Note: Descriptions are shown in the official language in which they were submitted.
- 13~8~
Mo-2789-ClP
A METHOD FOR THE MICROBIOLOGICAL PRODUCTION
OF NON-ANTIGENIC HYALURONIC ACID
FIELD OF THE INVENTION
This disclosure is concerned generally with the
preparation, purification and use of hyaluronic acid and its
salts and specifically with the preparation of hyaluronic acid
from a microbiological source.
BACKGROUND OF THE INVENTION
Hyaluronic acid is a naturally occurring high
o molecular weight polysaccharide typically recovered as its
sodium salt having an empirical formula of (C14 H20 N Na 11)n -~
where n > 1000. The general structure of hyaluronic acid is
illustrated ;n Merck Index, N;nth Ed. (3rd pr;nt;ng, 1978), at
page 624. It ;s well known that hyaluronic ac;d and ;ts salts,
hereafter collectively referred to as HA, can be obta;ned from
at least three sources: human umb;l;cal cords, rooster combs
and certain bacterial cultures such as group A and C hemolyt;c
streptococci. However, certain d;sadvantages are associated
with the former two sources (e.g. relatively low y;elds,
contaminat;on w;th chondro;tin sulfate, and labor ;ntensive ~ -
processing and purification steps). -
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Mo-2789-CIP -
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Since HA is found in aqueous and vltrecus humor
of eyes and the synovial fl~id of mammalian j oints,
there has been considerable interest in obtaining
p~ ified HA for use as a fluid replacement to co~~ect
5 pathological conditions in the eye and in joints. The
preparation of HA from rooster combs and h~man ~mbilical
cords and its use in eye and joint applications is
described in U.S. Patent 4,141,973 to E. A. Balazs.
That patent also provides a detailed review of the
10 technical lite ature describing the isolation,
characterization and uses of HA.
U.S. Patent 4,303,676, also to E. A. Balazs,
describes cosmetic formulations containing sodium
hy21uronate fractions in various molecular weight ranges
1~ made from rooster combs. U.S. Patent 4,328,803 to L. G.
Pape discloses the use of an ultrapure hyaluronic acid
salt in eye su~gery. The HA product used was a sodium
hvaluronate salt a~ailable under the registered
trademark ~YLARTIL from Pharmacia, Inc. and obtained in
20 commercial quantities from rooster combs.
Because the medical applications of ~A require
that the HA be in3ected into a mammalian body (e.g. as a
fluid replacement), it is very important that the ~ ~
injected products be as pure as possible to avoid -
2~ reactivity problems. This importance of purity is
described in U.S. Patent 4,141,973 which desc-ibes an
ultrapure HA prod~ct prepared from rooster combs or,
alternatively, from h~man umbilical cords. In addition
to purity, it appears that control of molecular weight
30 of an HA product is very important (e.g. the 4,141,g73
patent suggests an average molecular weight of at least
750,000 daltons and U.S. Patent 4,303,676 suggests
h2~ing two distinct fractions of contro'led molecular
weight, one low and one high). Although there is a
~o-2789-CIP
,,
132~8~1
- 3 -
description of a high molecular weight (1,200,000
daltons) HA preparation of very high purity (i.e. less -~
than 0.05 % protein) in a paper by swann, Arch. Opthal.
88, pp. 544 - 8 (1972), we are unaware of any description
of an HA product having the following advantages: (1)
derivable from a microbiological source at relatively low
costs, in high yields, and with low reactivity upon
injection; (2) having a desirably high and closely
controlled average molecular weight: and (3) being
substantially free of protein and nucleic acid
impurities.
The microbiological production of hyaluronic
acid is well known in the literature. A rather extensive
discussion is found in "The Biosynthesis of Hyaluronate
by Group A Streptococci", a 1955 doctoral thesis on file
at The University of Minnesota. Japanese Published
Patent Application 83-56692 teaches greatly enhancing the
production of hyaluronic acid from StrePtococcus
zooepidemicus and streptococcus equi cultures by adding
high levels of glucose to a protein (yeast extract)
containing culture and continuously aerating while
shaking. Although a 1976 paper of Kjem in Acta. Pathol.
Microbial Scand. is acknowledged as teaching
microbiological production of hyaluronic acid no
explicit mention of a chemical defined medium (CDM)
or a protein free medium is made. There is no indication
that its technology could be applied to such a
specialized medium. U.S. Patent No. 2,975,104 to Warren
teaches a techni~ue for increasing the acceptable
incubation time of streptococci in producing hyaluronic
acid by the use of a particular medium which contains a
hyaluronidase inhibitor. The growth of Group A ~
streptococcal strains in a chemically defined medium -~ - -
(CDM) with the production of a hyaluronic acid capsule
Mo-2789-CIP
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is disclosed in "Growth Characteristics of Group A
Streptococci in a New Chemically Defined Medium" by Van
de Rijn and Kessler at pages 444 to 448 of Volume 27,
Number 2 (February 1980) of Infection and Immunity. The
5 recovery of hyaluronic acid from a somewhat different CDM
by heat killing the culture, filtering the medium, ~-
precipitating with ethanol, centrifuging the precipitate,
suspending the precipitate in an aqueous sodium chloride
solution, reprecipitating with cetyl pryidinium chloride,
o centrifuging the reprecipitate, dissolving this
reprecipitate in an aqueous sodium chloride solution and
finally precipitating once again with ethanol is
described in "Isolation of Hyaluronic Acid from Cultures
of Streptococci in a Chemically Defined Medium" by Kjems
15 and Lebech at pages 162 to 164 of Section B, Volume 84
(1976) of Acta Path. Microbiol. Scand. This procedure
has some significant disadvantages. The heat killing of
the microorganisms will result in the hyaluronic acid's
becoming unnecessarily contaminated with proteins,
20 nucleic acids and other internal cell components which
are difficult to separate from the hyaluronic acid and
can provoke immune reactions in mammals. Thus, some of
the benefit of utilizing a chemically defined medium to
avoid protein contamination and achieve non-antigenicity
is unnecessarily lost as compared to the contamination
suffered merely from the natural death of cells in a
grow.ing culture. Furthermore, this CDM itself has
limited utility because it will not support the growth of
most streptococcal strains. The recovery of hyaluronic
acid of a mean molecular weight of about 55,000 daltons
from an anaerobically grown culture of Streptococuss
pvoqenes inactivated with trichloroacetic acid by
filtering using a 0.22 micrometer pore size, -
Mo-2789-CIP
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diafiltering with a filter ha~ing a nominal retention of
30,000 daltons molecular weight until the conduct-vi~y
of the filt~ate is 0.5 mega-ohms (believed to mean
inverse ohms per centimeter times 104), precipitating
S with ethanol, resuspending in an aqueous sodium chloride
solution, precipitating with CETAB (belie~ed to be
hexadecyl trimethyl ammoni~m bromide), coarse filtering,
resuspending in an aqueous sodium chlo~ide solution and
diafiltering with a filter ha~ing a nominal molecular
10 weight retention of 30,000 daltons until the
conductivity is "0.5 mega-ohms" is described in U.S.
Patent No. 4,517,295 to Bracke and Thacke-. This
procedure also involves the troublesome killing of the
cult~re cells. In this case, the trichloroacetic acid
15 not only kills these cells ~ith the resultant
contamination problem, b~t it also is relied on for an -
effective separation of the cells from the broth.
Furthermore, this broth is only semi-defined and
contains casein derived proteins which may also
20 contaminate the obtained hyaluronic acid.
Non-antigenic hyaluronic acid is also discussed
in the literature. A process of obtaining hyaluronic ,~
acid free from proteins, antigens and pyrogens by
treating an aqueous alkaline suspension to denature the
25 protein, subjecting the suspension under appropriate
condi~ions to proteolytic ferments, removing amino acids
and mineral salts with ion exchangers, and acidifying to
pH 3 to 4 to precipitate the remaining impurities with
some of the hyaluronic acid is described in U.S. Patent
30 Number 3,396,081 to Billek. A reportedly ultrapure
hyaluronic acid suitable fo~ injection Lnto the human
eye or animal joints and obtained by an involved
extraction procedure including a five day chloroform
extraction under mixing is described in U.S. Patent No.
