Production of hyaluronic acid

A process for the production of hyaluronic acid by continuous fermentation of Streptococcus equi in a chemostat culture gives high yields of high molecular weight hyaluronic acid uncontaminated by toxic impurities. The process is advantageous in that it solves the problem of traditional batch culture in which degradation enzymes can begin to break down the cell walls of Streptococcus releasing cell contents into the fermenter broth, leading to purification difficulties.

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 approaches stationary phase, various degradation enzymes 
start to break down the cells releasing cell contents into the fermenter 
broth and this leads to purification difficulties. This does not occur if 
a continuous fermentation process is used since the fermentation medium is 
maintained in a steady state so that the expression of such enzymes is 
reduced. A further advantage of the steady state obtained with continuous 
fermentation is that the cell wall turnover is reduced which is 
advantageous because it has proved extremely difficult to separate HA from 
cell wall components which have been released into the fermentation 
medium. Finally, the use of continuous culture avoids the expression of 
various toxins which are expressed during the stationary phase of batch 
cultures. The use of a continuous fermentation process therefore allows 
for the production of a much purer product. 
The HA produced by the process of the invention has an average molecular 
weight of from 1 to 3 million and under preferred conditions, the 
molecular weight is from 1.6 to 2.5 million. High molecular weight HA in 
solution has visco-elastic properties which make it extremely useful in a 
variety of clinical fields including wound treatment, ophthalmic surgery 
and orthopaedic surgery. HA is also potentially useful in a variety of 
non-medical fields. 
If the HA produced by the process of the invention is to be useful in 
medicine, it is of course important that any contaminants should not be 
toxic. The species of Streptococcus used in the fermentation process 
should therefore preferably be one which is not a human pathogen in order 
to minimise the risk that any bacterial contaminants remaining in the 
product will then be toxic. In addition, the safety of the manufacturing 
facility is increased if a non pathogenic Streptococcus is used and an 
accidental leak from a fermenter will not cause serious health risks. A 
particularly suitable species for use in this fermentation process is S. 
equi although other species could of course be used. 
Suitable strains of S. equi for use in the process will easily be selected 
by those skilled in the art using long term selection in the chemostat 
culture and choosing a stable, high yielding phenotypic variant suitable 
for long term culture by isolating from the culture, individual cells to 
use as seed for further fermentations. A sample of the culture may be 
streaked on to solid medium and colonies originating from individual (or a 
small number of) cells allowed to grow. We have found that the starting 
strain of S. equi may be improved by selecting fast growing colonies with 
large mucoid capsules having a stringy appearance when pulled with a loop. 
These colonies may be used to seed the fermenter for the next run. 
Preferably, they are first subcultured onto further plates and the same 
selection criteria applied to select seed-colonies. 
A particularly suitable strain has been deposited by us under the Budapest 
Treaty at the National Collection of Industrial and Marine Bacteria 
(NCIMB), 23 St. Machai Drive, Aberdeen, Scotland AB2 IRY on 24 Oct. 1990 
under the accession No NCIMB 40327. 
The fermentation process of the invention takes place in a nutrient medium 
containing the following components: 
an assimilable source of carbon; 
a source of nitrogen; 
sources of phosphorus, sodium, potassium, magnesium, iron, zinc and 
manganese; 
sources of growth factors; and 
a source of sulphur. 
Carbon may be supplied in the form of a sugar, particularly glucose, 
although sucrose can also be used. The source of nitrogen may be a 
non-toxic nitrogen containing salt, particularly a water soluble salt, for 
example, an ammonium salt such as ammonium chloride. The metals and 
phosphorus may also be supplied in the form of water soluble salts. The 
necessary growth factors are all contained in a source such as yeast 
extract which may also be the source of the sulphur which is required. 
The nutrient medium may additionally contain sources of one or more of 
calcium, molybdenum, cobalt copper or boron. 
The growth rate of the bacteria in the continuous fermentation process may 
be controlled by limiting the availability of one essential component of 
the nutrient medium, thus limiting biomass production but not energy 
conversion or polysaccharide formation. The supply of any of the essential 
components listed above may be limited but it is preferred to limit the 
supply of the sulphur. 
The pH of the nutrient medium must, as mentioned above, be maintained 
within the range of 6.0 to 7.0. A preferred range is 6.0 to 6.4 and most 
favourable conditions are achieved when the pH is 6.2. 
The pH of the medium may be maintained within the desired range by the 
addition of an alkali such as sodium hydroxide during the fermentation 
process. Any foaming which results may be controlled by the addition of a 
suitable non-toxic foaming agent, for example an agent based on 
polypropyleneglycol. 
