Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-06-19
2002-12-03
Barts, Samuel (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S053000, C536S055100, C536S055200, C514S054000
Reexamination Certificate
active
06489467
ABSTRACT:
The present invention relates to a process for purifying high molecular weight high molecular weight hyaluronic acid or a salt thereof.
Hyaluronic acid is a mucoid polysaccharide of biological origin, which is widely distributed in nature. For example, it is known that hyaluronic acid is present in various animal tissues such as umbilical cord, synovial fluid, vitreous humor, rooster comb and various connective tissues such as skin and cartilage.
Chemically, hyaluronic acid is a member of glycosaminoglycans and it is constituted by alternating and repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, to form a linear chain having a molecular weight up to 13×10
6
Daltons.
In the meaning of the present invention, high molecular weight hyaluronic acid is hyaluronic acid having a molecular weight of not less than about 0.5×10
6
Daltons.
It is to be noted that the term “hyaluronic acid” in the present description and claims may mean indifferently hyaluronic acid in its acidic form or in its salt form such as for example sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or others.
Hyaluronic acid, at high molecular weight, is viscous and able to maintain a jelly state, acting as a lubricant, preventing the invasion of bacteria and retaining water.
Due to these properties, hyaluronic acid is able to retain the tonicity and elasticity of the skin.
Pharmaceutical use of hyaluronic acid or of a salt thereof is widely described in the literature.
Since hyaluronic acid is a non-immunogenic substance and has viscoelastic and hydrophilic properties, it is used, since several years, as an eye vitreous or joint fluid replacement or as a supportive medium in ophthalmic surgery, as disclosed for example in U.S. Pat. No. 4,141,973 of Balazs.
In joint fluids, the viscous hyaluronic acid solution serves as a lubricant to provide a protective environment to the cells, and for this reason, it is used in the treatment of inflamed knee joints.
EP-A-0 781 547 of Chemedica S.A. discloses a sodium hyaluronate based ophthalmic formulation for use in eye surgery.
EP-A-0 719 559 of Chemedica S.A. discloses sodium hyaluronate viscous solutions for use as masking fluid in therapeutic photokeratectomy by means of excimer laser.
EP-A-0 875 248 of Chemedica S.A. discloses the use of hyaluronic acid or of one of its pharmaceutically acceptable salts for the preparation of an aqueous solution useful as intra-articular lavage liquid.
EP-A-0 698 388 of Chemedica S.A. discloses an ophthalmic preparation for use as artificial tears containing hyaluronate as a viscosity thickener.
Thanks to its highly hydrophilic nature, hyaluronic acid may also be used in cosmetic products such as lotions and creams.
The pharmaceutical use of hyaluronic acid or of a salt thereof requires a highly pure product.
Hyaluronic acid can be extracted and purified for example from umbilical cords, from rooster combs or from group A and C Streptococci as disclosed for example in U.S. Pat. No. 4,141,973 of Balazs and U.S. Pat. No. 5,559,104 of Romeo et al.
Production of hyaluronic acid by Streptococci was first disclosed by Forrest et al. in 1937, (J. Biol. Chem. 118, 61 (1937)) and later it was demonstrated that hyaluronic acid from animal source is identical to hyaluronic acid from microbial source.
Processes for producing hyaluronic acid from microbial source are based on the property of Streptococci to have hyaluronic acid as the main component of their capsules.
Such microorganisms are able to transform the glucose present in their environment into D-glucuronic acid and N-acetyl-D-glucosamine and to produce hyaluronic acid as a secondary metabolite, to build their protective capsules.
The biosynthesis of hyaluronic acid by Streptococci is disclosed for example in U.S. Pat. No. 4,897,349 of Swann et al.
A number of processes are known for obtaining pharmaceutical grade hyaluronic acid or a sodium salt thereof from microbial sources.
For example, U.S. Pat. No. 4,780,414 of Nimrod et al., U.S. Pat. No. 4,517,295 of Bracke et al., and U.S. Pat. No. 5,563,051 of Ellwood et al. disclose processes for obtaining hyaluronic acid by continuous fermentation of Streptococcus bacteria and then purifying hyaluronic acid thus obtained up to pharmaceutical grade.
However, in all these processes, the purification of hyaluronic acid involves the precipitation of hyaluronic acid from microbial source by using large amounts of organic solvents such as ethanol, acetone, isopropanol, etc.
Some purifying processes are known wherein precipitation of hyaluronic acid from microbial source occurs by means of quatemary ammonium salts (U.S. Pat. No. 4,517,295 of Bracke et al.) or by means of anionic and cationic surfactants (U.S. Pat. No. 5,316,926 of Brown et al.).
However, the described procedures are quite complex and result in high production costs.
Some purifying processes are known wherein hyaluronic acid solution is diafiltered by using a filter having a nominal molecular weight cut-off of 10.000, 20,000 or 30,000 Daltons as disclosed for example in U.S. Pat. No. 5,563,051 of Ellwood et al. and U.S. Pat. No. 4,517,295 of Bracke et al.
However, due to the small nominal molecular weight cut-off used, these diafiltering procedures can be used only to discard small soluble molecules, and the diafiltered hyaluronic acid solution has to be treated by further processing in which various precipitation systems are involved, which render the whole purifying process complex.
An object of the present invention is to obtain a pharmaceutical grade high molecular weight hyaluronic acid or a salt thereof from any biological source, in particular from microbial sources, in high yield, at relatively low cost and preferably without the use of organic solvents or other added substances.
According to the present invention, this object has been achieved by a process for purifying high molecular weight hyaluronic acid from a biological source, characterized by:
a) a step of adjusting the pH of an aqueous solution containing high molecular weight hyaluronic acid from a biological source to a pH in the range from 1.7 to 3.3 and then diafiltering said aqueous solution at the same pH using a filter having a pore size in the range from 100,000 Daltons nominal molecular weight cut-off to 0.45 &mgr;m;
b) a step of removing cells from the aqueous solution containing high molecular weight hyaluronic acid from biological source, said step being carried out either before step a) or after step a);
c) a step of sterilization.
Advantageously, by means of a process including a step of diafiltration at a pH in a range of 1.7 to 3.3 and a step of removing cells it is possible to purify high molecular hyaluronic acid so as to obtain in high yield a pharmaceutical grade high molecular weight hyaluronic acid or a salt thereof from any biological source and in particular from a microbial source.
According to step a) of the process of the present invention, hyaluronic acid is diafiltered at a nominal molecular weight cut-off or pore size which allows the returning of the hyaluronic acid and the passing through the filter of all substances contained in the solution or broth.
The process of the present invention may be carried out by using any biological source of hyaluronic acid.
Advantageously, pharmaceutical grade hyaluronic acid may be obtained by the process of the present invention without traces of organic solvents or other added substances, since in the purifying process of the present invention, hyaluronic acid is not precipitated with organic solvents or other substances, but is maintained in aqueous solution during the whole process.
Other objects, characteristics and advantages of the present invention become apparent from the following detailed description.
Hyaluronic acid from any biological source may be purified up to a pharmaceutical grade by the process of the present invention.
For example, the aqueous hyaluronic acid solution containing high molecular weight hyaluronic acid which is firstly treated, either according to step a)
Carlino Stefano
Magnette François
Barts Samuel
Browning Clifford W.
Chemedica S.A.
Henry Michael C.
Woodard Emhardt Naughton Moriarty & McNett
LandOfFree
Process for purifying high molecular weight hyaluronic acid does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for purifying high molecular weight hyaluronic acid, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for purifying high molecular weight hyaluronic acid will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2917724