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US 4,707,543 AGranted
Process for the preparation of detoxified polysaccharide-outer membrane protein complexes, and their use as antibacterial vaccines
AssigneeThe United States of America as represented by the Secretary of the Army
InventorsWendell D. Zollinger, John Boslego, Ellen Moran, Brenda Brandt, Hugh Collins, Robert Mandrell, Patricia Altieri, Sanford Berman
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Abstract
Process for preparing a detoxified polysaccharide-outer membrane protein complex from bacterial envelopes; the so-obtained products which are useful as vaccines against infection by the same bacteria and method for protecting animals against the same infection by administration of a pharmaceutical composition containing the detoxified polysaccharide-outer membrane protein complexes.
Claims33
A process for preparing an immunogenic detoxified polysaccharide-outer membrane protein complex from bacteria wherein said complex is essentially free of toxic polysaccharide comprising the steps of:
- a. suspending the outer membrane proteins in a TEEN buffer solution containing 1% of the zwitterionic detergent Empigen BB, 0.01M ethylenediaminetetraacetic acid, 0.05M tris-HCl, and 0.15M NaCl, said solution having a pH of about 8.0;
- b. stirring the suspension formed in step a. for about 1 hour at about room temperature followed by mild sonication;
- c. centrifuging the suspension formed in step b. at about 20,000.times.g for about 15 minutes and separating the supernatant fluid from the insoluble residue;
- d. adding solid ammonium sulfate to the supernatant layer formed in step c. in an amount of 500 grams/liter of supernatant protein solution and stirred until the ammonium sulfate is dissolved, and then allowed to stand at about room temperature for about 1 to 2 hours to allow precipitation of the outer membrane protein;
- e. centrifuging the product of step d. at about 20,000.times.g for about 20 minutes and collecting the precipitated outer membrane proteins which form a top layer;
- f. dissolving the precipitated outer membrane proteins formed in step e. in a buffer solution of the type described in step a., adding ammonium sulfate, and separating the precipitate;
- g. repeating the process of step f.;
- h. dissolving the precipitate formed in step g. in a TEEN buffer to form a solution, then dialyzing said solution against TEEN buffer to remove (NH.sub.4).sub.2 SO.sub.4 ;
- i. centrifuging the solution formed in step h. at about 35,000.times.g for about 20 minutes and removing any insoluble material;
- j. filtering the centrifuged solution formed in step i. sequentially through 1.2.mu., 0.45 .mu., and 0.22.mu. membrane filters;
- k. sterile filtering the solution formed in step j. through a 0.22.mu. membrane filter;
- l. complexing the sterilized outer membrane proteins with a sterile filtered polysaccharide by combining the outer membrane protein, derived from one or more strains, and polysaccharide, derived from one or more strains, in a ratio within the range of 1:2 to 2:1 in TEEN buffer and coprecipitating by the cold, sterile absolute ethanol to 75% volume/volume;
- m. centrifuging, under sterile conditions, the ethanolic solution formed in step l. at about 20,000.times.g for about 15 minutes and separating the precipitate;
- and
- n. washing the precipitate formed in step m. with sterile absolute ethanol to remove essentially all components of the buffer solution yielding an immunogenic detoxified polysaccharide-protein complex.
12 more elements
A process for purifying outer membrane protein derived from bacterial wherein said protein is essentially free of toxic polysaccharide comprising the steps of:
- a. suspending the outer membrane proteins in a TEEN buffer solution containing 1% of the zwitterionic detergent Empigen BB, 0.01M ethylenediaminetetraacetic acid, 0.05M tris-HCl, and 0.15M NaCl, said solution having a pH of about 8.0;
- b. stirring the suspension formed in step a. for about 1 hour at about room temperature followed by mild sonication;
- c. centrifuging the suspension formed in step b. at about 20,000.times.g for about 15 minutes and separating the supernatant fluid from the insoluble residue;
- d. adding solid ammonium sulfate to the supernatant layer formed in step c. in an amount of 500 grams/liter of supernatant protein solution and stirred until the ammonium sulfate is dissolved, and then allowed to stand at about room temperature for about 1 to 2 hours to allow precipitation of the outer membrane protein;
- e. centrifuging the product of step d. at about 20,000.times.g for about 20 minutes and collecting the precipitated outer membrane proteins which form a top layer;
- f. dissolving the precipitated outer membrane proteins formed in step e. in a buffer solution of the type described in step a., adding ammonium sulfate, and separating the precipitate;
- g. repeating the process of step f.;
- h. dissolving the precipitate formed in step g. in a TEEN buffer to form a solution, then dialyzing said solution against TEEN buffer to remove (NH.sub.4).sub.2 SO.sub.4 ;
- i. centrifuging the solution formed in step h. at about 35,000.times.g for about 20 minutes and removing any insoluble material;
- j. filtering the centrifuged solution formed in step i. sequentially through 1.2.mu., 0.45.mu., and 0.22.mu. membrane filters;
- and
- k. sterile filtering the solution formed in step j. through a 0.22.mu. membrane filter thereby obtaining sterilized outer membrane proteins.
9 more elements
Dependencies are inferred from the claim text.