Processes for the production of chemically-modified heparins

Abstract

The present invention relates to a process for the production of heparin derivatives having an average molecular weight of from about 4.6 to about 6.9 kDa and an anti-factor Xa activity of less than about 10 IU/mg, comprising the steps of oxidation of unfractionated heparin, depolymerisation and reduction of resulting terminal groups.

Claims

1. A process for the preparation of a heparin derivative, the process comprising the consecutive steps of: (i) oxidising an acidic aqueous solution of unfractionated heparin by addition of an oxidising agent; (ii) depolymerising the oxidised heparin by subjecting the product of step (i) to alkali to form an alkaline solution; (iii) maintaining said solution from step (ii) at an alkaline pH for a period of time required to provide depolymerised heparin; and (iv) reducing terminal aldehyde groups of said depolymerised heparin by addition of a hydride reducing agent to the solution obtained from step (iii), wherein the heparin derivative obtained from said process has a predominantly occurring disaccharide as shown in formula I below ##STR00003## wherein ##STR00004## n is an integer from 2 to 20, corresponding to molecular weights between 1.2 and 12 kDa, and the heparin derivative displays signals in the 5.0 ppm to 6.5 ppm region in a .sup.1H NMR spectrum with an intensity (% ratio) of less than or equal to about 4% relative to the signal at 5.42 ppm of the .sup.1H NMR spectrum of unfractionated heparin, and has a weight average molecular weight of from about 4.6 to about 6.9 kDa and an anti-factor Xa activity of less than about 10 IU/mg; wherein the period of time between the completion of step (i) and the start of step (iv) is controlled in order to minimise the effect of residual oxidising agents; and wherein said period of time in step (iii) is determined by analysis of said solution, or by reference to a previously-performed substantially identical step (iii).

2. The process as claimed in claim 1, in which, in the heparin derivative, at least 70% of the molecules have a molecular weight of greater than about 3 kDa.

3. The process as claimed in claim 1, in which the heparin derivative comprises polysaccharides with a distribution of cumulative molecular weights as indicated in the table below: TABLE-US-00006 Molecular mass, kDa Cumulative weight, % >10 4-15 >8 10-25 >6 22-45 >3 >70.

4. The process as claimed in claim 1, wherein, in the heparin derivative, the signals in the .sup.1H NMR spectrum are present at about 5.95 ppm and about 6.15 ppm.

5. The process as claimed in claim 1, wherein the heparin derivative further has an anti-factor IIa activity of less than 10 IU/mg.

6. The process as claimed in claim 1, wherein, in the heparin derivative, the polysaccharide chains present in the heparin derivative are essentially free of chemically intact saccharide sequences mediating the anticoagulant effect.

7. The process as claimed in claim 1, wherein, after the oxidation step, at least about 90% of non-sulphated vicinal diol moieties in the starting unfractionated heparin have been converted to the corresponding aldehydes.

8. The process as claimed in claim 7, wherein the non-sulphated vicinal diol moieties comprise the iduronic and glucuronic acid residues of heparin.

9. The process as claimed in claim 1, wherein the oxidising agent used in step (i) is sodium metaperiodate.

10. The process as claimed in claim 1, wherein step (i) requires oxidising an aqueous solution of unfractionated heparin by addition of sodium metaperiodate, at a pH of from about 4.5 to about 5.5 and at a temperature below about 25 C.

11. The process as claimed in claim 1, wherein step (ii) requires depolymerising the oxidised heparin by subjecting the product of step (i) to alkali to form a solution having a pH of from about 8 to about 13.

12. The process as claimed in claim 1, wherein the process further comprises the step of: (iiia) subjecting the solution obtained from step (iii) to acid to form a solution having a pH of from about 5.5 to about 6.5.

13. The process as claimed in claim 1, wherein the reducing agent used in step (iv) is sodium borohydride.

14. The process as claimed in claim 1, wherein the process further comprises the step(s) of: (iva) quenching the reduction reaction by lowering the pH to form an acidic solution; and, optionally, (ivb) adjusting the pH of the solution from step (iva) to about neutral.

15. The process as claimed in claim 1, wherein the process further comprises the step of: (v) recovering the heparin derivative from the solution obtained from step (iv).

16. The process as claimed in claim 1, wherein the period of time between the completion of step (i) and the start of step (iv) is a maximum of 6hours.

17. The process for the preparation of a pharmaceutical composition comprising a heparin derivative having an average molecular weight of from about 4.6 to about 6.9 kDa and an anti-factor Xa activity of less than about 10 IU/mg, which process comprises the steps of: preparing a heparin derivative using a process as claimed in claim 1; and (b) combining the heparin derivative obtained in step (a) with one or more pharmaceutically-acceptable adjuvant, excipient or diluent.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows a graph of the molecular weight of depolymerised heparin obtained over time in Example 2a as provided herein.

EXAMPLES

(2) The present invention may be further illustrated by the following examples.

Example 1

(3) Processes as described herein may be performed using the following general methods.

