Procedure for the purification of hyaluronic acid and fraction of pure hyaluronic acid for ophthalmic use

• US 5,559,104 A
• Filed: 05/11/1995
• Issued: 09/24/1996
• Est. Priority Date: 04/19/1991
• Status: Expired due to Term

• Current Assignee: Fidia Farmaceutici s.p.a (P.&R. S.p.A)
• Original Assignee: Fidia S.P.A.
• Inventors: Romeo, Aurelio, Lorenzi, Silvana
• Primary Examiner(s): Kunz, Gary L.
• Assistant Examiner(s): Fonda, Kathleen Kahler
• Application Number:
  US08/439,437
• Time in Patent Office: 502 Days
• Field of Search: 536/53, 536/55, 536/55.1, 536/55.2, 536/55.3, 514/54, 514/62, 514/912
• US Class Current: 514/54
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates

Abstract

A highly pure fraction of hyaluronic acid is disclosed which is non-inflammatory and avoids post-operative complications in ocular surgery. Also disclosed is a process for the preparation of hyaluronic acid characterized by converting hyaluronic acid into a corresponding quaternary ammonium salt and, following purification procedures, reconverting the quaternary ammonium salt into a sodium salt of hyaluronic acid.

Claims

1. A hyaluronic acid fraction, or a salt thereof, having a mean molecular weight in the range of 750,000 Daltons to 1,230,000 Daltons and having the following characteristics:

a) limiting viscosity number ranging between 14.5 and 21 dl/g, when determined at 25°

C. in 0.15M NaCl at pH 7.0 using an Ubbelhode suspended level viscosimeter;

b) protein content not exceeding 0.2% expressed as albumin;

c) a U.V. absorbance at 257 nm and at 280 nm not exceeding 1.0 A.U., when measured on a 1% weight per volume aqueous solution;

d) a dynamic viscosity of a 1% weight per volume solution in 0.15 NaCl at pH=7.0, not exceeding the following limits at the defined shear rates, using a rotational viscosimeter at a temperature of 20°

C.;

space="preserve" listing-type="tabular">______________________________________ Shear rate Dynamic viscosity (mPa ·

s at 20°

C.) ______________________________________ 1 s.sup.-1 not more than 20000 mPa ·

s 10 s.sup.-1 not more than 2000 mPa ·

s 100 s.sup.-1 not more than 1000 mPa ·

s 350 s.sup.-1 not more than 500 mPa ·

s ______________________________________

e) a sulfated mucopolysaccharide content not exceeding 0.07% as sulfur;

f) an iron content not exceeding 10 p.p.m.; and

g) stability of isotonic buffered solutions with a physiological pH of the fraction, naturally aged and heat-sterilized, determined by assessment of the limiting viscosity number and expressed with the corresponding decrease in mean molecular weight, not exceeding the following limits;

the actual value after storage at 25°

C. for 6 months must not be lower than 97% of the initial value

the actual value after sterilization at 118°

C. for 32 minutes must not be lower than 75% of the initial value

the actual value after sterilization at 121°

C. for 16 minutes must not be lower than 80% of the initial value

the actual value after sterilization at 124°

C. for 8 minutes must not be lower than 90% of the initial value.

2. A hyaluronic acid fraction according to claim 1, wherein said mean molecular weight is between 925,000 and 1,230,000 Daltons.

22. A pharmaceutical preparation containing as the active ingredient the hyaluronic acid fraction according to claim 1 together with pharmaceutical excipients.

25. In a method of performing cataract operations or ocular lens implants, the improvement which comprises the use of the hyaluronic acid fraction of claim 1.

3. A process for the purification of hyaluronic acid or a salt thereof, which comprises:

a) converting hyaluronic acid or a salt thereof into a corresponding quaternary ammonium salt;

b) dissolving said corresponding quaternary ammonium salt in an organic solvent capable of dissolving such ammonium salts; and

c) filtering the solution and treating with a sodium halogenide to recover the hyaluronic acid in the form of a sodium salt.

4. The process according to claim 3, wherein said quaternary ammonium salt is formed by absorption of an aqueous solution of said salt of hyaluronic acid onto an acid ion exchanger salified with a quaternary ammonium base.

5. The process according to claim 4, wherein said ion exchanger is a strong cationic exchanger with sulfonic groups in a quaternary ammonium form.

