Method for the microbiological production of non-antigenic hyaluronic acid

• US 5,316,926 A
• Filed: 11/22/1991
• Issued: 05/31/1994
• Est. Priority Date: 11/25/1983
• Status: Expired due to Term

• Current Assignee: Miles, Inc. (Bayer AG)
• Original Assignee: Miles, Inc. (Bayer AG)
• Inventors: Ruiz, Linda L. C., Greene, Nathan D., Wilson, Curtis D., Trump, Sandy L., van de Rijn, Ivo, Brown, Karen K., Bryant, Sharon A.
• Primary Examiner(s): Marx, Irene
• Application Number:
  US07/796,178
• Time in Patent Office: 921 Days
• Field of Search: 435/101, 435/885, 435/172.1, 435/253.4, 536/55.1, 536/55.2, 536/123, 536/124
• US Class Current: 435/101
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: Y10S 435/885 Streptococcus

Abstract

The present disclosure is concerned with the production of high molecular weight hyaluronic acid suitable for medicinal administration to mammals without provoking an immune response from microbiological fermentation. The cultures may be prepared from specially developed strains of hyaluronic acid generating bacteria obtained by passaging in serologically negative host animal blood. The cultures are kept in log phase growth for an extended period by appropriate temperature, pH and glucose content adjustments. If the cultured strain is not hyaluronidase negative the hyaluronidase activity is inhibited. The hyaluronic acid is precipitated from the culture by the sequential addition of an anionic surfactant and then a cationic surfactant and extended from the precipitate with a high molarity aqueous calcium ion solution. The isolated aqueous hyaluronic acid solution may then be purified by passage through a nitrocellulose filter. Its pyrogenicity may be alleviated by treatment with a strong acid washed activated carbon.

Claims

1. A process for the production of high molecular weight hyaluronic acid with a narrow molecular weight distribution comprising

a) increasing the virulence and the hyaluronic acid generating ability of an existing strain of Streptococcus equi by passaging it through serologically negative horse blood,

b) growing a culture of the strain of a) in a medium free of extraneous protein in a defined medium,

c) maintaining the culture in log phase growth for at least about 24 hours by

i) maintaining the pH of the medium between about 7 and 7.2,

ii) maintaining the temperature at about 37°

C., and

iii) feeding at least about one weight percent of glucose, based on the total culture weight to the medium no less frequently than about every 24 hours,

d) separating the generated capsular hyaluronic acid from the cell walls by incubating the culture with at least about 0.01 weight percent, based on the total culture weight, or a sulphonate based anionic surfactant,

e) precipitating the generated hyaluronic acid from the medium containing said anionic surfactant by the addition of an aliphatic quaternary ammonium salt,

f) separating the precipitated hyaluronic acid from the anionic surfactant and ammonium salt by dissolving it in a high molarity aqueous solution of a calcium salt,

g) precipitating the hyaluronic acid from the calcium salt solution by combining the solution with a lower alcohol selected from the group consisting of ethanol and isopropanol,

h) repeatedly redissolving the reprecipitated hyaluronic acid in water, adding sufficient sodium chloride to provide at least about ten grams per liter and reprecipitating the hyaluronic acid by combining the solution with ethanol or isopropanol,

i) passing an aqueous solution of the repeatedly dissolved and precipitated hyaluronic acid through a nitrocellulose filter, and

j) treating an aqueous solution of the repeatedly dissolved and precipitated hyaluronic acid with sufficient strong acid washed activated carbon to remove any pyrogen from said hyaluronic acid as measured by the rabbit pyrogen test.

2. A process for the production of high molecular weight hyaluornic acid comprising

a) growing a microorganism having all of the identifying characteristics of the strain of Streptococcus equi ATCC Number 39506 in an aqueous chemically defined medium which is free of protein not released by the microorganism,

b) inactivating any extracellular hyaluronidase generated by the microorganism,

c) maintaining the microorganism to essentially log phase growth for in excess of about 24 hours by

i) maintaining the pH in the range of between about 7 and 7.2 by continuous or intermittent addition of base,

ii) maintaining the temperature in the range of about 37°

C., and

iii) adjusting the glucose content of the medium to at least one weight percent at least every 24 hours,

d) isolating the generated hyaluronic acid from the culture without disrupting the streptoccoccal cells, and

e) purifying the isolated hyaluronic acid without causing any significant molecular weight degradation.