3~ 4,141,973 to Balazs.
Mo-2789-CIP
i3288~1
-- 6 --
A useful procedure for the isolation of
hyal~ronic acid capsules from streptococci cult~res by
incubating the bacteria, ~Thich had already been isolated
by cetrifugation and washed with saline, with sodi~m
5 dodecyl sulfate in a saline suspension until the capsule
was released, centrifuging to ~eco~er the supernatant,
filtering the supernatant through a 0.22 micrometer pore
filter, precipitating the hvaluronic acid with
hexadecyltrimethyl amnonium bromide, recover;ng the
10 precipitate by centrifugation, redissolving the
precipitate in 2M calci~m chloride and clarifying the
solution by cen~rifugation, precipitating with ethanol,
redissolving ~th water followed by the addition of
sodium chloride and clarifying the solution by
15 centrifugation, and repeating the ethanol precipitation
five times is described in "Streptococcal Hyaluronic
Acid: Proposed Mechanism of Degradation and Loss of
Synthesis During Stationary Phase" by Van de Rijn at
pages 1059 to 1065 of volume 156, number 3 (December
20 1983) of the Journal of Bacte-iology. The use of sodi~m
dodecyl sulfate to separate another virulence factos,
M-protein, from Stre~tococcus equi is discussed in U.S.
Patent No. 4,582,798 to Bsown, Bryant and Lewis. These
latter two developments became publicly available after
25 many of the developments with which the present
disclosure is concerned.
There is a need for an efficient and
inexpensive procedure to obtain high molecular weight
hyaluronic acid suitable for injection into mammals. A
30 microbiological fesmentation which gives high yields of
a relatively uncontaminated high molecular weight
hyaluronic acid and a recovery procedure which does not
create un~ecessary purification problems and does not
adversely affect the molecula~ weight of the hyaluronic
35 acid are both desirable and would meet this need.
Mo-2789-CI~
: ::` 132~
-- 7 --
BRIEF DESCRIPTION OF FIGURES
Figures 1-4 are graphs showing HPLC determined
molecular weight distributions (retention times) of HA
made from four microbiological fermentations in
5 accordance with the disclosures herein.
Figures 5~7 are graphs showing HPLC determined
molecular weight distributions (retention times) of three
commercially available prior art HA preparations.
Figures 8-9 are graphs comparing the efficacies
lo as a joint fluid replacement of the HA products of this
disclosure with controls and/or, a commercially available
product.
Figure 10 is a graph showing the use of the
product of this disclosure as a fluid replacement in an
15 equine joint diseased by unspecified cause.
SUMMARY OF THE INVENTION
A procedure for culturing suitable micro-
organisms to give high yields of high molecular weight
antigen-free hyaluronic acid by growing these organisms
20 in a chemically defined medium free of protein, keeping
them in log phase growth by appropriate pH control and
addition of dextrose (glucose) for an extended period,
and isolating the hyaluronic acid without killing the
microorganisms has been developed. The microorganisms
25 are suitable if they produce extracellular hyaluronic
acid capsules and either do not produce extracellular
hyaluronidase or are grown in a medium treated by heat or
an appropriate chemical agent to inhibit the activity of
hyaluronidase. A procedure for separating hyaluronic ~-
30 acid capsules from the cells which produced them by
treatment with an anionic surfactant has also been
developed. Also developed is procedure of isolating
hyaluronic acid from the bacterial culture which produced
it by sequentail treatment with an anionic
Mo-2789-CIP ~c
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s~r,actant 2nd then a cationic s~rfactant. Additionally
developed is a procedure for isolating hyaluronic acid
from anionic/cationic surfactant complexes by dissolving
it in a hi~h molarity aqueo~s solution of a calci~m ion
5 such as calcium chloride. Another development is the
reduction of the protein content of an aqueous
hyaluronic acid solution by passin~ it through a nitro
cellulose filter.--An additional development is the
removal of pvrogens from high molecular weight
10 hyaluronic acid by treating an aaueous solution of it
with strong acid washed activated carbon. A preferred
procedure is to combine some or all of the above
developments with dissolution in water and precipitation
in a lower alcohol, such as ethanol or isopropanol, to
15 obtain high yields of a pyrogen free very pure high r
molecular weight hyaluronic acid. An especially
preferred procedure is to apply these developments in
such a way as to obtain a high molecular weight
hyaluronic acid which as an approximately l weight
20 percent aqueous solution has a protein content of less
than about 1.25 mg/ml, prefera~ly 0.10 mg/ml, ànd a
nucleic acid content of less than about 0.045 mgiml, ~-
preferably 0.005 mg/ml, as determined by W absorbance
at 280 and 257 nanometers, respectively. It is further
25 preferred to apply these developments so as to obtain
hyaluronic acid which displays a single significant
substantially symmetrical HPLC retention peak with ~
retention times representati~e of molecular weights F
between about l,lO0,000 and 4,000,000 daltons and with
30 at least-98Z of the distribution lying within this ,
single peak. It is particularly preferred to utilize
the extraction and purification developments to obtain a
hyaluronic acid which as an appro~imately 1 weight
percent aqueous solution has an am:no acid content by
Mo-2789-CIP
~328~
orthophthalaldehvde fl~iorescence (which inherently it
involves the hydrolysis of any protein present back to
its constit~ent amino acidic) o less than about 0.4
mg/ml and a nucleic acid content by ethidium bromide t;~
5 fluorescence of less than about 0.06 mg/ml and which has
a standardized ~PLC determiined average molecular weight
of at least abo~t l,100,000 daltons.
A hyaluronic acid which has a high molecular ---
weight, a narrow molecular weigh~ distribution and a ~ -
10 very high p~irity has also been developed. The process
developments have enabled the recovery and purification
of hyaluronic acid without the significant loss of -`
molecular weight and without subs~antial broadening of ~
the molecular weight distribution. A preferred ;
15 hyaluronic acid s pvrogen free, has a single ~
substantially symmetrical HPLC retention peak lying - -
between retention times representative of molecular
weights between about 1,100,000 and 4,000,000 daltons i
with about 98~ of the distribution encompassed by this -~
20 peak and has a W absorbance determined protein content '
of less than about 1.25 mglml and a W absorbance ~'?;,,
determined nucleic acid content of less than about 0.04
mg/ml, both determined on a one weight percent aqueous
solution. ç
Finally, a techniaue for creating strains of
hyal~ronic acid generating bactesia espPcially suitable -
for the production of hyaluronic acid by passaging it x
through the blood of an animal which is susceptible to
the bacteria but has not developed an immune response to ~,
30 the bacteria has been developed. A preferred procedure
is to passage a Streptococcus equi strain through horse
blood which gives no evidence, such as a detectable
antibody level, of prior exposure to this bacterium. A ~;
strain was developed in accordance with this technique ~-
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and is on deposit w th the American T~pe Culture ,
Collection (ATCC) under number 39,506. ~-
DETAILED DESCRIPTION OF T~E INV~NTION
The maintenance of hyaluronic a~id generating
5 mic~oorganisms in the log or eY~ponential growth phase
for extended periods is achieved by careful pH and
temperatllre control and peri~dically supplementing the
cult~re with additional dextrose. Neither shaking nor ~
con~inuous aeration are required. This procedure would, ;-
10 of course, be f~tile unless either the particular ~~
microorganism was hyluronidase negative, i.e. it did not
generate extracellular hyluronidase, or the activity of ~'r '
any such extracellular hyaluronidase had been inhibited
by heat treatment or a chemical inhibitor such as those
15 taught in U.S. Patent No. 2,975,104 to Warren. It is
preferred to conduct this growth in a chemically derined
media such as that taught in "Growth Characteristics of -
Group A Streptococci in a New Chemically Defined Medium" :
by Van De Rijn and Kessler at pages 444 to 448 of Yolume ;
20 27, N~mber 2 (February 1980) of Infection and ImmNnitY ~`
so as to avoid the presence of extraneous protein. This
simplifies the s~bsequent p~rification since only the
proteins released fr~m the microorganism being cult~red
then need to be removed. The temperature and pH of the ~--
2~ cult~re should be maintained in ~he range known to be
conducive to the growth of the particular microorganism
being cultured. In the case of Groups A and C -;
streptococci, which are preferred microorganisms, the
tempe-ature is advantageously around 37~C and the pH
30 should be maintained in the range of about 7 and
slightly above. In the case of the particularly
preferred Group C streptococci and the most preferred
Stre~tococcus eoui the pH is preferably maintained
between about 7.0 and 7.2 and the temperature is
Mo-2789-CIP
`- 13288~
1 1 ~ r
prefe~ably abo~t 37~C. Culturin~ fo~ between about 24
and 120 hours ~nder ~hese condi.ions has been found to
be ad~antageous. The pH control may be continuous or it
may be intermittent. In the latter case the p~ may be
5 readjusted each ti~e dextrose is added. For the Group C
strep.ococci i~cluding the Streptococcus eaui it is
prefe-zed to Teadi~st the pH to about 7.6 at each
de~trose addition.
