Table 3. Summary of the time points for the long-term stability studies at 2–8 °C (data shown in Figure 2)
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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
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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, lees4@baxter.com
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