High molecular weight sodium hyaluronate

• US 4,784,990 A
• Filed: 01/18/1985
• Issued: 11/15/1988
• Est. Priority Date: 01/18/1985
• Status: Expired due to Term

• Current Assignee: Bio-Technology General Corp.
• Original Assignee: Bio-Technology General Corp.
• Inventors: Nimrod, Abraham, Greenman, Benjamin, Kanner, Dov, Landsberg, Moshe
• Primary Examiner(s): Weimar, Elizabeth C.
• Application Number:
  US06/692,692
• Time in Patent Office: 1,397 Days
• Field of Search: 435/101, 435/253, 435/801, 435/803, 435/818, 435/885, 536/55.1, 536/123, 514/54, 514/62, 514/847, 514/915
• US Class Current: 514/54
• CPC Class Codes: A61K 31/715 Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12R 2001/46 Streptococcus ; Enterococcus; Lactococcus
• CPC Class Codes: Y10S 435/885 Streptococcus

Abstract

A novel mutant microorganism Streptococcus zooepidemicus HA-116 ATCC 39920, has been produced. The microorganism produces large amounts of high molecular weight hyaluronic acid. The invention provides a method of obtaining such microorganisms.

The invention also concerns a method of obtaining sodium hyaluronate which comprises growing with vigorous agitation a microorganism of the genus Streptococcus under appropriate conditions in a suitable nutrient medium containing a sugar component as a carbon source. The sugar component is present in a substantially constant concentration between 0.2 and 10 grams per liter. The medium has a substantially constant pH between about 6.0 and 7.5 and includes a substantially constant magnesium ion concentration above 0.05 grams per liter. The sodium hyaluronate excreted into the medium by the organism is purified using methods involving precipitation, redissolving and reprecipitating the hyaluronate. Composition of sodium hyaluronate which are characterized by an absence of pyrogenicity and skin irritation are obtained.

Claims

1. A composition characterized by an absence of pyrogenicity and inflammatory activity and by the absence of proteins of animal origin which comprises between about 88% to 92% by weight sodium hyaluronate and from about 8% to 12% by weight water, the sodium hyaluronate being characterized by a minimum viscosity of about 3.5 m³ /Kg, an average molecular weight from about 3.5×

10⁶ daltons to about 4.0×

10⁶ daltons, a glucoronic acid to N-acetyl glucosamine ratio of 1;

1, from about 4% to about 6% sodium ion, a specific optical rotation measured at 25°

C. and at a wavelength of 436 nm from about 155°

to 165°

, less than 0.01% by weight protein, less than 0.001% by weight sulfate, less than 0.02% by weight neutral sugar, less than 0.05 ng/ml of endotoxin, less than 0.2 mg/g of iron and less than 0.2 mg/g of copper.