Sufficient dextrose should be added to the
10 microo~~anism on 2 timely enough regimen to avoid
complete depletion of the dextrose from the culture
medium. The mic~oo-ganism utilizes the dextrose in
synthes.~ing its hyzluronic acid capsule and th~s
- continuously cor.sumes dextrose from the medium. In the -
15 case of the streptococci, especially the Group C and
most especially the Stre~tococcus eaui addition on no
more than a twenty four hour interval is preferred.
With these bacteria it is preferred to add approximately -
1 weight percent o dextrose, based on the total weight
20 of the culture including medium. An especially ~-
prefe_red procedure ~rth these bacteria is to add 1
weight percent dextsose every twenty four hours but to -
also add an additional 0.5 weight percent of dextrose
between the larger additions, preferably about s;xteen -
2S hours after the 1.0 percent addition. -~
The recove-y of hyaluronic acid is enhanced if
the pH cont~ol is d7scontinued s~fficiently before it is
to be harvested to cause a significant pH drop. A drop i-
to a value of between about 6.5 and 6.8 is preferred. A
30 desirable ~rop can be obtained by ceasing pH control
about 12 hours before harvest although ceasing berween
about 6 and 12 hours before harvest has been ound
suitable. Such ~ pIocedure facilitates centrifugation
and increases the yield of hyaluronic acid.
~ " ' ,~: ' '
Mo-2789-CIP
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rne ability to ma;n.ain the microorganis~ in ,~
log phase growth is surp-ising and makes it possible to
continuouslv culture the microorganism. In s~ch a ;:
procedure once some of the n~trients other than the
5 rapid~ consumed dextrose become exhausted, the depleted
medi~m may be separated from the cells of the
microor~anism and replaced ~-ith a freshly constit~ted
medium. Al,ernatively, the depleted nutrients can be
added to the medium in app-opriate amounts. However, a.
10 some point, .he waste products and the hyaluronic acid
not attached to the cells need to be extracted from the
culture. One convenient technique is simply to filter "
the medium using a pore size which retains the cells.
Then the cells can be pro~ided with fresh medium and
15 hyaluronic acid can be ext.acted from the depleted ~`-
medium.
In preparing the initial medium and, in the
case of con~inuous culturing, in preparing a replacement
medium it is preferred to sterilize the medium to avoid
20 the presence of undesirable microorganisms. A
convenient technique for such a sterilization is to pass
the medium through a fine pore filter, such as a 0.22
micr~meter pore filter. -
In initiating the culture,-it is also -
25 convenient to use an inocu'a.ion seed grown on the same -
CDM as the growth medium and to use between about l and
5 volume percent Oc inoculate. If the CDM described by
Van De Ri~n and Kessler, Supra, is used it is also
convenient to add the cysteine and bica_bonate of soda
30 to the medium just befo,e inoculation. These two :
nutrients have a tendency to fo~m salts which can
precipitate out of the mediu~.
The separation of hyaluronic acid, especially
in czpsular form, from the ce~ls of the microorganism
Mo-2789-CIP
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1328841
- 13 -
which generated it ~s conveniently a~fected by ~.
inc~bation with an anionic s~rfactanL. Among the
suitable surfactants are those compo~nds which are
available in sufficient puriLy to use in such a
5 fermentation bath and which carry one or more sulfate or
sulphonate groups. Among the particularly preferred
surfactants are sodi~m dodecyl sulfate and dioctyl
sodium s~lfos~ccinate, with the former being especially
preferred. The amount of sur~actant required is
10 dependent on the hyal~ronic acid content of the cultu-e
and is generally at least about 0.01 weight percent
based on the weight of the culture. In the case of
Group C streptococci and sodi~m dodec~l sulfate at a
0.01 wt. % le~el, an incubation time at 37~C of about 15 -
15 minutes has been found to be beneficial. In the case of
microorganisms force grown, i.e. kept in log phase, for
an extended period the increased content of hyaluronic
acid requires a higher content of su-factant. ~or - --
cultures containing between about 1 and 2.5 grams of
20 hyaluronic acid per 10 liters a surfactant content in
the neighborhood of 0.1 weight percent is usually found
suitable. A suitable range for such high yield culture
is between about 0.05 and 0.25 weight percent, based on
the weight of the culture including the medium. The
2~ separated hyaluronic acid can then be isolated from the
culture by any suitable means including filtration to
remove the larger cells and diafiltration to remove the -
lower molecular weight species. A particularly
preferred technique is to precipitate the hyaluronic
30 acid and anionic surfactant by the addition of a
cationic surfactant. :
The isolation of hyaluronic acid from the
medium in which it is grown can be effected by the
sequential addition o~ an anionic surfactant followed by
Mo-2789-CIP
-" 132~841
- 14 -
the addition of a cationic surfactant. The suitable
types and amounts of the anionic surfactant are discussed
hereinabove. The cationic surfactant is preferably an
ammonium salt and more pr~ferably a quaternary ammonium
salt. Especially preferred are quaternary ammonium salts
with four aliphatic substituents particularly those in
which at least one of the substituents is a long
hydrocarbon chain. Among these long chain substituted
aliphatic quaternary ammonium salts hexadecyl trimethyl
lo ammonium bromide is particularly preferred. A preferred
technique for precipitating the hyaluronic acid is to
cross titrate the anionic and cationic surfactants in
samples of the culture until a heavy readily separable
flock is obtained. A convenient manner to accomplish
this crosstitration is to prepare numerous one milliliter
samples of culture at each of several levels of anionic
surfactant, for example at levels between 0.05 and 0.25
weight percent in 0.05 percent steps, and then add
various amounts of the cationic surfactant to the samples -
for each level of anionic surfactant, for example amounts
between lO and 100 microliters of a 10% solution may be
added in lO microliter steps. As a double check after
the appropriate amount of anionic surfactant has been
added to the total culture a sample may be titrated
25 against the cationic surfactant to confirm the
appropriate level to be added.
The anionic surfactant is thoroughly mixed and
incubated with the culture before the addition of the
cationic surfactant. In general, a mixing time of at
30 least about fifteen minutes is preferred with a mixing
time of at least thirty minutes being more preferred,
especially with culture volumes in the neighborhood of
2000 liters or more.
Mo-2789-CIP
.
i328841
- .5 -
It has been fo~nd advantageous to use a 10
percent aq~eo~s solution of hexadecyl trimethyl ammonilm
bromide to precipitate the hyaluronic acid from a ten
liter culture to which at least about 0.01 weight
5 percent of sodium dodecyl sulfate had been added and
mixed fo- at least fifteen minutes. It was found that
between about 100 and 400 millilite-s of this solution
was suitable and that at least abo~t one hour sho~ld be
allowed ~or maximum floc formation.