(4) Oxidation of Glucuronic and Iduronic Acid (Residues), Deletion of Anticoagulant Activity

(5) A quantity of about 3000 grams of Heparin (mucosal heparin of Ph. Eur. and USP quality) is dissolved in purified water to obtain a 10-20% w/v solution. The pH of this solution is adjusted to 4.5-5.5. The sodium metaperiodate (NalO.sub.4) is subsequently added to the process solution, with the quantity of periodate being 15-30%, target 25% of the weight of heparin. The pH is again adjusted to 4.5-5.5. The reactor is protected from light. The process solution is reacted for 18-26 hours with constant stirring maintenance of the temperature at 13-17 C. (e.g. about 15 C.), with the temperature being reduced to 5 C. during the last two hours.

(6) Depolymerisation of Polysaccharide Chains By An Alkaline Beta Elimination Process

(7) While maintaining the temperature at 5-10 C., a NaOH solution is added until a pH of 10.5-11.5 is obtained. The de-polymerization reaction is thereby initiated which leads to a slow decrease of the pH since OH.sup. ions are neutralised. Thus, the pH is carefully increased and tightly controlled in the range 10.5-11.5 by addition of a NaOH or Na.sub.2CO.sub.3 solution. Simultaneously, in-process control is initiated to follow the degree of de-polymerization by repeated GPC-HPLC analysis. The reaction proceeds for up to 4 hours or when the optimal molecular weight is obtained. A target range of molecular weight at 5.7-6.3 kDa is preferred. A table (see Example 2) or graph (see FIG. 1) could be used to predict the reaction time needed to obtain the preferred molecular weight range.

(8) The reaction is stopped by slow addition of 4 M HCl until a pH of 5.5-6.5 is obtained.

(9) The molecular weight is determined by GPC-HPLC carried out with a TSK 2000 and TSK 3000 SW columns in series, calibrated using the 1.sup.st international standard for LMWH . Refractive index is used to monitor the concentration of the eluate.

(10) Reduction of Iodine Compounds to Iodide and Iodine, Stabilization of the Product By Conversion of Terminal Aldehyde Groups of Polysaccharide to Corresponding Alcohols

(11) While maintaining the temperature at 5-17 C., a quantity of 130-200 grams of sodium borohydride is then added in portions, this to avoid over heating by the exothermic reaction, and the pH will increase to 9 (e.g. 10) to 11. The reaction is continued for 14-20 hours. After this reaction time, a dilute acid is added slowly in order to adjust the pH to a value of 4, this degrades remaining sodium borohydride. After maintaining a pH of 4 for 45-60 minutes, the pH of the solution is adjusted to 7 with a dilute NaOH solution.

(12) Precipitation of Reduced Product and Initial Removal of Iodine-Containing Compounds

(13) Ethanol (95-99.5%) is added to the reaction mixture over a period of 0.5-1 hour, with careful stirring and at a temperature of 5-25 C. The volume of ethanol to be added is in the range 1-2 volumes of ethanol per volume of process solution. The oxidized heparin is then allowed to precipitate and sediment for 15-20 hours, after which the mother liquor is decanted and discarded. Next, the sediment is dissolved in purified water to obtain a 15-30% w/v process solution. NaCl is added to obtain a concentration of 0.15-0.30 mol/L in the process solution

(14) Purification of the Product

(15) One volume of process solution is then added to 1.5-2.5 volumes of ethanol (95-99.5%) followed by centrifugation at >2000 G, and at <20 C. for 20-30 minutes, after which the supernatant is decanted and discarded.

(16) The product paste obtained by centrifugation is then dissolved in purified water to obtain a product concentration 10-20% w/v. Then NaCl is added to obtain a concentration of 0.20-0.35 mol/liter. Further on 1.5-2.5 volumes of ethanol (95-99.5%) are added per volume of process solution which precipitates the product from the solution. Centrifugation follows at >2000 G, and at <20 C. for 20-30 minutes after which the supernatant is decanted and discarded.

(17) Next the remaining paste is added purified water to dissolve. The product concentration would now be in the range of 10-20% w/v. The pH of the product solution is now adjusted to 6.5-7.5. The solution is then filtered to remove any particulates. Then, to one volume of process solution is added 1.5-2.5 volumes of ethanol (95-99.5%). Centrifugation follows at >2000 G, and at <20 C. for 20-30 minutes after which the supernatant is decanted and discarded.

(18) Reduction of the Size and Water Content of the Precipitate Paste

(19) A reactor is then filled with ethanol, volume 2 liter. While stirring the ethanol, the precipitate paste is added. The mechanical stirring solidifies the paste and replaces the water present by the ethanol giving a homogenous particle suspension. The stirring is discontinued after 1-2 hours after which the particles are allowed to sediment, then the mother liquor is decanted. This procedure is repeated twice. The precipitate is isolated on a polypropylene (PP) filter cloth. This procedure is repeated two more times. After removal of excessive liquid, the particles are passed through a sieve to obtain smaller and uniform sized particles.