As discussed above, the nutrient medium is supplied to the fermentation 
zone at a dilution rate (flow rate per unit volume of the fermenter) of 
from 0.05 to 0.12 h.sup.-1. The most favourable conditions occur when the 
dilution rate is about 0.07 h.sup.-1. Effluent is withdrawn from the 
fermenter at a rate equal to the rate of supply of nutrient medium to the 
fermenter so as to maintain a constant volume of medium within the 
fermentation vessel. A constant physicochemical environment is maintained 
using automatic controllers which maintain constant optimum conditions for 
the selected strain of S. equi during long term continuous culture. The 
product is harvested from the effluent. 
The culture of Streptococcus is carried out under microaerophilic 
conditions. Air or other oxygen containing gas may be pumped into the 
culture medium at a rate sufficient to maintain a dissolved oxygen tension 
in the fermentation medium of less than 1% saturation and preferably in 
the range of 0.1 to 0.5% saturation. The gas supplied to the fermentation 
medium must be sterile and, in the case where air is used, a suitable flow 
rate may be from 0.1 to 0.5 v.v.m. (volumes per fermenter volume per 
minute). 
It is desirable that the fermentation is carried out with continuous 
agitation since the fermentation medium is viscous and thorough mixing of 
the medium is essential to eliminate "dead zones" in which growth of 
undesired non-HA producing strains of S. equi can occur. 
The temperature of the fermentation medium should be maintained in the 
range of 30.degree. to 40.degree. C. but a preferable range is from 
35.degree. to 40.degree. C. and the most suitable temperature is 
37.degree. C. 
Under the preferred operating conditions, the process of the invention has 
yielded as much as 2.5 g of HA for every litre of fermentation medium and 
it is possible that even higher yields may be obtained. Stable culture 
conditions can be maintained for over 500 hours. The process therefore 
represents a considerable improvement over prior art batch fermentation 
processes. 
After the fermentation process, the biomass is killed and the HA extracted 
with an aqueous medium containing an anionic surfactant. These two steps 
may take place either simultaneously or sequentially. A variety of means 
may be used to kill the biomass, including heat or a killing agent such as 
an antibacterial substance. A particularly suitable agent for killing the 
biomass is formaldehyde which may be used as the aqueous solution commonly 
known as formalin. A suitable concentration of the solution is from 0.5 to 
1.5% (v/v). The surfactant may be added at a concentration of from 0.01 to 
0.05% (w/v) and preferably at a concentration of 0.02% (w/v). A suitable 
surfactant for extracting the HA from the killed biomass is sodium dodecyl 
sulphate. 
The biomass is kept in contact with the killing agent and the surfactant 
until substantially all the HA has been released from the cellular 
capsules and this may take from 10 to 24 hours, usually about 16 hours. 
The residual biomass is then separated from the aqueous solution by 
filtration, for example using a plate and frame filter process and an 
appropriate filtration medium such as kieselguhr filter pads. It is 
necessary to clean or replace these filter pads during the fermentation 
process and a suitable time interval for this may be every 24 hours. An 
alternative filtration method which is particularly useful for large scale 
operation is to use a filter cartridge. Such cartridges have to be 
replaced at similar time intervals to the filter pads. After the 
filtration, a cell-free filtered solution of HA is obtained and this 
solution may be purified by diafiltration to remove low molecular weight 
impurities. These impurities are those derived from the production 
organism's metabolism, the residual components of the nutrient medium, 
residual killing agent and residual anionic surfactants. It is necessary 
in this step to use an ultra filtration membrane with an appropriate 
molecular weight cut off which is usually from 10,000 to 25,000 Daltons, 
and preferably 20,000 Daltons nominal molecular weight. Suitable membranes 
are based on polysulphones and are available commercially. The filtered 
solution containing the dissolved HA is diafiltered against from 8 to 20 
volumes, preferably about 10 volumes of purified water and the liltrate is 
continuously discarded. Water of suitable purity has a conductivity of 
less than 10 .mu.Scm.sup.-1. 
After the diafiltration, the molarity of purified solution of HA is 
adjusted to within the range of 0.18 to 0.24M, preferably 0.20M with 
respect to sodium chloride. The pH is also adjusted if necessary to a 
value of from 6.3 to 7.8, preferably from 7.0 to 7.5. The most favourable 
results are achieved when the pH is 7.2. The pH may be adjusted by the 
addition of a base such as sodium hydroxide or an appropriate buffer, 
particularly a phosphate buffer. 