The preclpitate formed by the sequential
addition of anionic and cationic surfactants may be
separated from the balance of the culture by anv common
liquid solid separation technique. Among the convenient ~~
and pre e-red techniques are fil,ration and
15 centrifugation. It is preferred to cool the entire
medium below the growth temperature and preferably below
about 10C and to store it for an extended period, in
the case of 2000 liter or larg~s cult~res for in excess
OI si~teen hours, before effecting this separation. The . ~-
20 most pre,erred separation technique is centrifugation.
Utilizing such a procedure resulted in a supernatant
almost completely free of hyaluronic acid.
Hyaluronic acid may be separated from an
anionic/cationic surfactant complex by dissolution in a
25 high molarity aqueous solution of a calcium ion. A
prefe-red solvent is a 2M solution of calcium chloride.
It is p-eferred to use a reduced volume of solvent
compared to the original culture and between about 5 and
10 volume percent has been found suitable although
30 between about 10 and 40 volume-percen, is preferred.
The solution is preferably ca- ied out at temperatures
between about 4 and 30C with temperatures between about
4 and lODC being preferred for 2 period of at least 6
hours w~th periods of be ween about 1 and 10 days being
Mo-2789-CIP
r
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132g841
- 16 -
preferred fo~ original cultures of 2000 liters o,
greater.
The dissolved hyaluronic acid can now be
isolated from the s~rfactant complex and other materials
5 which precipitated with the su~factant complex ~y any
typical solid/liquid separation technique including
filtration or centrifugation. Centrifugation is
particularly preferred.
The hyal~ronic acid can then be purified by a
10 va-iety of techniques which involve precipitation in a
lower alcohol followed by resolution in water.
Particularly suitable alcohols a,e ethanol and
isopropanol.
The protein and nucleic acid content of the
15 hyaluronic acid can be significantly reduced by passing
an aqueous solution of it through a nitrocellulose
filter. It is pseferred to conduct at least one and
preferably several ethanol precipitations and subsequent
water resolutions before effecting this filtration step.
20 This will reduce the protein load on the filter matrix
and reduce the probability that it will become saturated
with protein. The pore siz~ of the filter is not
critical but is somewhat dependent on the viscosity of
the solution being filtered. Pore sizes ~om 0.22
25 micrometers to 2~ micrometers m~y readily be utilized
~th pore sizes of 8 micrometers and greater being
preferred for the more viscous solutions to minimize the
back pressure. With a single thickness of a standard
nitrocellulose filter it is preferred to utilize no more
30 than about 20 mg/cm2 of protein per unit area of filter
with a ratio of no more than about 10 mg/cm2 being more
preferred and a ratio of no moFe than about 2 mg/cm2
being especia~ly preferred. ~lthough nitrocellulose is
known to ha~e affinity for n~cleic acids it is -
o-2789-CIP
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~32~8~1
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s~rpris ng that it is able to extract both nucleic acids
and protein from an aq~eo~s solu~ion of hyaluronic acid,
which itsel~ is known to have an affinity for these
materials. There does not appear to be any basis for
5 predicting that the eauilib~ium between water and
nitrocellulose, let alone water con~ainin~ an a~tractant
for protein, would lead to substantial extraction of
protein. In this reg2-d treating a 4.5 liter solu,ion
by ~assage thro~gh an approximately 670 cm
10 nitrocellulose filte- ~esulted in an about 78% red~ction
in protein from 0.028 ~. % to 0.006 wt. ~. Such an ~-
equilibrium pa-tition;ng was totally unpredictable.
Further filte, thicknesses proportionall~
;ncrease the protein ~e~ unit area of filter which can
15 be remo~ed. For inst2nce with two thicknesses, it ls
prefe-red to utilize no more than aboùt 15 mg/cm of
protein per unit filte~ area.
Hyaluronic acid may be freed of pyrogens by
contact as an aqueous solution with acid washed
20 activated carbon. The pyrogenic agent is evidently
independent from the protein and nucleic acids which may
provoke an im~une response in a m~mal. A pyrogenic
response has been detected in rabbits iniected with
hyaluronic acid which has a very low amino acid content
25 (b~ the orthophthalaldehyde fluorescence which
hydroiyzes any proteins present and detects them as
their constituent amino acids) and a very low ethidi~m
bromide fluorescence detected nucleic acid content.
This ,esponse was observed using the established -~
30 protocol of injecting into the ear vein of a frequently
handled ~abbit and observing any resultant increase in
body te~perature. A r~se of greater than 0.6C o. a
total rise of greater than 1.2C from this rabbit and
two additionally injécted rabbits is an indicatior. of
Yo-2789-CIP
''~ '
13288~1
- 18 -
pyro~enicitv. When the same batch of hval~ronic acid is
tested according to the same protocol after exposing it
to a strong acid washed carbon no pyrogenic indication
was observed. It is prefe~red to ~se s~fficient
5 activated carbon to give 2000 pre:Eerably 10,000 and most 1,
preferably 20,000 m of s~face area per liter of
aq~eous 1 weight percent sol~tion being treated. An
especially preferred technique is to utilize a fairly
high s~rface area ac~ivated carbon of more than abo~t
10 500 m2/g and to ~til-ze in excess of abo~t 10 grams pe-
liter of one weight percen. aqueous hyal~ronic acid
solution.
The acid washed active carbon treatment can be
either continuous or batch. In the former case,
15 treatment may conveniently be effected by circulation
th~ough a cartridge. In elther case treatment can be
repeated until negat;ve pyrogenicity is achieved. In
view o. the minimal Emo~nt of pyrogen present, ~ypically
in the nanograms per milliliter range, the concern is
20 not saturation o the adsorbent but rather adequate ,
contact with the pyrogen.
The aqueous hyal~ronic acid solution obtained
by the use of the calcium ion treatment of the '
anionic/cationic surfacta~t precipitant may be further
25 p~rified by repeated prec;pitations ;nto a lower
alcohol, such as ethanol or isopropanol and resolutions
in water. In a prefer~ed procedure the supernata~t from
the calci~m ion ~reatment (preferably a 2M aqueous
calcium chloride solution) is extracted with 2 volumes
30 of a suitable alcohol, preferably 9~% ethanol or 97Z
isopropanol and cen rifuged or sieve filtered after at
least one hour. Then this precipitate is solubilized
overnight at between abo~t 4 and 10C in deionized,
distilled wate. uSing between abou~,l/lO and 1/20 of the
Mo-2789-CIP
- 19 - 13288~1
original vol~e and the precipitate is removed by sieve
filtration or centrifugation. This is followed ~y the
addition of s~fficient sodium chloride to ~ive a one
percent solution by weight, i.e. 10 grams per liter of
5 solution. This solution is then extracted with two
volumes of an appropriate alcohol to reprecipitate the
hyaluronic acid (actually at this point more precisely
sodium hyaluronate). The precipitate is then isolated
by either sieve filtration or centrif~gation. The
10 resolution in water, addition of sufficient salt (sodi~m
chloride) to give 1% (10 grams per liter) and
precipitation with two volumes of an appropriate alcohol
are continued until the aqueo~s solution is clear using
;ncreasingly smaller volumes of water with each
1~ resolution and consequently s~aller volumes of alcohol
(twice the volume of the solution being extracted).
This typically requires four additional alcohol
p~ecipitations. An outline of this proced~re is as -:
follows:
OUILINE O~ PROCESS ~OR EXTRACTION -
OF BACTERIAL H~ALURONIC ACID -
1. Grow Streptococcus-orga~ism
2. 1 ml/l SLS 10~.
3. 10 - 40 ml/l.
Hexadecyltrimethylammo~ium bromide 10~.
4. Collect ppt.
. Solu~ilize in 2M CaC12.
6. Collect supernate.
7. 2 Vol. alcohol
(ppt. HA, some nucleic acids, some protein).
