The meningitis vaccine (NeisVac-C) comprises the de-O-acetylated form of group C meningococcal polysaccharide conjugated to tetanus toxoid. The polysaccharide purification process includes base treatment and subsequent diafiltration to remove hydrolyzed cell impurities. During development, a 100 kilodalton (kDa) nominal molecular weight cut-off (NMWCO) ultrafiltration membrane was used for the diafiltration, and process yields ranged from 87 to 135%. Unexpectedly, the yield from the first 12 production lots varied from 18 to 100%. Investigation revealed that ultrafiltration membrane porosity varied with different manufacturers' lots. The corrective action included changing from a 100 kDa to a 50 kDa NMWCO membrane; this change occurred just before license approval. To support the change, a process stream sample spiked with >200% of cell impurities was purified with the tightest 50 kDa membrane available. Even under this worst-case condition, the impurities were removed to meet the original specifications achieved by the 100 kDa membrane. The change was implemented and >100 lots have since been produced with satisfactory yield and purity.
During polysaccharide purification development, we encountered a unique challenge with the ultrafiltration (UF) membrane used in the purification process. This article describes the problem and the solution.
MATERIALS AND METHODS
The antigen component of the vaccine, de-O-acetylated GCMP, was purified from the culture supernatant of Neisseria meningitidis serogroup C, strain C11. The carrier protein, TT, was obtained from Statens Serum Institut, Denmark. The conjugation of GCMP to TT used reductive amination technology.2
The GCMP content was determined by a resorcinol-HCl method. This method measured GCMP monomer (sialic acid) by a colorimetric assay using N-acetyl neuraminic acid (sialic acid) as a standard.3
The protein impurity content in the process was determined by the Bradford method using BSA as a standard.4
The nucleic acid impurity content was determined by absorbance at 260 nm, assuming an absorbance of 1 (in a 1-cm wide cell) for 50 µg/mL of nucleic acid.5
The molecular weight (MW) of GCMP 100 kDa retentate was determined by a laser-light-scattering method (Wyatt Technology Corp) after fractionation on an HPLC with Superose-6 column (GE Healthcare).6