Results
The Phase 1 formulation consisted of 20 μg/mL GCMP–TT (μg refers to GCMP) in 150 mM NaCl (saline), 0.01 % thimerosal and 1
mg/mL aluminum (added as aluminum hydroxide), and filled in single-dose vials with a deliverable dose of 0.5 mL. The 3 mL
vial was Type 1 Flint glass and the closure was 13 mm chlorobutyl rubber stoppers. After four years of storage at 5 °C, the
pH of the vaccine rose from 5.5 to 7.3. The slow rise in pH was caused by gradual leaching of alkaline from the glass vials.
Phosphate buffered saline (PBS, 10 mM sodium phosphate and 150 mM NaCl, pH 7.4) was chosen for the Phase 2–3 clinical formulation
because of pH stability concerns in the unbuffered vaccine.
The adsorption kinetics were studied at small scale and GCMP ELISA (method A) was used to measure percent adsorption. Adsorption
of GCMP–TT to aluminum hydroxide gel in saline reached completion instantaneously (>99.9% adsorbed). Adsorption in PBS went
slowly and in an hour only 98% was adsorbed. For clinical production, we formulated in PBS for one hour and used a tentative
specification of ≥90% for percent adsorption. The formulated vaccine was filled into 1-mL syringes with a 0.5-mL deliverable
dose. The syringe barrels were silicon lubricated European and US
Pharmacopeia type 1 borosilicate glass (Becton Dickinson). The closures were siliconized bromobutyl plunger stoppers. Medical grade DC
360 silicone helped stopper placement and movement and was consistent with US standards. Tip caps (chlorobutyl natural rubber
blend) maintained sterility of the closure. Thimerosal preservative was not necessary for single-dose fills and was eliminated.
The formulation consisted of 20 μg/mL GCMP–TT and 1 mg/mL aluminum in PBS.
Two lots (49801 and 49802) were produced for Phase 2–3 trials and placed on stability studies. The stability indicating methods
included percent adsorption, pH, potency, sterility, general safety, and appearance.
After completing formulation, the percent adsorption samples were taken from the formulated bulk and determined to be 97%
and 98% (method A) for lots 49801 and 49802, respectively. Both lots passed specification.
Table 1. Desorption of conjugate vaccine from the aluminum hydroxide adjuvant in PBS
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Before the start of clinical trials, percent adsorption dropped for both lots as determined by GCMP ELISA. Because of the
decrease in percent adsorption, the supernatant contained detectable amounts of GCMP and protein that allowed us to confirm
the percent adsorption by chemical assays measuring sialic acid and protein, respectively (Table 1).2,3
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