8. Collect ppt.
9. Solubilize ppt. in DI-H2O.
10. Discard undissol~ed ppt.
11. Collect supernate.
Y.o-2789-CIP
- 20 - 132884~
12. ~ ~aCl
Vol. alcohol
(ppt. HA).
13. Collect ppt.
5 14. Solubilize in DI-H2O.
15. Discard ppt.
Collect supernate.
l~. 1% NaCl
2 Vol. alcohol
(ppt. HA).
17. Collect ppt.
18. Sol~bilize in DI-H20.
19. Discard ppt.
Collect supernate.
15 20. Filter - protein binding type (e.g., nitro-
cellulose) (remove some of the minimal protein
remaining.
21. l~ NaCl
2 Vol. alcohol
~ppt. HA).
22. Collect ppt.
23. Solubilize 0.15M phosphate burfered saline pX 7.2.
24. ~djust to lZ HA by spectrophotometric assay. -
25. Ste_ilize with 0.1Z betapropiolactone
4 - 10 C 24 - 48 hours.
26. Hydrolvze betapropiolactone
37 C 24 - 48 hours.
27. Fill syringes.
The ~Lnal steps of product pTeparation may involve
30 washi~g with g5% EtOH and 99% acetone followed by drying
unde_ vacuum. The dried ~A is resuspended in 0.15M
sodiu~ phosphate buffer to a concentration of 1.0~.
This m2y be filter sterilized throu~h a final 0.45
mic~o~eters nitrocellulose type f~lter and/or sterilized
35 in fir.al bulk form with 0.1% betap opiolactone. The
betapIopiolactone sterilization is conducted at 4 C for
24 - 48 hou~s followed by hydrolization of the
Mo-27~9-CIP
13288~
- 21 -
betapropiolactone at 37~C for 24 - 48 hours. The final product
contains 10 mg/ml HA in 0.15M sodium phosphate buffer. When
these steps are followed, HA of the highest purity is obtained
in high yield (> 99.90% HA).
A more preferred procedure to isolate sodium
hyaluronate from the supernatant of the calcium ion treatment
involves the use of diafiltration, and reverse precipitation
with a winding device. This supernatant may be diafiltered
against a membrane which allows species with molecular weights
less than about 1~0,000 to pass through. The water to the feed
side of the membrane may then be cut off and water flow on the
pass side of the membrane continued until the volume is between
about 10 and 40% of the initial volume. This typically gives a
hyaluronic acid (or calcium hyaluronate) concentration of
between about 0.4 and 0.7 weight percent. This is followed by
three ethanol precipitations with a winder apparatus and
resolutions utilizing the procedure described in copending
Canadian Patent Application Serial Number 521,989 filed
November 3, 1986 by Karen K. Brown et al. In particular, the -
sodium chloride content of this diafiltered concentrate is
adjusted to ten grams per liter and it is fed to three volumes
ot 95% ethanol in which a device with vertical fingers is
slowly rotating. The precipitating sodium hyaluronate attaches
itself to these moving "fingers". The precise configuration
and speed of rotation of this "winder" is adapted to the
particular vessel used and the batch size but a cage like
general configuration is preferred. The ethanol is then
drained from the vessel and the precipitated hyaluronate is
redissolved by the addition of water to the vessel. The winder
should preferably be rotated for at least about 30 minutes
after the last of the diafiltered hyaluronate solution is added
and may also be rotated after the water addition to aid in
Mo-2789-CIP
~/
~ .
13288~
-- 22 --
in resolution. The third resolution may conveniently be in the
final desired product formulation buffer such as an aqueous
0.15M sodium phosphate solution. In such a case it is
advisable to assay the hyaluronate after the second resolution.
The fairly pure sodium hyaluronate solution may now
be further purified and sterilized. In this regard, it is
convenient to drop the viscosity of the solution in accordance
with copending Canadian Patent Application Serial No. 521,989
filed November 3, 1986 by Karen K. Brown et al. In particular,
o it may be heat treated, preferably at a temperature in excess
of about 50C for in excess of about 72 hours in an open
vessel, or forced through a filter with a pore size less than
about one micrometer, until the 37C viscosity is less than
about 250 centistokes. At this point, the solution may be
conven;ently tested for pyrogenicity in rabbits and, if a
positive result is obtained, appropriately treated with strong
acid washed activated carbon. Finally, the now low viscosity
pyrogen free solution may be filtered through a 0.45 or 0.22
micrometer pore nitrocellulose filter for both sterilization
and final protein and nucleic acid reduction.
The former procedure was used to obtain sodium
hyaluronate for further evaluation. The material from four
different fermenters was initially evaluated after
solubilization in the 2M calcium chloride. One of these lots
was then further purified in accordance with this procedure and
evaluations made at various stages.
The typical 10 1 fermenter of Streptococcus equi
produces 5 9 to 7 g dry weight of cells and 1.0 g to 2.5 9 dry
weight of HA. Yield is therefore between 14.3% and 50% (w/w).
Yields of HA from extraction of rooster combs as in U.S.
Mo-2789-CIP
~ . ,.
- 132~8~1
-- 23 --
Patent 4,141,973 are reportedly around 0.079%. -
It should be noted that a latter-stage filtration
through a suitable protein-binding filter (for example a
nitrocellulose filter) is necessary in order to remove
reactivity of the final product HA. Other types of filters
(plain cellulose and cellulose acetate) do not adequately
remove reactivity as observed in the horse joint injection
test. It is thought that this step removes the minute quantity
of reactive proteinaceous material remaining in the HA.
The purity of this bacterial-derived HA was
determined via a colorimetric protein assay, U.V.
spectrophotometry, HPLC, and slab gel electrophoresis. Initial
experiments involved quantitation of protein contamination as
measured via the BIO M D* Protein Assay. This method can
5 detect levels of protein as low as 200 ug/ml. Table I lists
the results of testing aqueous 1.0% solutions of hyaluronic
acid extracted from four different fermenters of Streptococcus
equi.
TA~LE I
BIO RAD* Protein Assay Results
Concentration
SampleO.D. at 595 nm of Protein
Ferm 10.00,0.000 < 200 ug/ml
Ferm 20.00,0.010 < 200 ug/ml
Ferm 30.00,0.005 < 200 ug/ml
Ferm 40.00,0.005 < 200 ug/ml
According to such data, the protein content of a 1.0%
bacterial-derived HA solution may be as high as 0.02%.
A second method of determining protein, peptide,
and/or amino acid content is UV absorption at 280 nanometers.
A known concentration of bovine serum albumin was used as a
control. Table II reports these
*Trademark
Mo-2789-CIP
"'' ~,
. i ~ ''i .,. . , . .,, . ~ ~ .. .
~32~8~1
- 24 -
results. Als~ reported are the W absorptions of some f
comparative sol~tions at 257 nm. Absorption at 257 nm
~epresents contamination with n~cleotides or nucleic
acid such as DNA and ~'A. It is noted that '.,
5 spectrophotome~ric absorption at 280 nm detects more
protein contamination than the Bio RAD assay. The l.OZ
sol~tions of bacterial-derived HA contain at most O.lZX
contaminants which absorb at 280 nm. Since these ~ame !,
solutions contain almost no nucleic acid contamination,
10 ~he purity is in the range of at least 99.88X. In this
_espect it is notable that amino acid analysis of
s;milarly extracted HA indicated the presence of ~ 0.04%
protein. This would mean that the HA purity is as high
as 99.96%. This is compared with the p~-ity of
1~ commercially available rooster comb derived XA
(HYLARTIL~ available from Pharmacia or HyalovetT~
available from Trans Bussan) which, according to o~r
tests, have purities in the range of 99.78Z to 99.86X
respectively.
