At the time of the yield investigation, the marketing authorization application had been submitted for approval to the Medicines
Control Agency (MCA) of the UK. There were communications between MCA and Baxter to finalize the product specifications. The
100 kDa membrane yield problem and the 50 kDa membrane change proposal were communicated to the MCA and the change proposal
The 50 kDa membrane change was implemented in production. For the five subsequent lots, the yield returned to the expected
range and the purity was well within the specifications established by the 100 kDa membrane (Table 4).
Table 4. Results from production lots after 50 kDa membrane implementation
The size of the GCMP after 100 kDa processing was evaluated as part of the low yield investigation. Based on absolute MW determination
by laser light scattering, the MW of GCMP was determined to be 200–300 kDa. A rule of thumb in UF is that the membrane of
choice should have a NMWCO one-third to one-fifth of the MW of a product that is to be retained.9 According to this rule, if GCMP sizing data had been available during process development, the 50 kDa membrane would have
been a better choice than the 100 kDa membrane.
Since the UF membrane was changed from 100 kDa to 50 kDa, over 100 batches of commercial vaccine have been produced with satisfactory
yield and purity.
Throughout the production history, the 50 kDa UF membranes have been reused up to 11 times. Other UF steps have reused UF
membranes for up to 26 cycles. Most of the time, UF membranes are retired because they exceed our internal specification of
24-h dirty membrane hold time. Most of the NWP values following cleaning appear to be stable. Because meningitis vaccine purification
involves multiple UF steps and each UF step uses multiple UF membranes cassettes, the ability to reuse UF membrane represents
a substantial savings in production cost.
The key lesson learned from the UF yield investigation is that in designing a UF process and validation program, one should
consider the sensitivity of the desired separation to variation in the retention profile of different lots of membrane.11 In this case, the UF step was used for concentration and purification of GCMP. The impact of membrane variability on product
yield was significant. The impact of this variability on product yield and purity should be evaluated as part of process development
and confirmed during process validation.
Shwu-Maan Lee, PhD, is a technical director, Bob Kruse, PhD, is a research scientist, and Amy Robinson, PhD, is a senior research manager, all at Baxter Healthcare, Beltsville, MD, 301.419.8587, email@example.com
1. Borrow R, Findlow J. Prevention of meningococcal serogroup C disease by NeisVac-C. Expert Rev Vaccines. 2009;8:265–79.
2. Jennings HJ, Lugowski C. Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates.
J Immunol. 1981;127:1011–8.
3. Svennerholm L. Quantitative estimation of sialic acids II. A colorimetric resorcinol-hydrochloric acid method. Biochimica
et Biophysica Acta. 1957;24:604–11.
4. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem. 1976;72:248–54.
5. Frasch CE. Production and control of Neisseria meningitidis vaccine. Bacterial Vaccines. 1990;123–145.
6. D'Ambra AJ, Baugher JE, Concannon PE, Pon RA, Michon, F. Direct and indirect methods for molar-mass analysis of fragments
of the capsular polysaccharide of Haemophilus influenzae type b. Anal Biochem. 1997;250:228–36.
7. Michon F, Huang CH, Farley EK, Hronowski L, Di J, Fusco PC. Structure activity studies on group C meningococcal polysaccharide-protein
conjugate vaccines: Effect of O-acetylation on the nature of the protective epitope. Dev Biol (Basel). 2000;103:151–60.
8. Pellicon and Pellicon-2 cassette filters maintenance procedures. 1998;1–19.
9. Millipore Corporation. Protein concentration and diafiltration by tangential flow filtration. Millipore technical brief
10. Tkacik G, Michaels S. A rejection profile test for ultrafiltration membranes and devices. Bio Technol. 1991;9:941–46.
11. Parenteral Drug Association. Industrial perspective on validation of tangential flow filtration in biopharmaceutical
applications. Parenteral Drug Association Technical Report No. 15. 1992 Suppl; 46:S1–13.