Formulation Studies of an Adsorbed Conjugate Vaccine

How a thorough formulation study ensured the development of a stable and effective vaccine.
Oct 02, 2008


NeisVac-C is a polysaccharide-protein conjugate vaccine for use against Neisseria meningitidis serogroup C infection. The Phase 1 clinical formulation consisted of physiological saline, thimerosal, and aluminum hydroxide. In an attempt to maintain pH for the commercial vaccine, phosphate buffered saline (PBS) replaced saline in a subsequent clinical formulation. In addition, thimerosal was eliminated and the vaccine was filled in single-dose syringes. Before the clinical trial began, the amount of group C meningococcal polysaccharide tetanus toxoid (GCMP-TT) conjugate adsorbed to aluminum hydroxide (percent adsorption) dropped from >90% to about 80% in 1–4 months. The PBS formulation was determined to be unacceptable and new lots were produced with the saline formulation. These lots were used for Phase 2–3 clinical trials. The saline formulation continued to be used for commercial product. The long-term stability data for both the PBS and saline formulations are presented in this article.

(Baxter Healthcare)
NeisVac-C was introduced in the United Kingdom in 2000. Currently, it is licensed in 35 countries. The vaccine is used to prevent the invasive disease caused by N. meningitidis serogroup C. The active ingredient of the vaccine is a polysaccharide–protein conjugate. Each dose contains 10 μg of De-O-acetylated group C meningococcal polysaccharide (GCMP) conjugated to 10–20 μg of tetanus toxoid protein (TT) and adsorbed to 0.5 mg of aluminum in saline. This report summarizes the formulation studies performed during product development and demonstrates the importance of formulation in vaccine stability.

Materials and Methods

The antigen component of the vaccine, De-O-acetylated GCMP, was purified from culture supernatant of N. meningitidis serogroup C, strain C11. The carrier protein, tetanus toxoid, was obtained from Statens Serum Institut, Denmark. The conjugation of GCMP to TT used reductive amination technology.1 The adjuvant, aluminum hydroxide, was obtained from Brenntag Biosector, Denmark.

Figure 1. Percent adsorption by competitive GCMP ELISA: standard curve
Percent adsorption by GCMP ELISA (method A): In a formulated sample, the aluminum hydroxide phase was removed by centrifugation and a competitive ELISA measured GCMP in the supernatant. In the competition assay, sample and serum containing antibody against the analyte were incubated together in a coated plate. The 96-well microtiter plates were coated with GCMP–human serum albumin conjugate and blocked with 2% casein. The plates were incubated with rabbit anti-GCMP–HSA antiserum, which was mixed with the supernatant from the formulation sample. Goat anti-rabbit IgG (H+L)–horseradish peroxidase conjugate was used as the probing antibody with TMB (3,3', 5,5'-tetramethylbenzidine) as the substrate. The color development was inversely proportional to the amount of GCMP in the sample. The parent lot of conjugate was used as the standard. Calculations were based on the maximum optical density (ODmax), measured using wells containing no GCMP, and the OD from the standard. Figure 1 shows a typical standard curve. Percent adsorption was calculated as 100% – percent in the supernatant.

Percent adsorption by resorcinol-HCl method (method B): This method measured GCMP monomer (sialic acid) by a colorimetric assay using N-acetylneuramic acid (sialic acid) as a standard.2 GCMP in the aluminum hydroxide phase (precipitate) and in the supernatant were separated by centrifugation. The amount of GCMP in the precipitate and in the supernatant and the whole formulated sample were measured separately. The interference from aluminum hydroxide was minimal because OD was measured by extracting the color substances into the organic phase while aluminum hydroxide was left in the aqueous phase and discarded. The percent adsorption was determined directly by dividing the amount of GCMP in the precipitate by the total GCMP.

The protein content in the adsorbed supernatant was determined by the Bradford method using human immunoglobulin G (Pierce) as a standard.3

Potency of the formulated vaccine was evaluated using Swiss Webster mice (Harlan, Indianapolis, IN). Mice, 10 in each group, were injected with 0.2 mL of the diluted vaccine sample at 10 μg/mL, a negative control, or a positive control, on days 0, 14, and 28. The animals were exsanguinated on day 38, and sera were collected and stored frozen until they were analyzed. The immune response of the murine serum was determined by serum bactericidal antibody titer, which reflected functional antibodies.4 The assay used baby rabbit complement (Pel-Freeze Biologicals, Rogers, AR) and strain C11 of N. meningitidis. Test samples consisted of serum pools prepared from equal aliquots of individual mouse sera in each group.

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