~o-2789-CIP
:
- 25 - 1 3 2 ~
TABLE I I
PROTEIN AND NUCLEIC ACID CONTAMINATION OF 1. 0%
HYALURONIC ACID AS MEASURED BY W SPECTROPHOTOMETRY
W Absorbence Concentration
Nucleic
O.D. at O.D. at Protein Acid
Sample 280 nm 257 nm mg/ml ua/ml
Ferm 1 0.45 - - 0.66 - -
Ferm 2 0.83 - - 1.22 - -
10 Ferm 3 0.75 - - 1.10 - -
Ferm 4 0.47 - - 0.69 - -
Miles Labs
(Rooster Comb)
low purity1.16 1.31 1.70 48.5
15 Pharmacia
HYLARTIL~ 0.91 1.63 1.33 60.3
Trans Bussan
Hyalovet TM1.27 >2.0 1.86 >74<300
1/30 dil. = 0.27
Further studies on purity were conducted with
the bacterial-derived HA. Effectiveness of two alcohol
purification processes were followed
spectrophotometrically at 280 nm, 257 nm, and 195 nm.
Absorbance at 195 nm represents the actual absorbance of
HA and is linearly related to concentration of HA. Table
III shows optical density results whereas Table IV
converts all readings to concentrations of protein,
nucleic acid and HA. These two more purified lots
yielded bacterial-derived HA which was 99.99% pure using -
30 either 95% ethanol (ETOH) or 99% isopropyl alcohol -
extraction. ~
' ~' '
-:. '
Mo-2789-CIP ~ -
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1328841
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Mo-278g-CIP - "~
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- 28 - 1 3 2 8 8 ~ 1 d
Slab gel electrophoresis was used to further r
analvze the various 1.0~ hyal~ronic acid preparations
listed in Table II for nucleic acids. Such a technique
can differentiate DNA from RNA. A O.~Z low endosmosis
5 agarose containing 2 ug/ml e~hidi~m bromide was used in
conjunction with short w2ve length W light in order to
visualize the nucl~ic acids after electrophoresis. DNA
being of-a m~ch larger molecular weight remains near the
origin whereas RNA migrates with the buffer front.
10 Twenty-five ul samples were electrophoresed 18 hours at
90 volts in a Canalco Slab Gel apparatus. Results
indicated no detec,able nucleic acids in any of our fo~r
preparations or the HYLARTIL~ product. The Hyalovet
product showed a significant amount of nucleic acid in
15 the form of RNA.
High Performance Liquid Chromatography (HPLC)
was used to analyze the molecular weight of the various
~A preparations. This is a newer a~d more accurate
method than viscometry as mentioned in the Patent No.
20 4,141,973. A Waters Micro Bondagel/E-High ~ column was
used for molecular weight determination. In this column
it was impossible to run aqu~ous standards in the
molecular weight range required along with test samples
in order to determine accurate molecular weights.
25 However, relative molecular weights based upon retention
times on the column were dete mined. Theoretically,
with this procedure, the earlier the time of peak
detection the higher ~he molecular weight. The col~mn
had a retention time of 10 min. with minimum and maximum
30 molecular weight capabilities between 15,000 2nd -
7,000,000 daltons. Figures 1 - 4 show the 'HPLC tracings
for our first fou~ fermenter preparations. Miles
Hyaluron, Ph macia HYLARTIL, and Trans Bussan Hyalovet
are shown in Figuses 5 - 7. Ihe retention tLmes of the
~o-2789-CIP
. . " ~, . ,, . . . . . . -.. . . .-.
x ~Y ~ ~ q r~ i?~ .h~P:.. q.. ,.. ~.q,~.:i~.. I r~
- ~ -
13288~
- 29 -
peaks and shoulders were determined and relative
molec~lar wei~hts calculated based on a linear
relationship between molecular weight (15,000 to
7,000,000 daltons) and retention time (0 - 10 minutes).
5 Such relative molec~lar weights are listed in Table V.
As can be seen in Figures 1 - 4, sodium hyal~ronate
obtained according to this disclosure gives an
essentially single, substantially symme. ical high
molecular weight (avg. higher than about 2,000,000
10 dalton) HPLC distribution peak. In Figures 1 - 4 at
least about 9~Z of the HA molecules are within the
single peaks shown. Such close control of high
molecular weight distribution is not shown in existing
commercial products as illus~rated in Figures 5 - 7.
15 Figure 5 (Prior Art ~1) illustrates the HPLC tracing for
the Miles Hyaluron HA product. Figure ~ (Prior Art ~2)
represents the Hyalovet product and Figure 7 (Prior Art
~3) represents the HYLARTIL~ product. It is though~ -
that this close control of final molecular weight -ange
20 may be due to the simplicity of the extraction procedure
requiring minimal shear-producing steps as well as to
the lack of hyaluronidase which could degrade high
molecular weight HA. -
. .
Mo-2789-CIP
_ 30 _ ~ 32 88 4
TABLE V
Relative Molecular Wei~hts of Hyal~ronic
Acid Moieties in Various Pre~arations
Relative Molecular wei~ in Millions
5 Sample Range Avera~e
Fermenter 12.2 - 3.9 2.8
Fermentes 22.5 - 4.0 3.8
Fermenter 31.7 - 2.8 2.4
Fermenter 41.1 - 3.9 2.6 :~
lO Hvaluron0.015 - 3.0 0.015
HYALOVET~ 0.010 - 3.7 .015, l.8, 3.7
HYLARTIL~< O.OlO - 3.8 1.9
As noted above the relative molecular weight
range of HA moieties fo~nd in bacterial-derived HA is
15 narrow with the majority (98~) measu-ing between about
1,100,000 or 2,200,000 and 4,000,000 daltons. Via the
same method, Hyalovet contains three distinct moleculaT
weight moieties of 2,700,000; 1,700,000 and 300,000
daltons. Finally, the HYLARTIL~ product contained an
20 array of EA molecular weights from ~10,000 to 3,700,000 -~
daltons. As shown, the ~Y~ RTIL~ product contains the
widest variation of molecular weight sizes.
Fr~m the various analytical tests described
here;n, it has been determined tha~ HA extracted from
25 bacteria ~ia a simple method is purer than three
commercial products made from either rooster combs o-
~mbilical co~ds. The latter are produced via a complex
process which is inefficient yielding only 0.079~ ~A.
This is compared with H~ extracted from streptococci
30 which can reach yields as high as 50% w/w.
Joint Fluid Re~lacement ~
Hyaluronic acid prepared from bacteria as ~~ -
described herein has been tested for reactivity in
tibiotarsal and radial-caspal joints of horses. rne
Mo-2789-CIP
'''^'`"` '
- . r~ i
- 1 3 ~
- 31 -
following cl~nieal index test was devised in order to
measure reactivity of HA prepara.ions post ,~
intra-articula- injection of horses.
The test protocol is as follnws:
1. Assess normal movement of joint to be
iniected. Assign læmeness indices from 0 to 5 according
to the following definitions.
Lameness Index
0 = No lameness
1 = S;i~ht lameness - moderate ,~
2 = Noticeable lameness - moderate
3 = Obvious lameness
4 = Severe lameness - reluctant to move o ;
bea_ weight
5 = Cannot bear weight. If down, animal
is unable to rise.
2. Sedate horse (e.g. with Rompun~ sedative).
3. -Shave hair around the joint area to be
in3ected. -:~
4. Determine Swelling Observation Index
according to the following definitions.
Swellin~ Obser~ation Index
0 = No Swelling ~-
Nothing obvious - palpable fluid
2~ 2 ~ Slightly noticeable - palpable fluid ~-
3 c~Noticeable s~elling of entire joint `r':4-~ Severe swelling at injection site
Severe swelling involving more than
the joint alone. i~
5.~ With cloth tape measure, measure joint ;i;
ciscumference ~mmiediately anterior to the anterior --~
aspect of the third metatarsal (tibiotarsal joint) or
imnediately distal to the protuberance of the accessory
ca:rp~l bone that~is aro~nd the~rad~ial carpal bone
35 (c2:~al joint).
Mo-2789-CIP
~'
- 32 - 1328~1
The exact circ.u~ference o~ the joint (in ~
millimeters) before and after iniection is recorded. A t.-"
difference between the circumference each day post
in~ection and the original circumference is calculated. ',7
5 If the difference is greater than 1.0 cm, the exact
measurement is added t~ the other twc, index values in
order to determine the clinical index.
