METHODS OF SYNTHESIS AND TECHNOLOGY OF DRUG PRODUCTION

PRINCIPLES OF EXTRACTION AND PURIFICATION OF HYALURONIC ACID (REVIEW)

E. Yu. Ignatova and A. N. Gurov UDC 615.31:547.955.15].012

Hyaluronic acid (HA) was first isolated by Meyer and Palmer in 1934 [52], and identi-
fied chemically by Meyer and coauthors in 1936 [53, 54]. Since then, comprehensive informa-
tion on the extraction, purification, and fractionation of HA, and the investigation of
its structure, properties, and biological role has accumulated in the scientific and patent
literature [26, 47, 48]. The HA is found in many tissues and fluids of vertebrates, as
well as in the extracellular matrix formed by some bacterial species (e.g., hemolytic
streptococci groups A and B) [33]. It is present at the highest concentrations in syno-
vial fluid, brain, umbilical cords, cocks' combs, integument, and the vitreous body of the
eye [9, 15, 25].
Since then, interest in this polysaccharide has increased significantly as the problem
of the search for substitutes of the vitreous body was formulated at the end of the 1950's,
and the expediency of the utilization of HA solutions for this purpose was substantiated
[2, 26]. However, in spite of the numerous methods for the extraction and purification
of HA described up to that time, it was not possible to develop the technology for its
isolation in a form which could be utilized in medicine, and particularly in ophthamology,
for a long time. Moreover, the conception according to which "a component of the extra-
cellular matrix may be utilized as a factor controlling its own regenerative process in
the tissue environment in which it is present naturally" was not immediately perceived for
a long time in medicine [26]. In the process of the investigations which developed from
this, there arose a completely unexpected problem: the sterile apyrogenic solutions of
human and animal tissues induced an unusual inflammatory reaction of a non-immune charac-
ter after being introduced into the eye or a joint [26]. A whole decade was required for
the development of a method for the isolation of the "HA fraction not causing an inflamma-
tory reaction" [ii].
The patent [Ii] served as the basis for the development of the first pharmaceutical
preparation of HA which has found wide application in medical practice, and especially in
ophthalmic surgery, under the commercial name "Hea Lon" ("Pharmacia," Sweden). Its utiliza-
tion as an operational medium protecting the internal tissues of the eye from mechanical
damage (as a "viscoprotector") sharply increased the effectiveness of eye operations. The
preparation "Hea Lon" remains one of the best viscoprotectors. It is also most expensive:
the package prepared for use in one operation (0.4 ml) and containing the 1% solution of
sodium hyaluronate cost 66 dollars in 1985 (this corresponds to about 17 million dollars
on the basis of 1 kg of HA) [70].
With the appearance of the preparation "Hea Lon" in the world market (from 1980), the
terms "viscosurgery" and "viscomicrosurgery" also appeared. Intensive work for the produc-
tion of other viscoelastic materials based both on HA as well as other polysaccharides (in
particular, cellulose derivatives) commenced from this time [70]. Apart from ophthalmology,
HA is utilized in rheumatology (for the substitution of synovial fluid in the treatment
of arthroses, in arthroplasty and osteotomy for the protection of cartilaginous surfaces
and the peripheral nerve [I0]), in dermatology (for the protection of skin wounds, the
treatment of eczema and trophic disorders of the skin), and in the production of cosmetics
[72].
In the development of methods for the isolation of new pharmaceutical preparations
based on HA, it is unconditionally important to make use of the experience on the extrac-
tion of this polysaccharide which has accumulated in the scientific and patent literature.
An attempt to systematize the corresponding literature data has been made in the present

All-Union Scientific Research Institute of Eye Diseases, Ministry of Public Health of

the USSR, Moscow. Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 24, No. 3, pp.
42-46, March, 1990. Original article submitted April 21, 1989.

0091-150X/90/2403-0211512.50 9 1990 Plenum Publishing Corporation 211

review. It is addressed to specialists in the area of biochemistry, biotechnology, and
pharmaceutics concerned with the production and investigation of preparations based on HA
and HA-specific enzymes.

