Materials and methods
Figure 2: GCMP purification process.
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The antigenic component of the vaccine, the de-O-acetylated GCMP, was purified from the culture supernatant of Neisseria meningitidis serogroup C, strain C11. The cells and spent culture medium were circulated through 0.2 µm hollow fiber cartridges using
a circumferential piston pump. The permeate from the filtration contained the polysaccharide (see Figure 2).
This filtrate was concentrated and diafiltered across a 300 kilodalton (kDa) nominal molecular weight cutoff (NMWCO) ultrafilter
(UF), (Millipore Pellicon 2) (see Figure 2, step 4), which retains the GCMP. The concentrated GCMP was then chemically modified
with a saponification reaction to remove acetyl groups (Figure 2, step 5). Base treatment is the major purification step with
high temperature incubation for several hours in NaOH.
Deacetylation removed all the acetyl groups from the O-positions and most of the acetyl groups from the N-positions of the GCMP. It also hydrolyzed cell impurities and saponified any fatty acids, which were removed by subsequent
diafiltration with water for injection (WFI) across a 50 kDa NMWCO UF (Pellicon 2, Millipore) (see Figure 2, step 6). The
GCMP remain in the UF retentate.
The N-acetyl groups are believed to be immunologically important, and were restored in a subsequent chemical reaction (see Figure
2, step 7). After reacetylation, the GCMP was diafiltered and concentrated with a 30 kDa NMWCO UF (Pellicon 2, Millipore)
that retain the GCMP. The GCMP was then tested for concentration and purity.
The GCMP content was determined by a colorimetric resorcinol-HCl method (1). This method measured GCMP monomer (sialic acid)
using N-acetyl neuraminic acid as a standard.The protein imputity content was determined by the Bradford method (2) using
BSA as a standard.
The nucleic acid impurity content was determined by the absorbance at 260 nm, assuming an absorbance of 1 (1-cm light path)
for 50 µg/mL of nucleic acid (3).
The white, waxy precipitate was analyzed by Energy dispersive x-ray spectroscopy (EDXS), micro-Fourier transform infrared
(FT-IR) spectroscopy, proton nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry (LC-MS) to determine
its composition. LC-MS analysis of the precipitate was performed on a Waters 2695 HPLC system with a C18 column and a Waters
Q-TOf API-US mass spectrometer.
The fatty acids were identified at Microbial ID (MIDI). Samples were saponified in NaOH at elevated temperature and methylated.
The fatty acid methyl ester was extracted in an organic solvent prior to injection into the gas chromatograph. Fatty acid
identification is based on their retention times compared to a library of standard (4).
Results and discussion
Eight successive purification lots produced in a recent campaign had an atypical appearance (see Figure 1). These lots were
cloudy liquids with a white, waxy precipitate in the 50K retentate (see Figure 2, step 6) and/or the 30K retentate (see Figure
2, step 8). The precipitate was identified as mostly the sodium salt of palmitic acid (C16:0). This amount of precipitation
had not been noted in past commercial production and its appearance caused cessation of manufacturing while this issue was
investigated.
It is believed that most of the GCMP isolated from the fermentation is a lipidated molecule that is able to aggregate either
with itself or with other macromolecules such as lipopolysaccharide (5). The aggregates are small enough to pass through the
0.2 µm harvest filters but are retained by the 300 KDa UF (see Figure 2, step 4).
Table I: Fatty acid content (%) of Neisseria meningitidis.
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In the deacetylation, many macromolecules are hydrolyzed and the sodium salts of fatty acids are generated. The most common
fatty acids in Neisseria are palmitic and palmitoleic acids (see Table I). The fatty acid analysis of our working cell banks was consistent with the
literature (6-8) (see Table I) and allowed the authors to conclude that the source of the precipitate was not exogenous but
was derived from the cells in our fermentation.
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