Process for converting lipid-containing bacterial capsular polysaccharide into lipid-free polysaccharide
AssigneeMerck & Co., Inc.
InventorsAnn L. Lee, Mark S. Rienstra, Walter E. Manger, Robert D. Sitrin
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Abstract
A process for converting lipid-containing bacterial capsular polysaccharide, such as lipo-polyribosyl ribitol phosphate, lipo-PRP, into lipid-free, endotoxin-free polysaccharide, such as polyribosyl ribitol phosphate, PRP, by solubilizing polysaccharide-containing powder derived from culture media of bacteria, such as Haemophilus influenzae type b, cleaving covalently bound fatty acids from the polysaccharide, and removing the lipids, and endotoxin.
Claims14
A process for preparing lipid and endotoxin free capsular polysaccharide from a crude or purified preparation comprising both lipid-free and lipo-capsular polysaccharide, derived from bacteria, without substantial loss of the capsular polysaccharide, which comprises converting substantially all the lipo-capsular polysaccharide into lipid-free capsular polysaccharide by cleaving the lipid from the lipo-capsular polysaccharide, removing free lipid and endotoxin contaminants, and recovering the lipid-free capsular polysaccharide.
A process for preparing lipid and endotoxin free capsular polysaccharide from a crude or purified preparation comprising both lipid-free and lipo-capsular polysaccharide, derived from bacteria, without substantial loss of the capsular polysaccharide, which comprises converting substantially all the lipo-capsular polysaccharide into lipid-free capsular polysaccharide by cleaving the lipid from the lipo-capsular polysaccharide, removing free lipid and endotoxin contaminants, and recovering the lipid-free capsular polysaccharide wherein the lipid is cleaved from the lipo-capsular polysaccharide by a phospholipase.
A process for preparing lipid-free and endotoxin-free polyribosyl ribitol phosphate, PRP, without substantial loss of PRP, which comprises reacting lipo-PRP present in a crude or purified preparation of polysaccharide derived from a culture of Haemophilus influenzae type b with phospholipase D at a ratio of about 0.3 weight-percent of phospholipase D to PRP, in a reaction mixture comprising an ether:organic solvent mixture at a concentration of between 30% and 60% of the reaction volume, wherein the organic is an ether, selected from diethyl-ether, butyl-ether, or methyl-tert-butyl-ether, in admixture with a second organic selected from hexane, ethanol, or methanol, in the presence of a detergent selected from deoxycholate or Triton X-100, present at a concentration of between 0.1% and 0.4%, with addition of about 0.1 to about 10 mM CaCl.sub.2, in a buffer compatible with the afore-mentioned reagents at a pH between about 7.0 and 8.0, a temperature between about 20.degree. C. and 45.degree. C., for between about 30 minutes and about 4 hours.
A process for preparing lipid-free PRP without substantial loss of PRP, which comprises the steps of:
- a) culturing Haemophilus influenzae type b in a suitable culture medium;
- b) killing the Haemophilus influenzae type b with thimerosal or phenol;
- c) clarifying the culture medium of killed Haemophilus influenzae type b;
- d) concentrating the clarified culture medium to obtain a workable volume;
- e) precipitating contaminants in the culture medium by adding ethanol to a final concentration of about 48% ethanol to obtain a supernatant containing PRP and a contaminant pellet which is discarded;
- f) precipitating the PRP by adding ethanol to a final concentration of about 61% to obtain a crude-PRP pellet;
- g) adding water to the PRP pellet to solubilize it, followed by adding calcium chloride to a final concentration of 1.0M;
- h) adding ethanol to a final concentration of 23% to obtain an insoluble pellet of contaminants and a supernatant containing the PRP;
- i) adding ethanol to a final concentration of 37% to precipitate the PRP;
- j) triturating the PRP pellet with absolute ethanol and drying to obtain a PRP pre-phenol powder;
- k) phenol extracting a solubilized preparation of the PRP powder;
- l) precipitating the PRP by adding CaCl.sub.2 to 0.05M and alcohol to about 67%, triturating in absoulute ethanol to obtain post-phenol powder;
- m) removing endotoxin by hydropohobic adsorption from a solubilized preparation of post-phenol powder;
- n) reacting the PRP in crude-PRP, pre-phenol powder, or post-phenol powder, with a phospholipase, before proceeding with subsequent steps.
Dependencies are inferred from the claim text.