Levels of Localization of HA
Several levels of the localization of HA can be isolated as follows: i) tissue: integu-
ments, cocks' combs, connective tissue (synovial medium, vitreous body, umbilical cord); 2)
cellular: intercellular space (matrix), integumentary cells of the synovial membrane (syno-
vicytes), vacuoles, Golgi complex, hyalocytes (fibroblasts), streptococci; 3) supermolecular
and molecular: proteoglycans and free HA.
The HA in living organisms is found at the tissue level in the eyes, the combs, and
the brood spot of the birds, the dorsal fin of amphibia, reproductive skin of monkeys,
and the sex organs o~ mammals, birds, and fish [59, 68]. In the tissues of the cock's comb,
the HA is distributed in the mucoid fibers of the subcutaneous layer, which is most wide
at the base of the comb. The cocks' combs contain up to 7500 ~g/mg of HA [47]. The vitre-
ous body of man contains up to 200 ~g/ml of HA [47]. The highest concentration of the HA
in the vitreous body is at the base adjoining the flat part of the ciliary body and the
retina [18]. For example, the concentration of HA in the vitreous body of the eye of the
ox comprises 0.034 g/liter in the cortical layer, and decreases fivefold to tenfold on ap-
proaching the center [22]. The content of HA in the vitreous body of mammals and birds
increases with age, whereby it is far lower in rabbits, pigs, and birds than in other
mammals.
The eyes of fish contain a polysaccharide - ichthyosan A - which is similar to sodium
hyaluronate [26]. This is also a non-sulfated polysaccharide containing the equimolar amount
of hexosamine and hexuronic acid; the ratio of glucosamine to galactosamine in it is 2:1.
The concentration of ichthyosan A in different fish varies from 80 to i000 ~g/ml [24].
We will consider the localization of the HA at the cellular level taking the example
of fibroblasts and hyalocytes in the vitreous body, as well as cells of the synovial fluid
and streptococci. The vitreous body contains the fibrillar framework of interweaving col-
lagen fibers and the liquid mucopolysaccharide complex, the main part of which is the HA.
The cells of the vitreous body - the fibroblasts and hyalocytes, which probably produce
the HA, are found in the fibrillar framework [17]. There is coincidence in the concentration
distribution of the hyalocytes and HA. Individual authors regard the hyalocytes and fibro-
blasts as different cells; others assume that they are the same cells at different stages
of development. It is possible that the hyalocytes contain enzyme systems converting glucose
to glucosamine, and then the latter into the HA or higher molecular mass. The proportion
of cells in different sections of the vitreous body is not the same for different mammals.
The concentration of the hyalocytes is especially high in the cortical layers [6] (where
the highest concentration of HA occurs). The region adjoining the hyaloid tract is generally
free of hyalocytes [8]. At the serrated line of the eye of the pig, the mean number of
cells reaches 200 per 1 mm= of the surface, and decreases to 80 per 1 mm= in the direction
of the posterior pole; it is 60-70 cells per 1 mm2 in man. The proportion of cells is higher
in calves than in cows. The enlargement of the vitreous body during the growth period of
the ox is accompanied by the proportional decrease in the number of hyalocytes and fibro-
blasts producing HA [17].
In the synovial medium, the HA is localized in the synovicytes - cells of the integumen-
tary layer of the synovial membrane situated at varying depth in relation to the surface
of the layer. In the normal uninflamed joint, the hyaluronate appears in the cells of the
integumentary layer of the lateral walls. The synovicytes are filled with hyaluronate in
all parts of the joint cavity during inflammatory processes [I0]. According to the features
of the ultrastructure, the synovicytes are divided into the A- and B-cells [49]. In the
opinion of individual authors, a leading role in the synthesis and secretion of HA pertains
to the A-cells. Hyaluronate is found in the Golgi apparatus and the vacuoles of the A-cells
[28]. On the other hand, the B-cells are designated as "synovial fibroblasts" [43], and
a series of authors confirm that HA is produced by the B-cells [27]. The content of HA
in the basic substance - the matrix of the synovial membrane - comprises 40-120 ~g/ml.
The HA synthesized by hemolytic streptococci is found in the free state, forming the
protective capsule of the cellular bacteria [5]. Yet it is ~ynthesized in the form of the
peptidoglycan, from which it is released in the form of the pure polysaccharide.

212
 At the molecular level, the HA is localized in different complexes or in the free
state. For example, the proteoglycans of the extracellular matrix are complexes containing
HA in the connective tissue. The structure of the proteoglycans was studied and described
in detail in many reviews and monographs (e.g., in [71]).