6. Remcive joint fluid tl.0 - 2.0 cc) prior to `
in,ection with a 3.0 cc syrin~e with a 20 - 22 ga. X 1" ;
10 needle.
7. Inject joint with 2.0 cc of a 1
prepa.ation of hyaluronic acid being evaluated for
reactivity. ~or this injection, use a 3.0 cc syringe
with the same needle ~exchanging sy_inges only) as used
15 to remove joint fluid. This is done so as to reduce
trauma to the joint as m~ch as possible. .
8. Apply digital pressure to the injection
site for 1 to 3 minutes after injection. This is done
to pre~ent backflow of the very viscous HA. ~
9. Observations and measurements are made for .s -
four consecutive days post injection, then on day 7. -~
10. The Clinical Index (CI) is calculated as ~-
follows: ~ -
Total Lameness Index (TLI) - Sum of Daily ~,
Lameness Indices
Total Swelling Indeg (TSI) = Sum of Daily -
Swelling Observations . Sum of Joint
Circumference Measurements Greater Than 1.O
; ~ :
cm.
CI = TLI + TSI -
11. Interpretation :
Joint injection alone causes trauma with
development of some swelling and lameness. -
This was proven by evaluating numerous
Mo-2789-CIP -
,
,J
_ 33 _ 1 3 28 ~ 4
joints injected with phosphate buffered
saline (PBS) and some joints in which only
fluid was removed. CIs were calculated on
these traumatized joints. They varied from
0 to 18.7 a~fon~ 56 joints. However, the
average CIs in the three separate studies
of traumatized joints sho~ing these wide
individ~al ~ariations were 0.7, 5.3, and
3.4. It is thus suggested that an average i`.
CI value of 6.0 or less in at least lO
joints could be expected from injection
trauma. On this basis, we have assigned a
10 - joint average CI value of 6.0 or less ~,A'
~ as acceptable in the horse joint reactivity .
1~ test for evaluation of HA preparations.
Any product showing a lO - joint average of
CI of '6.0 is unacceptable. Using these ,~
criteria, several HA prepasations were
tested. Results are shown in Table VI. i
TABLE Vl ;
Evaluation of Hyaluronic Acid Prep æations ,
by the Horse Joint Reactivi tv Test :~
No. of Average AcceptabilitY ''
Pre~aration ioints CI of Preparation
25 Microbiological 14 4.6 Acceptable
Source HA Filtered
~ ~` th~ro~gh~itrocellu- 13 ~ Acceptable "
-~ lose --
, . ,~
Microbiological 11 11.4 Unacceptable '
30 Source HA Non- ~
filtered 6 17.4 Unacceptable --;.
Prior Art
Purified;and 10 6.2 Unacceptable
Filtered ---
The microbiological source HA listed in Table -~
. VI was that obtained from fermenters 1 through 4 as
Mo-2789-CIP
~ ` ~'.'.
.
-. :
: : .
1~288~1
- 34 -
described previo~sly. It is noteworthy that this ~,
material is acceptable for joint inj~ction arter
nitrocell~lose filtration b~t not prior to s~ch `.
filtration. On the other hand, Prior Art ~' (see Fig~re ;
5 5) is on the borderline of being ~nacceptable even after
nitrocellulose ~iltration. Evidently, the ~eactive
proteinaceo~s load in the Prior Art 1,1 prepa,ation is
too great to be removed via the protein binding
filtration step.
The same Clinical Index can be used to evaluate
efficacy of treatment of diseased joints wi-.h HA. In
this test system clinical symptoms are ind~ced in joints
wi~h intraarticular injection of complete o. incomplete
Fre~nd's adjuvant. This adjuvant produces rirst an
15 acute and then a chronic pathology of the joint
characterized by extreme lameness and swelling which
does not appear to reverse itself within two months.
Some such efficacy studies have been conducted
on the bacterial-derived HA.
Experiments were designed to eval~ate the
effect of semoving some of the joint fl~id ,rom adj~vant -~
induced pathological joints and replacing it with
bacterial-derived HA. This was done with both acute
joints (HA injection within three days of the Fre~nd's
25 Co~plete Adj~vant injection) and with chro~ic joints (HA
injected within 12 to 34 days of Freund's ;~jection).
Clinical Index evaluation was beg~n the day of adj~vant
injection and continued for fo~r days following the HA
injection a~te which weekly observations were made for
30 three weeks. Figures 8 and 9 display the ~es~lts over
30 day periods.
Fig~re 8 represents the acute situation. The
zero day readings were all adj~sted to seven on the
relative index scale so that comparisons could be better
o-2789-ClP
_ 35 _ 13288~1
~is~alized. Zero day repr~sents three days post
Fre~n~'s injection in the acute joints. Figure 8 then
portr~ys the change in Clinical Index for the first 30
days post injection with HA from fermenters 1 and 2 and ~
5 Prior Art ~1 after f~rther purification. These res~lts
are compared with similar adi~vant injected joints left
untreated (control). It is notable that the control
horses contin~ally worsen thro~gh day 4 post Fre~nd's
injection before showing some improvement on their own.
10 Howeve., this improvement does not ~each the starting
level by day th ee and by day four appears to be
plateauing. This is the t~pical picture for induction
of chronic pathology. A significant improve~ent in ';~
acute symptoms is observed after injection of HA.
The chronic situation is represented by Figure -
9. Horses which ha~e been injected with ~reund's
Complete Adj~vant 12 - 34 days prior to HA treatment can
serve as their own controls since these horses had been `-
stable for at least seven days prior to day zero. The
20 control line represents these control index levels.
Again, immediate clinical impro~ement is noted after -
treatment with HA. Longer term observation of these ~r,
horses has indicated that the improvement tends to
plateau. Therefore, it is expected that more than one .
25 treatment may be necessary.
One horse entered the study with a diseased '~
io:nt of unspecified cause. As indicated in ~ig~re 10,
two injections of bacterial-derived HA were a~ministered r~
to this joint 30 days apart. In this case, the zero day --
30 readings were adjusted to 10 in order to display the `:
complete treatment response. Immediate improvement was i~-
noted after both the first and second injections of HA.
The improvement after the first injection was followed
by some return of pathology as indicated by s~elling
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only. After the secon~ HA injection joint swelling was
eliminated and has not ret~rned within three months post r
treatment.
From the data presented herein it is obvio~s
5 that ~ltra p~re bacterial-derived HA is nonreactive in
horse joints and displays efficacy for reversing
lameness and/or swelling in diseased joints. Since
bacterial-derived HA as described herein is p~rer than
any commercially available product, incl~ding those used
10 in opthalmalogical trea~ments, it is highly probabl~
that these HA preparations could also be ~sed to replace
vitreous h~mor of the eve during surgery. They sho~ld
be able to substitute for any other use applied to the
rooster comb or umbilical cord HA.
The invention is further illustrated, b~t is
not intended to be l;m; ted by the following examples in
which all parts and percentages are by weight unless
otherwise specified.
EXAMPLE 1
The viruience of a Stre~tococcus equi and
therefore its hyaluronic acid production, which is a
virulence factor, was enhanced by passaging throu~h
serologically negative horse blood. In particular, so~e
pus from an abscess of a horse showing the symptoms of a
25 Stre~tococc~s eaui infection, which was later
established by differential s~gar testing and staining
to contain this microorganism, was inc~bated with some
serologically negative horse blood (blood having no
detectable levels of antibody against this
30 microorganism) and the CDM described in the Van de Rijn
and ~essler article, s~pra, for three ho~rs at 37C. A
small aliquot was then placed on a blood agar plate
(smsll petsi dish ~ith solid agar and small amount of
medi~m containing blood) o~ernight at 37~C. The large
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mociod colony was then transferred into a fresh mixt~re ~,
of serologically ne~ative horse blood and CDM for three
ho~rs at 37C. After a total of four inc~bations in
horse blood/CDM the final isolated strain was grown in
j~st CDM and a sample was submitted to the ATCC and
received deposit n~mbe~ 39,506. This strain was
s~bstantially ~ore virulent than the initial nat~rally
occ~rring strain.
