Hyaluronan Purification

After extraction, hyaluronan must undergo purification as the next production operation. The extract from the animal tissue usually contains the following impurities proteins, peptides, lipids, nucleic acids, mucopolysacchrides and low molecular weight precursors. The first purification stage involves the precipitation of HA from the primary extract using ethanol or acetone or acetic acid, or a double volume of ethanol with sodium acetate at 2 °C [4]. Sometimes the dissolution-precipitation cycles are repeated several times in order to help remove low-molecular weight compounds and lipids that are soluble in acetone and ethanol. The proteins (which are free and connected with the polysaccharides) are removed 

Papain ultrafiltration in 40% water-ethanol mixture. Lyophilized powder of sodium hyaluronan [5] 

Extract heating at 90-100°C lipid removal filtration treatment with activated carbon. Lyophilized powder of hyaluronan [6] 

Treatment with activated carbon then cellulose filtration. Lyophilized powder of hyaluronan, protein content below 0,05% 17] 

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N. Forsberg and S. Gustafson, Characterization and purification of the hyaluronan-receptor on liver endothelial cells, Biochim. Biophys. Acta 7078 12-18 (1991). 

Heparin/heparan, hyaluronan, and chondroitin are three prevalent glycosaminoglycans. Vertebrates use glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Chemical synthesis of naturally occurring polysaccharides is considered to be impractical. Most polysaccharides, especially those from bacteria origins, are obtained by purification from natural sources or from cell culture, enzymatic approaches have been increasingly applied to obtain some structures. 

In the discovery and development of hylan A and hylan B, of course, the most important criteria were their purity and biocompatibility. We could never have used these two molecules as therapeutics without the availability of the test that I developed in the late 1960s to monitor the purification process for the native hyaluronan, and producing NIF-NaHA. This test method was accepted by many regulatory agencies as a standard way of proving the biological compatibility of elastoviscous fluids used in ophthalmic... 

Bacterial polysaccharides represent a large variety of polymers biosynthesized by bacteria. Their chemical structures and also their physical properties in solution or in the solid state may vary widely. They often contain uronic acid and then become polyelectrolytes (190). Many new polysaccharides have been developed from bacteria for industrial purposes. Exocellular polysaccharides are produced on a large scale by the usual techniques of microbiology and fermentation. This procedure allows good control of the characteristics of the polymers and allows purification of the polysaccharides more easily than from other natural sources (191-194). Extension of such production also allows reducing the price and extends the range of applications. A good example remains the hyalmonan previously produced by extraction from animal somces but in which some fraction of proteins remained. Bacterial hyaluronan can be prepared in a very pme form (195). 

Until recently, the most economically viable way of obtaining HA was by its extraction from chicken combs [1,2]. A full procedure includes subsequent stages of tissue homogenization, extraction, purification and preparation of the final commercial HA product, which can be in the form of dry powder, solution, granules or in medicinal substances. The novelty of the various patent-protected methods is that they each involve different extraction technologies and/or purification procedures. Next is the typical procedure for the the extraction and purification of hyaluronan from chicken combs. 

Then a solution of papain (200ml of 0.1% solution of papain per 1kg of material) is added. Enzymatic hydrolysis of proteins is carried out for 20-40h. Every 6-8 h the pH must be adjusted to 6-7 (to optimize enzyme activity). The HA mixture is purified by ultrafiltration and precipitation with 96% ethanol. The final product, sodium hyaluronate, is dried by lyophilization and the precipitate is then dissolved in a water-alcohol mixture containing 40% ethanol. The final purification of hyaluronan is performed by membrane filtration of a solution containing 30-50% ethanol. 

The patent [24] described the method of production of hyaluronan fractions with the average molecular mass from 250000 to 350000Da. The proteins, which were still found in the extracts, were hydrolysed with papain, and then the resulting solution underwent ultrafiltration through a membrane. In addition to purification, ultrafiltration afforded separation from the fraction of 30 000Da and lower, since such a low HA molecular mass could activate inflammation processes for when apphed parenterally. The membrane can hold the HA fractions above 30000. 

During lyophilization of hyaluronan solutions, polysaccharide degradation is initiated with phosphate ions [50]. For purification from proteins, proteolitic enzymes could also be used to reduce the viscosity of the biopolymer, particularly papain SH-groups that are reducing agents and accelerate the decomposition of hyaluronan [34], Trypsin, which contains Fe " ions, could also be used for the purification of hyaluronan and to initiate the depolymerization process. The use of 8-hydroxyquinoline prevents HA viscosity reduction [25]. In order to maintain the polymerization level of hyaluronan, the initial tissue must be thoroughly washed from blood, which contains ions of iron, copper and phosphate. 

At the present time, the market share of commercial hyaluronan of bacterial origin is significantly higher than the products extracted from animal sources, the reason being that microbiological synthesis affords a significantly higher yield the fermentation of the strain Streptococcus sp. resulted in a polysaccharide concentration of 5-6 g per 11 of culture medium. Comparatively, the yield of HA in the extraction from the chicken combs is from 0.5 g to 6 g per 1 kg of raw material. Because the purification of bacterial HA from proteins and peptides is easier, so it is therefore easier to obtain the HA product with a molecular mass of 1 million Da and protein content of less than 0.05%. The HA products from... 

The natural biopolymers, to which HA belongs, are not polydispersed polymers due to the matrix nature of their synthesis. The nature of the biochemical synthesis is determined by the matrix the enzyme upon which the triopolymer is synthesized. Nevertheless, during biopolymer extraction and purification processes they degrade in one way or another. For example, polygalactomannan, different types of cellulose (wood or cotton), chitosan and hyaluronan are isolated as a wide range of the relatively narrow dispersed macromolecule fractions. 

The problems of production and purification of hyaluronic acid are discussed in detail in Chapter 3. The comparable data of the commercial products are presented in Chapter 3 as well. The structure and rheological properties are discussed in Chapter 4. The discussion is united by a general idea about cooperative conformational transformations as the main phenomena that manifest bio-specificity of hyaluronic acid as well its unique effect of the relationships of biological properties from molecular mass. The physical and chemical methods of hyaluronan structural modification are presented in Chapter 5. It presents reactions of the macro-molecule cross-linking with bi-functional reagents as well as highly... 

OTH, ovine testicular HAase (560 units/mg) BTH,bovine testicular HAase (500 units/mg) Determined by SEC calibrated with hyaluronan standards. Isolated yields after purification... 

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