Purification of polysaccharide-protein conjugate vaccines by ultrafiltration with ammonium sulfate solutions

#The present invention relates to the removal of unbound polysaccharides from conjugated polysaccharide-protein vaccines using the method of ultrafiltration whereby the diafiltration solution contains saturating levels of ammonium sulfate.

#Several publications are referenced in this application. Full citation to these publications is found where cited or at the end of the specification, immediately preceding the claims; and each of these publications is hereby incorporated by reference. These publications relate to the state of the art to which the invention pertains; however, there is no admission that any of these publications is indeed prior art.

#In recent years, a number of polysaccharide-protein conjugate vaccines have been developed for use in protection against bacterial infections. One such vaccine, the Haemophilus influenzae type B conjugate vaccine is now licensed for use in humans throughout the world. This vaccine is administered to infants along with their other routine pediatric vaccines. The Haemophilus influenzae type B conjugate vaccine has proven to be quite effective in protecting against Haemophilus influenzae type B disease (Santosham, M., 1993). The polysaccharide-protein conjugate vaccines are prepared by covalently attaching purified bacterial capsular polysaccharides to protein molecules using a variety of chemical methods. Upon completion of the conjugation reactions, the unreacted polysaccharide molecules are separated from the polysaccharide-protein conjugates using an assortment of separation techniques. The techniques that have been proven to be most effective in purifying the polysaccharide-protein conjugates include gel filtration chromatography, hydrophobic interaction chromatography, ultracentrifugation, liquid-liquid extraction, and ammonium sulfate precipitation/fractionation.

#FIG. 1 shows washout kinetics of adipic acid dihydrazide derivatized Neisseria meningitidis serogroup C from a conjugation reaction mixture ultrafiltered against 10 volumes of 50% (of saturation) ammonium sulfate using a Millipore Prep/Scale 30,000 MWCO spiral wound fitted with a regenerated cellulose membrane. Removal of free polysaccharide (PS) by ultrafiltration is displayed graphically as Filtrate ug PS/ml vs. Volumes Ammonium Sulfate Diafiltered.

#The invention has been applied to purify a number of distinctly different polysaccharide-protein conjugates that are derived from a variety of bacterial capsular polysaccharides, although the invention need not be limited to only these polysaccharide-protein conjugates. The bacterial capsular polysaccharide-protein conjugates that have been purified by this process include Streptococcus pneumoniae serotypes 1, 3, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, and Neisseria meningitidis serogroups A, C, W-135 and Y. Such polysaccharides are available from the American Type Culture Collection, or can be readily obtained from any of a number of potential sources. For example, such polysaccharides can be extracted and purified directly from microorganisms. See, e.g., Porro, U.S. Pat. No. 5,153,312 and EP 0477508 B1 for a discussion of such preparation procedures. The polysacharides may also be partially depolymerized or fragmented, using chemical or mechanical means, some of which are detailed by Porro. Hagopian et al. EP 497,525 and references cited therein also provide guidance as to methods of preparing and characterizing polysaccharides. See also, Chu, et. Al. (1991) Infect. Immun. 59: 4450-4458 for a description of lipopolysaccharide-based conjugate vaccines.

#According to the method of the present invention, the derivatized polysaccharide is first mixed with the protein carrier of choice in a solution of physiological saline (0.85% sodium chloride), and the pH of the mixture is adjusted to 5.0.+-.0.1. The conjugation reaction is initiated by adding 1-ethyl-3-(3-dimehtylaminopropyl) carbodiimide (EDAC) which serves to activate the carboxyl groups on the carrier protein allowing for reaction from a nucleophilic site that is present on the polysaccharide chain. The pH of the reaction is maintained at 5.0.+-.0.1 during the course of the reaction, normally for two hours at 18 to 25.degree. C. After the reaction time is complete, the pH of the reaction mixture is adjusted to 7.0.+-.0.1, and the reaction is allow to stand for 12 to 18 hours at 2 to 8.degree. C. to allow for hydrolysis of the unreacted EDAC.

#The polysaccharide-protein conjugate is purified by first allowing the mixture to equilibrate to 18 to 25.degree. C. The mixture is connected to a spiral wound ultrafiltration unit that is equipped with a Millipore Prep/Scale 30,000MWCO regenerated cellulose membrane. Ammonium sulfate is added to the reaction mixture at a specified level of saturation (in general, 50 to 60% of saturation). The conjugate mixture is diafiltered against 20 volume exchanges of 50 to 60% (of saturation) ammonium sulfate solution. The ammonium sulfate is removed from the polysaccharide-protein solution by diafiltration against 10 volume exchanges of physiological saline (0.85% sodium chloride). The purified conjugate is filtered through 1.2 and 0.45 micron membranes, and then sterilized by filtration through a 0.22 micron membrane.