Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1993-07-06
1995-05-02
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
4352521, 435885, 435101, 536 551, C12P 1926, C12P 1904, C12N 120
Patent
active
054118743
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for the production of hyaluronic acid (HA) by bacterial fermentation.
HA is a member of a class of polymers known as glycosaminoglycans. HA is a long chain linear polysaccharide and is usually present as the sodium salt which has a molecular formula of (C.sub.14 H.sub.20 NNaO.sub.11)n where n can vary according to the source, isolation procedure and method of determination. However, molecular weights of up to 14.times.10.sup.6 have been reported.
HA and its salts can be isolated from many sources including nearly all connective matrices of vertebrate organisms. However, HA is also a capsular component of bacteria such as Streptococci as was shown by Kendall et al, (1937), Biochem. Biophys. Acta, 279, 401-405.
HA is non-immunogenic and therefore has great potential in medicine. HA having a high molecular weight (over 1 million) has been found to be particularly useful because of its visco-elastic properties. The HA which is at present commercially available is generally obtained from avian sources such as rooster combs but problems with this material include the likelihood of it being contaminated by viruses. Complex purification procedures are therefore needed and a suitable process is described in U.S. Pat. No. 4,141,973. However, the need for this extensive purification clearly adds to the production cost of the material.
Because of the problems associated with the isolation of HA from avian sources, attempts have been made to develop fermentation processes in which HA is produced. Although all species of Streptococcus produce HA, it is important to choose a species which is a good producer of HA and which is free of hyaluronidase activity.
U.S. Pat. No. 4,517,295 describes a fermentation process using S. pyogenes but the product has an average molecular weight of only 55,000. EP-A-0144019 describes an alternative fermentation process using S. equi which claims to produce a high molecular weight HA but the molecular weight is calculated by a non-standard method and cannot therefore easily be compared with molecular weights calculated by other methods. WO-A-8604355 and U.S. Pat. No. 4,897,349 both describe fermentation processes in which HA of high molecular weight is produced in good yield but in both of the processes, a pathogenic species of Streptococcus is used and so the HA product is likely to be unsuitable for use in medicine because of contamination by the bacterial toxins.
In addition, all of the prior art processes described are batch fermentation processes. There are various problems with batch fermentation processes and these include production of a contaminated product which is difficult to purify.
It would therefore be particularly advantageous to develop a fermentation process which is free from the usual disadvantages of batch fermentation and in which HA having a high molecular weight (for example several million) could be produced.
In a first aspect of the invention, therefore, there is provided a process for the production of HA by fermentation of Streptococcus, characterised in that the process comprises continuous fermentation of Streptococcus in a chemostat culture which is maintained at a pH of from 6.0 to 7.0, a dilution rate of 0.05 to 0.12 h.sup.-1 and a dissolved oxygen tension of less than 1% saturation.
Continuous fermentation processes are known and the theory has been described by Herbert et al (1956) J. Gen. Micro., 14, 602-622. The number of commercial continuous fermentation processes are limited because of the perceived difficulty of continuous fermentation processes over traditionally based batch processes. Also, continuous fermentation processes have traditionally been considered to be suited only for large production output, low product value facilities whereas batch culture has always been used for low production output high product value facilities such as those used to make HA.
The process of the invention overcomes various problems associated with traditional batch culture technique. In batch culture, as the Streptococcus
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Dunn Geoffrey M.
Ellwood Derek C.
Evans Charles G. T.
McInnes Neil
Smith Keith J.
Fermentech Medical Limited
Lilling Herbert J.
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