If a product of medical grade is required, the process may include an 
optional step of precipitating nucleic acids from the solution. This is 
achieved by the addition of a cationic surfactant for example a quaternary 
ammonium compound such as cetyl pyridinium chloride. The cationic 
surfactant may be added as a dilute aqueous solution; for example a 1% 
(w/v) solution of cetyl pyridinum chloride may be added in a volume ratio 
of 1:60 to the solution. The precipitated nucleic acid may then be removed 
by filtration through an appropriate filter medium ranging in pore size of 
from 3.2 .mu.m to 0.2 .mu.m but preferably from 1.2 .mu.m to 0.2 .mu.m. 
If this step is used, subsequent processing must be carried out under 
sterile conditions using pyrogen-free equipment. 
After this optional stage, or, if the optional stage is not used, after the 
adjustment of the molarity and pH of the solution, HA is precipitated by 
the addition of a non-solvent, for example a lower alcohol such as 
isopropyl alcohol. The precipitated HA is filtered off and the filtrate 
discarded. 
Further purification of the HA product can be achieved by redissolving the 
HA in sodium chloride solution and then reprecipitating by addition of a 
non-solvent in the same way as described above. The sodium chloride 
solution must have a molarity in the range of 0.18 to 0.24M, the most 
favourable value being 0.20M. The pH of the solution must be from 6.3 to 
7.8 but is preferably 7.0 to 7.5 and most preferably 7.2. The solution may 
be buffered, for example with a phosphate buffer. 
In a second aspect of the invention there is provided hyaluronic acid 
produced by the process of the first aspect. This HA has an average 
molecular weight of at least 1 million and the range of molecular weights 
of the product is preferably from 1.6 to 2.5 million. 
In a third aspect of the invention, there is provided Streptococcus equi of 
the strain NCIMB 40327.

The invention will now be further described with reference to the following 
examples. 
EXAMPLE 1 
A growth medium for S. equi is formulated as follows: 
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Glucose 60.00 g 
Yeast extract 6.25 g (Oxoid L21) 
Sodium dihydrogen phosphate (2H.sub.2 O) 
2.02 g 
Ammonium chloride 2.14 g 
Potassium chloride 0.71 g 
Citric acid 0.42 g 
Magnesium oxide 0.40 g 
Calcium carbonate 0.10 g 
Sodium molybdate (2H.sub.2 O) 
2.42 mg 
Ferrous chloride (6H.sub.2 O) 
10.80 mg 
Cobalt chloride (6H.sub.2 O) 
0.95 mg 
Copper chloride (2H.sub.2 O) 
0.32 mg 
Zinc oxide 0.81 mg 
Manganese chloride (6H.sub.2 O) 
4.00 mg 
Boric acid 0.12 mg 
Conc. Hydrochloric acid 
0.178 mL 
______________________________________ 
This medium is made up to 1 L with purified water. It is then sterilized by 
filtration through an 0.22 .mu.m absolute rated filter. 
The fermentation medium is pumped continuously into the fermenter at a flow 
rate, in relation to the fermenter volume, of 0.07 h.sup.-1. The medium is 
aerated with sterile air which has been filtered through an 0.2 .mu.m 
absolute rated filter. The air flow rate is maintained at 0.2 v.v.m. and a 
dissolved oxygen .tension is maintained in the fermenter broth at 0.2% 
saturation. Streptococcus equi is grown in this culture medium at 
37.degree. C. The pH is maintained at 6.2 by automatically controlled 
additions of sodium hydroxide. Foam generation is controlled by addition 
as necessary of a polypropylene glycol based antifoam. 
Fermentation medium is continuously withdrawn from the fermenter at the 
same rate as the fresh medium is fed in. This effluent contains about 2.5 
gL.sup.-1 of hyaluronic acid. Sodium dodecyl sulphate and formalin 
solution are continuously fed into the effluent from the fermenter to 
achieve final concentrations of 0.025% (w/v) of sodium dodecyl sulphate 
and 1% (v/v) of formalin. Mixing of the effluent and the sodium dodecyl 
sulphate/formalin streams takes place in an in-line static mixer. The 
contact time is 16 hours and, after mixing, the stream passes through a 
vessel designed for this residence time. 
After release of the hyaluronic acid into the aqueous medium, the residual 
biomass is removed continuously by depth filtration in a cartridge filter 
using filters of appropriate pore size. Duplicate filter units are used to 
allow periodic diversion of the product flow to a clean filter, thus 
allowing cleaning and replacement of used filters. The filter units are 
sized to allow up to 24 hours operation before flow diversion is required. 
