Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-01-07
2000-07-18
Chin, Christopher L.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 41, 435 84, 435170, 4352521, 4352534, 435885, 514 54, 536 551, C12P 1926, C12P 1904, C12N 120
Patent
active
060905964
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention is related to a method for the production of high molecular weight hyaluronic acid by fermentation using supercapsulated strains of streptococci. The invention also relates to a method for the selection of supercapsulated mutants and to mutants producing such hyaluronic acid in high yield.
BACKGROUND ART
Hyaluronic acid (HA) or hyaluronan is a glycosaminoglucan consisting of repeating disaccharides of alternating D-glucuronic acid and N-acetylglucosamine molecules. These molecules are joined by a .beta. (1,3)-D linkage while the glucosamine to glucuronic acid linkage is .beta. (1,4)-D.
There are several sources of hyaluronic acid and its molecular weight varies considerably depending on the source. The HA found in synovial fluid has a molecular weight of about 1 to 8 million, that in human umbilical cord has a molecular weight around 3.6-4.5 million and HA in rooster combs may reach very high values, for instance up to 12-14 million, or even higher. The chemical composition of hyaluronic acid is the same regardless of its source and since it is non-immunogenic it has found several applications in medicine (Brimacombe and Webber (1964)). The effectiveness of HA is a result of an unique combination of elastic and viscous properties, which are correlated to the molecular weight. Therefore, there was early an interest in obtaining as high molecular weights as possible.
Accordingly, the literature contains numerous examples of very high values of the molecular weight of HA but these values very often refer to the source material. It should be noticed, however, that since the HA as produced in biological systems like rooster combs, is associated with proteins and other glycosaminoglycans, for example chondroitin sulphate, it has to be extensively purified. Even if very sophisticated methods for purification and sterilization have been developed it is inevitable that the molecular weight decreases during these steps and the final product in most cases has much lower molecular weight.
The major HA product on the market today is Healon.RTM. (Pharmacia AB, Uppsala, Sweden) which has a molecular weight around 3.5 million. This product is prepared from rooster combs according to a method based on the disclosure of U.S. Pat. No. 4,141,973. From the same source is prepared a HA product with a molecular. weight around 5 million, Healon.RTM. GV (Pharmacia AB). These moleular weights refer to the sterilized products and this means that the product before the sterilization step must have molecular weights around 5 and 7 million, respectively.
There are very few high molecular weight HA products on the market, in spite of the well-documented usefulness of HA in several medical indications, for instance in ophthalmology. One reason for this is probably the complex purification procedures required in order to obtain a pure product from the sources mentioned above, especially rooster combs, without too much degradation of the molecular chains. Therefore, there is a need for alternative sources or production systems which are well controlled and which allow a simplified purification procedure.
Numerous articles and patent applications have been published which relate to the production of HA in various bacterial systems. The use of bacteria for biotechnological production of HA has been advocated for several reasons, technical, economical as well as ethical. The production by Streptococcus spp. has been known for more than 50 years and most of the systems disclosed seem to refer to group A and C streptococci, for instance encapsulated strains of Streptococcus pyogenes (group A), which is a human pathogen (Kendall et al (1937)), and Streptococcus equi and Streptococcus equisimilis (group C), which are animal pathogens. The synthesis of hyaluronic acid as the major capsular polysaccharide in these pathogens is a way to evade host defenses (Roberts et al (1989)).
The biochemistry of HA synthesis in bacteria involves the action of two, sofar known, genes, has A coding for synthase, which is a
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Wirt et al., 1992. New aspects of surgical treatment of glaucoma. Comparison of viscoelastic substances in chamber angle surgery, Ophthalmologe (Germany) 89:218-222, BIOSIS Abstract #94126491.
Chin Christopher L.
Grun James L.
Pharmacia AB
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