6. The process according to claims 3, 4 or 5, wherein said organic solvent used to dissolve the quaternary ammonium salt of hyaluronic acid is an aprotic solvent.

7. The process according to claim 6, wherein the aprotic solvent is a lower dialkylsulfoxide or dialkylcarboxylamide.

8. The process according to claim 7, wherein said aprotic solvent is dimethylsulfoxide or N-methyl-pyrrolidone.

9. The process according to claim 3, wherein said salt of hyaluronic acid to be converted is a sodium salt and wherein said organic solvent is an aprotic solvent.

10. The process according to claim 9, wherein conversion of the ammonium salt of hyaluronic acid into the corresponding sodium salt of hyaluronic acid is conducted by the addition of sodium bromide to the solution of the ammonium salt of hyaluronic acid in said aprotic solvent.

12. The process according to claim 10, wherein an aqueous solution of said sodium salt of hyaluronic acid is subjected to molecular filtration to obtain a desired molecular weight fraction of said sodium salt of hyaluronic acid.

13. The process according to claim 12, wherein said acid ion exchanger is a sulfonic resin in the form of a quaternary ammonium salt.

14. The process according to claim 13, wherein said quaternary ammonium salt is a tetraalkylammonium salt having an alkyl group with a maximum of 6 carbon atoms.

15. The process according to claim 14, wherein said tetraalkylammonium salt is a tetrabutylammonium salt.

19. The process according to claim 12, wherein said sodium salt of hyaluronic acid is subjected to molecular filtration with a membrane having a molecular exclusion limit of 30,000 Daltons.

11. A process for the preparation of a fraction of hyaluronic acid which comprises

a) extracting hyaluronic acid from an animal organ with a first organic solvent in the presence of an iron-chelating agent to obtain a hyaluronic acid-containing extract which is substantially free of iron ions;

b) treating said hyaluronic acid-containing extract with a proteolytic agent;

c) contacting the thus treated hyaluronic acid-containing extract with an acid ion exchanger in the form of a quaternary ammonium salt;

d) dissolving the extract from the ion exchanger in a second organic solvent which is capable of dissolving a quaternary ammonium salt of hyaluronic acid;

e) treating the mixture of step (d) with a sodium halogenide to convert the quaternary ammonium salt of hyaluronic acid to the corresponding sodium salt; and

f) isolating the desired sodium salt of hyaluronic acid.

16. The process according to claim 11, wherein said first organic solvent is ethanol or acetone;

and said iron chelating agent is 1,10-phenanthroline.

17. The process according to claim 11, wherein said sodium salt is treated with a sterilization agent.

18. The process according to claim 17, wherein said sterilization agent is cetylpyridinium chloride.

23. In a method of performing ocular surgery, the improvement which comprises the use of the hyaluronic acid fraction of claim 11.

24. In a method of performing procedures in ophthalmology, the improvement which comprises the use of the hyaluronic acid fraction of claim 11.

20. A process for the preparation of a fraction of sodium hyaluronate which comprises:

a) extracting organic material from hen crests with an extraction solvent comprised of acetone or ethanol and phenanthroline or phenanthroline to produce an extract which is substantially free of iron ions;

b) treating said extract with a proteolytic agent selected from the group consisting of papain, pepsin, trypsin, pronase and cysteine hydrochloride;

c) contacting the thus treated extract with a column filled with a sulfonic acid resin in the form of tetrabutylammonium salt;

d) eluting the column to obtain a solution containing the tetrabutylammonium salt of hyaluronic acid;

e) drying the solution to obtain a residue comprised of the tetrabutylammonium salt of hyaluronic acid;

f) dissolving said residue in an aprotic solvent;

g) filtering the solution of step (f);

h) washing the solution of step (g) with methylene chloride;

i) treating the solution of step (h) with a sodium halogenide to produce sodium hyaluronate;

j) precipitating said sodium hyaluronate;

k) dissolving the precipitate in water and subjecting the solution to dialysis;

l) precipitating sodium hyaluronate from the dialyzed solution; and

m) subjecting said precipitate to molecular filtration with a membrane having a molecular exclusion limit of 30,000 Daltons, to produce a solution containing the sodium salt of hyaluronic acid having an average molecular weight of between 750,000 and 1,230,000 Daltons.

21. The sodium hyaluronate fraction prepared by the process of claim 20.