained potent in animals with both formulations.

6. Iyer S, HogenEsch H, Hem SL. Effect of the degree of phosphate substitution in aluminum hydroxide adjuvant on the adsorption of phosphorylated proteins. Pharm Dev Tech. 2003;8(1):81–6.

Discussion


Table 4. Specifications established for clinical and commercial lots
Desorption of antigen from aluminum hydroxide adsorbed vaccine by phosphate has been reported.5 Rinella, et al., reported desorption of negatively charged ovalbumin from aluminum hydroxide adjuvant by the addition of phosphate. Phosphate anions were adsorbed to aluminum hydroxide and lowered the isoelectric point of the adjuvant. This in turn decreased the electrostatic interaction with negatively charged antigen. The extent of desorption depended on the ionic strength, the phosphate concentration, and the age of the vaccine. As the antigen adjuvant complex aged, the ability of phosphate to desorb the antigen decreased. It was believed that the antigen might undergo conformational changes to optimize its interaction with adjuvant. Our data supported this hypothesis. The desorption of GCMP–TT from aluminum hydroxide occurred only in the first few months and leveled off during the two-year storage.

Iyer, et al., treated aluminum hydroxide adjuvant with phosphate ion, which resulted in ligand exchange of phosphate for surface hydroxyl groups.6 This ligand exchange decreased the isoelectric point of the adjuvant. In addition, the adsorptive capacity and adsorptive coefficient for a negatively charged antigen were reduced.

The antigen in the current study, GCMP–TT, is negatively charged because of functional groups on the sialic acid monomer. The lowering of percent adsorption in PBS was caused by the ligand exchange of phosphate for hydroxyl groups on the aluminum and decreasing electrostatic interaction between aluminum and GCMP–TT. In the adsorbed vaccine formulation, phosphate buffer should be avoided. If buffering capacity was required to maintain the stability of the vaccine, we would recommend using Tris buffer instead of phosphate buffer.

Acknowledgement

The authors would like to thank Professor Stanley Hem for his interpretation of the PBS adsorption data.

SHWU-MAAN LEE, PHD, is a technical director, BOB KRUSE, PHD, is aresearch scientist, and CHRIS DONALDSON is a research associate, all at Baxter Healthcare, Beltsville, MD, 301.419.8587,

References

1. Jennings HJ, Lugowski C. Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J Immunol. 1981;127:1011–8.

2. Svennerholm L, Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochimica et Biophysica Acta. 1957;24:604–11.

3. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

4. Michon F, Huang CH, Farley EK, Hronowski L, Di J, Fusco PC. Structure activity studies on group C meningococcal polysaccharide-protein conjugate vaccines: effect of O-acetylation on the nature of the protective epitope. Dev Biol. 2000;103:151–60.

5. Rinella JV Jr, White JL, Hem SL. Effect of anions on model aluminum-adjuvant-containing vaccines. J Colloid Interface Sci. 1995;172:121–30.


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