The solution from which the cells have been removed is then processed by 
diafiltration against purified water to remove residual materials from the 
culture medium, sodium dodecyl sulphate and formalin. This diafiltration 
is operated using a polysulphone based ultrafiltration membrane with a 
20,000 Dalton nominal molecular weight cut off. The solution is 
diafiltered against ten volumes of water having a conductivity of less 
than 10 .mu.S cm.sup.-1, and the filtrate is continuously discarded. After 
diafiltration, sodium chloride (final concentration 0.2M) is added to the 
solution obtained and the pH adjusted to 7.2 by addition of phosphate 
buffer (Na.sub.2 HPO.sub.4, 0.22 gL.sup.-1 ; NaH.sub.2 PO.sub.4.2H.sub.2 
O, 0.045 gL.sup.-1). A 1% (w/v) solution of cetyl pyridinium chloride is 
then added in a ratio of about 1:60 by volume. The nucleic acids thus 
precipitated are removed by pumping the solution through 1.2 .mu.m and 0.2 
.mu.m (absolute rated) depth filters arranged in series. The filtered 
solution of hyaluronic acid thus obtained is then continuously mixed in 
line with a metered flow of isopropyl alcohol at a flow ratio of 1:.2. The 
mixing is performed in a static mixer and the precipitated hyaluronic acid 
is separated from the aqueous solution in a basket filter. The filtrate is 
discarded. The recovered hyaluronic acid is redissolved in 0.2M sodium 
chloride solution buffered at pH 7.2 with phosphate (Na.sub.2 HPO.sub.4, 
0.022 gL.sup.-1 ; NaH.sub.2 PO.sub.4.2H.sub.2 O, 0.045 gL.sup.-1) to give 
an HA concentration of 0.2% w/v). The hyaluronic acid is again 
precipitated from this solution by addition of isopropyl alcohol in the 
same way as previously. 
The precipitated hyaluronic acid is washed with isopropyl alcohol and the 
washings are discarded. Final traces of isopropyl alcohol are removed by 
drying in air under sterile conditions. All the purification procedures 
are carried out at ambient temperature. 
A medical grade solution may be made by dissolving hyaluronic acid produced 
in the manner described in 0.15M sterile saline solution buffered with the 
phosphate buffer mentioned above, pH 7.3 to give a 1% (w/v) solution of 
hyaluronic acid. The sodium hyaluronate solution so prepared has an 
average molecular weight of 1.6 to 2.5.times.10.sup.6 Da as determined by 
low angle laser light scattering techniques and viscometry. The solution 
has a protein content of less than 0.2% (w/w) and a nucleotide level of 
less than 0.15% (w/w). The 1% (w/v) solution shows a U.V. absorption of 
0.14 AU at 260 nm and 0.1 AU at 280 nm. The viscosity of the solution is 
159 Pa.s at zero shear falling to less than 1 Pa.s at 1000 s.sup.-1. 
The following example demonstrates a method of selecting suitable strains 
of S. equi for use in the fermentation process. 
EXAMPLE 2 
A solid medium for growth of S. equi is formulated as follows: 
______________________________________ 
Glucose 20 g 
Yeast extract 5 g (Oxoid L21) 
Agar 15 g (Oxoid L 13 
.sup. Agar No. 3) 
Di-potassium hydrogen 
1.706 g 
orthophosphate anhydrous 
Potassium dihydrogen ortho- 
1.388 g 
phosphate 
Sodium dihydrogen orthophosphate 
2.92 g 
Ammonium chloride 5.01 g 
Potassium chloride 372 mg 
Citric acid 420 mg 
Magnesium oxide 50.4 mg 
Calcium carbonate 10 mg 
Sodium molybdate 2.4 mg 
Ferrous chloride (6H.sub.2 O) 
270 mg 
Cobalt chloride (6H.sub.2 O) 
2.37 mg 
Copper chloride (2H.sub.2 O) 
0.85 mg 
Zinc oxide 2.05 mg 
Manganese chloride (4H.sub.2 O) 
10 mg 
Boric acid 0.3 mg 
Conc. Hydrochloric acid 
0.24 mL 
______________________________________ 
The glucose is dissolved in 50 mL of water, the yeast extract in 100 mL and 
the agar/salts dissolved in 820 mL of water, sterilized separately by 
autoclaving at 121.degree. C. for 15 minutes and then mixed together prior 
to pouring the plates. 
Samples of S. equi from a fermentation experiment performed as described in 
Example 1 were obtained for strain improvement. Strain improvement was 
carried out by selecting large fast growing domed colonies with large 
mucoid capsules having a stringy appearance when pulled with a loop. These 
colonies were subcultured onto further plates where the same selection 
criteria were applied. 
It can therefore be seen that the process of the invention provides an 
economically viable fermentation process for obtaining pure HA of high 
molecular weight and therefore represents a significant improvement over 
prior art processes.