Chemical and Physicochemical Properties of HA

i. Structure. Hyaluronic acid in an anionic linear polysaccharide with the molecular
mass 5-104-8"106 (the molecular mass 13"106 was communicated) [19]. Its chain consists
of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine connected in se-
quence by ~-(l~4)-glucosaminide and B-(l~3)-glucuronide bonds. In other words, HA is a
homopolymer of the disaccharide hyalobiuronic acid [71]. The HA macromolecule has the con-
formation of the randomly convoluted knot in solution [21]. It does not contain residues
of other carbohydrates or amino acids, and does not have covalent intramolecular bonds [19].
2. Enzymatic Hydrolysis. The breakdown of the bonds in HA occurs by the action of
hyaluronidases which are present in the membranes of pathogenic bacteria, sperm, snake
toxin, spider toxin, toxin of bees, salivary secretions of leeches, and rapidly growing
tumors [37]. The hyaluronidase of microbes and toxins, which breaks down the HA of the
intercellular substance, favors the distribution of infection in deep-seated tissue. The
hyaluronidase of sperm, which dissolves the follicular layer of the egg cell, creates the
conditions for its fertilization.
Depending on the mechanism of action, three types of hyaluronidase are discerned. The
type I comprises the testicular hyaluronidases (from sperm, and the toxin of scorpions and
snakes); they break down HA, hydrolyzing the B-(l~4)-bond, and giving a series of oligo-
saccharides (from four disaccharide units and higher). The rate of reaction decreases
rapidly with the decrease in the length of the chain of the HA. The type II comprises the
bacterial hyaluronidases. They also break down the ~-(l+4)-bonds of the HA. intramolecular
hydrolysis thereby occurs with the separation of water; this results in the formation of
unsaturated disaccharides. The chain length of the substrate does not influence the rate
of the reaction. An example of the hyaluronidases of the type III is the hyaluronidase
of leeches. It cleaves the B-(l+3)-bonds of HA; this results in the formation of tetra-
saccharides with glucuronic acid at the end of the chain. The hyaluronidase of leeches
is an endopolyglucuronidase [37].
In order to prevent the uncontrolled enzymatic hydrolysis of HA, it is recommended
that it should be isolated in the presence of chloroform at a low temperature [ii, 20].
3. Oxidoreductive Destruction. The HA is degraded under the influence of oxidation-
reduction systems. In the vitreous body, it decomposes by the action of ascorbic acid;
oligosaccharide is not thereby recovered as in the case of the action of hyaluronidases
[62]. The reaction is speeded up in the presence of hydrogen peroxide or traces of copper,
and only proceeds in the presence of oxygen. Moreover, the hydrogen peroxide itself
causes a decrease in the molecular mass of HA; this appears as the decrease in the vis-
cosity of its solutions. The reaction of ascorbic acid with HA proceeds virtually to com-
pletion in the presence of diethyidithiocarbamate [64].
The cleavage of HA by the action of oxidation-reduction systems proceeds by a free-
radical mechanism. Thus, the free radicals are formed from ascorbic acid in the reaction
with HA and hydrogen peroxide in the presence of peroxidase, as well as in the reaction
with oxygen in the presence of ascorbic acid oxidase [64]. It was also established that
HA is decomposed by the action of Fe 2+ and Fe 3+ ions in the presence of reducing agents
[29].
The oxidation-reduction destruction of HA may occur in the isolation of the poly-
saccharide. This is specified by the fact that the HA prepared in an atmosphere of nitro-
gen has a higher viscosity than that prepared in air. It is also proposed that the oxida-
tion-reduction depolymerization catalyzed by heavy metals may also occur in the process
of the isolation of the polysaccharide. For example, when HA was purified from proteins
by using trypsin containing Fe 3+ ions, the application of 8-hydroxyquinoline, inhibiting
the spontaneous decrease in the viscosity of the solution, was shown to be expedient [29].
Degradation in the presence of phosphate ions often occurs when solutions of HA are lyo-
phiiically dried [64]. The utilization of papain in the purification of HA by the breaking

213
 TABLE i. Different Methods for the Separation of Proteins and the Breakdown
of the Mucopolysaccharide Complexes

Denaturation and Literature Breakdown of complexes Literature

separation of source with mucopolysacchar- source
protein ides and removal of
the latter
Extraction by chloro-
form with amyl alcohol [3] Hydrolysis with 18%
Extraction with 90%
hydrochloric acid [53]
phenol [36, 42, 62] Treatment with cetyl-
pyridinium chloride [35, 61, 63]
Enzymatic hydrolysis
with papain [55, 57] Addition of heavy
metal salts [3, 49]
Ultrafiltration [56, 57]
Enzymatic hydrolysis:
Extraction with
sodium acetate [32] with pepsin [3, 13]
Precipitation with
with pronase [65]
cetylpyridinium [46, 53]
with trypsin [58]
lon-exchange chroma-
Adsorption on
tography [4, 38, 45]
activated carbon [34]
Electrophoresis [30, 66,.67]
Electrodialysis [58]

