RESULTS AND DISCUSSION
The Neisseria meningitidis production strain, C11, is an O-acetyl positive strain. It produces GCMP with O-acetyl groups at either the C7 or C8 position,
but not both.7 De-O-acetylation is accomplished by incubating native GCMP with NaOH at high temperature. Under these conditions, the acetyl
groups at the O-positions and at most of the N-positions are removed (Figure 1). The N-acetyl groups are believed to be an
important epitope of the vaccine, and therefore, are restored with acetic anhydride in a subsequent chemical reaction. The
NaOH treatment also hydrolyzes the cell impurities, which must be removed. Diafiltration with a plate and frame system (Pellicon
2, Millipore) containing multiple 5 ft2 cassettes of polyethersulfone-based membrane (Biomax) was used to remove these impurities. The UF step used 10 process volumes
of diafiltration with water for injection (WFI), followed by concentration of GCMP to a targeted volume. Diafiltration with
a 100 kDa UF membrane was the method of choice during process development because the MW of the native GCMP was believed to
be at least 300 kDa and the cell impurities had been degraded into low MW substances.
Figure 1. The de-acetylation of group C meningococcal polysaccharide (GCMP)
During the early phase of this process development, we encountered a serious transmembrane plugging problem. When the process
stream was cooled to ambient temperature after base treatment at elevated temperature, a large amount of insoluble material
soon blocked the UF membrane. Attempts to neutralize or to filter the polysaccharide before diafiltration were not successful.
The final UF protocol involves maintaining the process stream at 50 °C (the highest temperature the membrane can tolerate)
while performing the diafiltration.
After use, the UF membranes were cleaned according to the manufacturer's recommendation: recirculation of 0.1 N NaOH at 40–50
°C for 45 min and 0.1 N phosphoric acid at 40–50 °C for 45 min.8 The membranes were rinsed with WFI and tested for integrity and normalized water permeability (NWP).8 An NWP specification is established before the first use of a membrane; and after cleaning, the NWP must be at least 60%
of the NWP of the new membrane for it to be reused.
Figure 2. Ultrafiltration step yield for development and production lots
During development, the yield for the 100 kDa UF step was excellent and ranged from 87 to 135% (Figure 2). There are two possible
reasons for the yield being higher than 100% in many lots: the crude starting material contains a high level of impurities
and some of them may inhibit the sialic acid assay, or the de-O-acetylated polysaccharide may have a higher light absorbance
than native polysaccharide in the sialic acid assay.
When the process was transferred from development to manufacturing, the production yield dropped drastically after the 5th
lot (Figure 2). At that point, production was halted and an investigation started.
The diafiltration step is modeled with the following equation:9
GCMP Recovery (%) = 100 × e(R-1)(In VCF + N)
in which VCF is the volume concentration factor (the amount that the feed stream has been reduced in volume from the initial volume).
For constant volume diafiltration, VCF = 1. N is a measure of the extent of washing that has been performed during diafiltration and is based on the volume of diafiltration
buffer introduced into the unit operation compared to the retentate volume. Ten volumes of WFI were used and N = 10. R is the retention coefficient (the fraction of polysaccharide that was retained by the membrane). R = 1 – (GCMP concentration in the permeate/GCMP concentration in the feed).