Connaught Laboratories Limited

Other

Forintek Canada Corporation

Larry U Tan

Gail E Jackson

Raafat Emil Fahim

Michel Henri Klein

Pele Chong

John Vose

Michel H Klein

Ernest K Yu

John N Saddler

Po S Wah

Andrew Herbert

Luis Barreto

John Thipphawong

Dirk Alkema

John R Vose

Pertactin (formerly 69 kDa protein) is recovered in stable biologically pure form having no detectable adenylate cyclase activity from fermentation broth from the fermentation of as well as from the cells. The broth is processed to selectively remove pertussis toxin (PT) and filamentous haemagglutinin (FHA), the pertactin is precipitated by ammonium sulphate and the precipitate is dissolved in buffer at pH 6.0 to 8.5, the solution then is passed through hydroxyapatite and ion-exchange chromatograph columns before final ultrafiltration. Cells are extracted with urea and the extract ultrafiltered and diafiltered. The pertactin is precipitated from the extract and the precipitate processed as above. In a variation, the broth is contacted with ammonium sulphate to precipitate pertactin, PT and FHA, the precipitate is dissolved and the PT and FHA selectively removed, before the solution is passed to the chromatograph columns.

Purification of a pertussis outer membrane protein

A fimbrial agglutinogen preparation is prepared from a bordetella strain, particularly a B. pertussis strain, by a multiple step procedure involving extraction of the fimbrial agglutinogens from cell paste and concentrating and purifying the extracted material. The fimbrial agglutinogen preparation may be used to prepare acellular pertussis vaccines with other pertussis antigens, including pertussis toxin or toxoid thereof, the 69 kDa protein and filamentous hemagglutinin and other Bordetella antigens.

Acellular pertussis vaccines and methods of preparing thereof

Pertactin (formerly 69 kDa protein) is recovered in stable biologically pure form having no detectable adenylate cyclase activity from fermentation broth from the fermentation of Bordetella pertussis as well as from the cells. The broth is processed to selectively remove pertussis toxin (PT) and filamentous haemagglutinin (FHA), the pertactin is precipitated by ammonium sulphate and the precipitate is dissolved in buffer at pH 6.0 to 8.5, the solution then is passed through hydroxyapatite and ion-exchange chromatograph columns before final ultrafiltration. Cells are extracted with urea and the extract ultrafiltered and diafiltered. The pertactin is precipitated from the extract and the precipitate processed as above. In a variation, the broth is contacted with ammonium sulphate to precipitate pertactin, PT and FHA, the precipitate is dissolved and the PT and FHA selectively removed, before the solution is passed to the chromatograph columns.

Lymphocytosis promoting factor (LPF) and filamentous hemaglutinin (FHA) are isolated from the growth medium of the Bordatella pertussis organism and purified by selecting adsorbing the LPF and FHA on a selective adsorbing medium, such as filter aids or gel filtration media, at low ionic strength and subsequently removing the adsorbed LPF and FHA at using an aqueous medium of high ionic strength, either simultaneously or sequentially. Prior to desorbtion of the LPF and FHA, the adsorbing medium may be contacted with an aqueous solution of a non-ionic detergent, which enables the LPF and FHA subsequently desorbed to be substantially free from contamination by lipopolysaccharides (LPS). The LPF and FHA may be further purified on hydroxyapatite. The LPF and FHA may be detoxified separately or together by contacting with a cross-linking agent, such as glutaraldehyde or formaldehyde, in the presence of an anti-aggregation agent. The resulting purified and detoxified LPF and FHA may be used to formulate a vaccine against pertussis.

Purification of pertussis toxins

The present invention provides for the use in the production of cellulolytic and xylanolytic enzymes, particularly xylanase and cellulase, of the microorganism Thermoascus aurantiacus in a culture medium containing at least one of a cellulose or hemicellulose substrate whereby to produce thermostable enzymes, particularly cellulase and xylanase.

Production of thermostable xylanase and cellulase

The separation of xylanases from mixtures thereof with other hemicellulases, particulary cellulase produced by the culturing of hemicellulolytic microorganisms, particularly the fungus Trichoderma harzianum E58 and Trichoderma reesei by ultrafiltration through an ultrafiltration membrane having a low molecular weight cut-off point in the range of about 1,000 to 20,000 daltons to obtain a cellulase rich retentate and xylanase rich ultrafiltrate. The dilute xylanase rich filtrate from the ultrafiltration is concentrated and purified by adsorption and elution from an insoluble matrix, e.g. a cationic exchange resin. The xylanase obtained is suitable for use in the hydrolysis of hemicellulose for which it is selective, particularly in the presence of cellulose and the cellulase rich retentate is suitable for the hydrolysis of cellulose.

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Mississauga, Canada

Larry U Tan