down of its complexes with protein also lowers the viscosity of the product, since the SH-
enzyme papain is a reducing agent and induces the penetrating cleavage of HA [23].
4. Acid-Alkaline Hydrolysis of HA. The HA macromolecule is very sensitive to the
pH of the medium. Even the weak acidification of the extract with acetic acid causes the
irreversible lowering of the viscosity by a factor of 2.5 [20]. The HA is completely
hydrolyzed by mineral acids with the formation of glucosamine, acetic acid, carbon dioxide,
and glucuronic acid. The action of dilute sulfuric acid for a short time leads to the
formation of disaccharide crystals [31].

Principles of the Extraction and Purification of HA

The HA was first isolated in 1934 by Meyer by aqueous-acetone extraction from the
vitreous body of cattle [52]. In the same year, Sevag isolated the acid from human umbili-
cal cords by applying an aqueous-chloroform extraction~ The HA was isolated from the vitre-
ous body of the eyes of pigs and from the solutions of bovine blood in 1936 [53]. The HA
was first isolated from the groups A and B of the hemolytic streptococci in 1937 by Kendall
[29]. The acid was isolated from cocks' combs by Boas in 1949 [32]. Up to the present
time, different methods, in which various forms of extraction, enzymatic hydrolysis, frac-
tionation, etc. are applied, have been described.
Any procedure for the isolation of HA includes the sequential breaking down of the
structures at each level of localization (see above) with the directed utilization of deter-
mined factors for this.
Thus, the breaking down of the structures of the tissue level is achieved by the crush-
ing, homogenization, and other procedures to guarantee, first of all, the maximal contact
with the extractants. The breaking down of the cellular structures is usually achieved
in disintegrators, and also by the action of enzymes and organic solvents. The release
of HA from the complexes with other polysaccharides and proteins is achieved using enzymes,
organic solvents, detergents, and the directed effect on one or the other of the bonds
retaining it in the complexes.
For laboratory investigations, sources which are utilized for the isolation of HA are
the vitreous body [40], pig skin [13, 14], the pericardial fluid of the rabbit [39], carti-
lage of sharks [12]. The most widespread and accessible sources for the isolation ef HA
are cocks' combs, as well as human umbilical cords, the vitreous body of cattle, an4 syno-
vial fluid.

214
 Freshly frozen tissues for the removal of traces of blood are extracted with acetone
5-10 times until a clear solution is obtained [67], with 95% ethanol with added chloroform
(i kg of tissue to 4 liters of ethanol) [ii], with 90% acetic acid, and with sodium acetate.
The tissues retained after washing off the blood to avoid oxidative destruction may be "con-
served" for an extended time (up to 24 months) at the temperature of 4-22~ in 95% ethanol
[67], or dried in a stream of acetone [49]. For further treatment, the tissues are crushed
in disintegrators [7, 41], and in ball mills [3]. The dried tissues are swollen in 95%
ethanol or water. The synovial fluid and the vitreous body, purified from connective tissue,
are first filtered [48, 51, 60]. For the purpose of the separation of the protein and the
release of the HA from its complexes with proteins and other mucopolysaccharides, the pre-
pared tissue is treated by different methods (Table i). The HA is reprecipitated several
times from the filtered centrifuged final solutions with ethanol [3, 40] and cetylpyridinium
chloride [16], and washed with sterile acetone [ii, 50]. The residue is concentrated in
a lyophilic dryer [44] or over phosphorus pentoxide [68] in a vacuum [i, ii]. Further,
depending on the designation, the HA is stored in the dry form at a temperature not exceed-
ing -18~ [69], or it is dissolved in a physiological buffer solution and packed in a con-
venient packing, for example, in syringes [ii].
Independent of the way in which transactions proceed in the market of pharmaceutical
preparations, the majority of western companies agree on the fact that the area of applica-
tion of HA will broaden continuously, and that its production will remain profitable~ It
is confirmed in the prediction of the market for preparations based on HA published in 1984
[70] that the volume of selling in 1990 comprises about 200 million dollars; this includes
150 million dollars for eye surgery, i0 million dollars for the cosmetic industry, and 40
million dollars for other purposes (including materials for wounds, preparations for rheuma-
tology, etc.).
There are no doubts that HA is becoming one of the main components for the pharmaceuti-
cal and cosmetic industry, and the mastering of its production is an important scientific
and technical task.

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