XAMPLE 2
The strain developed in Example 1 was ~sed to
grow cult~res for 22 and 64 ho~rs and the hyal~ronic
acid generated was harvested and isolated. In
partic~lar these c~lt~res were,grown in a stirred vented
vessel with air feed lines which s;mply compensate for
15 the oxygen cons~med by the microorganisms b~t do not
"aerate" by vigorously adding air or oxygen in the same
CDM as ~sed in Example 1 at 37C with the pH initially
set at 7.6 and thereafter controlled at a value of abo~t
7.2. In the 64 ho~r c~lture 1 weight percent of
20 dextrose, based on the total weight of c~lt~e, was
added every twenty-fo~r ho~rs. For both c~lt~res the p
control was terminated abo~t sixteen ho~rs before
harvest.
Both c~ltures Temained in the log or
25 exponential growth phase for at least the period of pH
control. This was appaTent from the pH controller
' recosd which indicated additions approximately every
'~ five min~tes and from optical density meas~rements which
increased during the entire cu}turing peri~d as iolloas:
. :
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ITEM 22 Hour C~lture 64 Ho~r C~lture 6'
In HoursOPtical Density Optical Density }
3 0.13 0.07
6 0 ~ 17 0 ~ 12 r
22 0~18 0~22 ~ -
28 0.22
0.22
33 0.235 -
38.5 0.5 -
64 0.675
(at 1:10 dil~tion)
The la~ in the sixty fo~r hour culture was attri~uted to
its exhausting the glucose in its medium before the next
addition. It was s~rprising that a streptococcal
15 organism could be kept in log phase in a CDM for an ';
extended period. The only p-ior reported extended log
phase growth for this genus of microorganisms appears to
be an aerated-Shaker Culture in a protein rich medium
which is reported in Japanese Published Patent
20 Application ~6692-83.
Sodium hyaluronate was then harvested and
isolated from each culture by the first techniq~e
describe~ hereinabove in~olving ethanol precipitation
followed by centrifugation and redissolution in water. ~ -
25~However, in this case five precipitations with ethanol
were utilized. The ~dry cell weight of each culture was ''
estimated by designating one centrifugation pellet as a --
standard and then estimating the dry cell weight content ~--
fs the pellet weight. The d-y cell content and
30 hyaluronic acid yield were as follows:
.'
' . "',,
. '.' ' ~'.
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`,:
':
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Yield
Hyaluronic Based on
Acid Dry Cell ~.
Culture Drv Cell WeightObtained Wei~ht
5 2~ Hour 2.9 grams/10 11.1 g/10 1 37.9%
64 Hour 3.79 ~rams/10 12.06 g/10 154.4Z
Thus, the longer term culture is substantiall~ more .
efficient in producing hvaluronic acid. However,
subsequent experiments indicated that culturin~ for
10 bevond 72 hours results in only ma-ginal increases in
~ield evidently because nutrients other than glucose
became depleted from the CDM.
EXAMPLE 3
.:
Hyaluronic acid was harvested and isolated from
15 2 one hundred liter culture prepai-ed in a manner similar
to .hat described in Example 2. However, it was
2nalyzed for protein content by W absorption at 280
na~ometers and nucleic acid content by UV absorption at .
260 nanometers immediately before and immediately after
20 passage t~rough a single thickness 1.2 micrometer pore ~-
293 mm diameter nitrocellulose filter. The 4.5 liters !
OI aqueous sodium hvaluronate solution being treated had
the following pre and post filtration contents:
Pre Fil- Post ~ t~
tration tration Percent
ContaminateContent Content Reduction
Protein0.28 mg/ml 0.06 mg/ml 78% ; ~:
Nucleic acids 0.09 mg/ml 0.023 mg/ml 74%
The protein load on the filter was about 1.88 ~
30 mg/cm2 and it remo~ed approximatelv 1.48 mg/cm2 of
protein to effect 2 protein reduction of 78%. After one .
further ethanol precipitation and resolution this lot
was found to contain 20.08 grams of sodium hyaluronate
for a yield of about two grams pel ten liters of :~.
3~ o~i~inal culture. - -
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The degree of contaminate removal appeared to be
related to the contaminate load on the nitrocellulose
filter. In a similar experiment 7.5 liters of an aqueous
sodium hyaluronate solution were passed through a similar
filter. The protein reduction appeared to be about 43%
and the nucleic acid reduction seemed to be about 29% but
the protein load was about 4.25 mg/cm2. The absolute
removal was about 1.85 mg/cm2 in reducing the
lo concentration from 0.38 to 0.215 mg/ml. The absolute
reduction in nucleic acid content was also similar
between the two experiments with 0.045 mg/cm2 removed
from the 4.5 liter batch and 0.052 mg/cm2 removed from
the 7.5 liter batch.
EXAMPLE 4
An aqueous approximately one weight percent
sodium hyaluronate solution displaying pyrogenicity in
the rabbit test was rendered non-pyrogenic by treatment -
with strong acid washed activated carbon. The solution
was contacted with 29 grams per liter Gelman 12011 a
strong acid washed activated carbon with a surface area
of about 700 m2/g (thus giving 20,000 m2/liter of surface ;:
area) for one and sixty minutes. A reference solution of
lipopolysaccaride (LPS), the commonly suspected pyrogenic
agent, was similarly treated. The LPS content of both
was evaluated by Limulus amoebacyte lysate (LAL) analysis
with the following results:
Post Post
Initial 1 minute 60 minute
ContentTreatmentTreatment
H~ solution 15.5 ng/ml12.08 ng/ml 8.12 ng/ml
LPS solution 30 ng/ml 13.35 ng/ml 12.75 ng/ml
The results with the hyaluronate solution was
35 confirmed by testing of similarly treated samples in the
rabbit test.
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1328~1
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The ability of ac~ivated carbon to extract LPS
from an aqueous sodium hyal~ronate sol~tion appeared to
be uniq~e. Although other materials such as ion
exchange resins and affinity binding colf~mns marketed
5 for LPS adsorption extracted LPS from a water solution
they were unable to extract from an aqueous hyaluronate
solution.
EXAMPLE 5
Hyal~ronic acid was prepared using the st-ain
10 of microorganism prepared in accordznce with Example 1 -~
by culturing a five hundred liter batch for forty-eight
hours in accordance with the procedures of Example 2
except that a second glucose addition of 0.5 weight
percent hased on the total culture weight was made
15 sixteen hours after each one percent addition by
isolating and purifying the product by the second
preferred procedure utilizing the "winder" which is
discussed hereinabo~e. The procedure included the
especially preferred features of lowering the ~iscosity
20 by heat treatment, and filter sterilizing bv passage
through a 0.2 micrometer pore filter as well as a
subsequent pyrogen~treatment by passage at between 37 ,:
and 56C through a Gelman 12011 strong acid washed
acti~ated carbon filter pro~iding 20,000 m s~rface area
25 per liter of solution being treated. The final aqueous
solution had a sodium hyaluronate cor.tent of 1.19 ~eight
percent. This hyaluronic acid had a FPLC (fast prot~in
liquid chromatography) dete-mined average molecular
weight of 1.88 x 106 daltons ~th a narrow essentially
30 symmetrical single distribution peak, a 37DC visc~sity
of 147 cSt, a nucleic acid content of less than 0.003
mg/ml by ethidium bromide fluorescence and a total afino
acid content of less than 0.005 mg/ml by
orthophthalaldehyde fluorescence. lt caused no readily
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apparent antigenic reaction when injected into horses
and ~ave a negative indication in the rabbit pyrogen
test.
Although the invention has been described in
5 detail in the foregoing .or the purpose of illustration,
it is to be ~nderstood that such de~ail is solely for
that purpose and that variations can be made therein by
those skilled in the art witho~t departing from the
spirit and scope of the invention except as it mav be
10 limited by the claims.
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