(20) Vacuum Drying and Sieving

(21) The product is distributed evenly onto two pre-weighed trays, and placed in a vacuum cabinet. The pressure is reduced with a vacuum pump, the pressure actually obtained being noted, and the trays are heated to 35-40 C., with constant recording of the temperature. A stream of nitrogen is passed through the drier at this time while maintaining the low pressure in the dryer. After 2-3 days, the trays are removed from the drying cabinet and their weights measured. The drying is then continued for an additional 24 hours, thereafter the trays are taken out and weighed. This procedure is performed to monitor the progress of the drying. When a constant weight is obtained, i.e. no further evaporation is noticed, the drying is considered complete. The dry product is dispensed in 2 layer plastic bags covered by laminated plastic/aluminum foil. Storage is performed in a dry area at a temperature of 20-25 C.

Example 2

(22) The process was performed under three sets of conditions (Examples 2a, 2b and 2c), using two different batches of unfractionated heparin (referred to herein as Batch A and Batch B), under the following general conditions.

(23) Oxidation of Glucuronic and Iduronic Acid (Residues), Deletion of Anticoagulant Activity

(24) A quantity of about 25 grams of Heparin was dissolved in purified water to obtain a 15% w/v solution. Then 6.25 gram of sodium metaperiodate (NalO.sub.4) was added to the process solution and the pH was the adjusted to 4.9-5.0. The reactor was protected from light. The process solution was reacted for 22 hours with constant stirring maintenance of the temperature at 15 C., after which the temperature was reduced to 5 C. during the last two hours. The total reaction time was 24 hours.

(25) Depolymerisation of Polysaccharide Chains By an Alkaline Beta Elimination Process

(26) While maintaining the temperature at 5-10 C., a NaOH solution was added until a pH of 11-11.5 was obtained. The pH was continuously monitored and adjusted to the pH indicated in respect of Example 2a, 2b and 2c (below) over a period of 250 minutes. The reaction was stopped by slow addition of 4 M HCl until a pH of 5.5-6.5 was obtained. The time needed for adjusting the pH was approximately 15 minutes.

(27) During the reaction time, eleven samples were taken at specified time points during the reaction time. The samples were immediately diluted by a 15 mM phosphate buffer solution and pH adjusted to 7 to stop the ongoing reaction. The samples were then subjected to analysis of the molecular weight by GPC-HPLC, as discussed below. A table was created for each process (see below). The molecular weight was determined by GPC-HPLC carried out with TSK 2000 and TSK 3000 SW columns in series, calibrated using the 1st international standard for LMWH (as discussed herein). A refractive index was used to monitor the concentration of the eluate.

(28) Reduction of Iodine Compounds to Iodide and Iodine, Stabilization of the Product By Conversion of Terminal Aldehyde Groups of Polysaccharide to Corresponding Alcohols

(29) While maintaining the temperature at 5-15 C., a quantity of 1.75 gram of sodium borohydride was then added in portions during 30 minutes, this to avoid over heating by the exothermic reaction, and the pH increased to 10. A sample was withdrawn from the solution after completed addition of sodium borohydride and was analysed for molecular weight. The results confirmed that the molecular weight remained unchanged. The reaction continued for 20 hours. After this reaction time, a dilute acid was added until a pH of 4-4.5 was obtained, which degraded remaining sodium borohydride. The formation of hydrogen gas bubbles was noticed, which confirmed that NaBH.sub.4 had been initially added in excess of required amount. After maintaining a pH of 4 for 45-60 minutes, the pH of the solution was adjusted to 7 with a dilute NaOH solution.

(30) Precipitation of reduced product and initial removal of iodine-containing compounds

(31) Ethanol (95-99.5%) was added to the reaction mixture over a period of 0.5-1 hour, with careful stirring and at a temperature of 5-25 C. The volume of ethanol added was 1.5 volumes of ethanol per volume of process solution. The product precipitated out of solution and was separated by centrifugation at approximately 5000 G for 20 minutes. Then the mother liquor was decanted. Next, the product paste was dissolved in purified water to obtain a 15-30% w/v process solution. NaCl was added to obtain a concentration of 0.15-0.30 mol/liter in the process solution. Next, ethanol was added, amount 2 volumes of ethanol per volume of process solution. The product precipitated out of solution and was separated by centrifugation at approximately >2000 G for 20 minutes. Again the mother liquor was decanted.

(32) Next the remaining paste was added purified water to dissolve. The product concentration was now in the range of 15-30% w/v. The pH of the product solution was then adjusted to 6.5-7.5 and then the solution was then filtered to remove any particulates. Then, to one volume of process solution was added 2 volumes of ethanol (95-99.5%). Centrifugation followed at >2000 G, and at <20 C. for 20-30 minutes after which the supernatant was decanted and discarded. The paste was then dehydrated by twice repeated additions and decanting of ethanol and manual grinding of the paste.

(33) Vacuum Drying and Milling

(34) The dehydrated paste was then transferred to a glass flask connected to a vacuum dryer. Drying followed under vacuum at a temperature of 38-40 C. The drying was stopped after approximately 48 hours of drying. Then milling followed after which the final product was dispensed in airtight glass vials.

(35) Analysis of the Depolymerisation Step

(36) The progress of the depolymerisation step was analysed by GPC-HPLC using TSK 2000 and TSK 3000 SW columns in series, calibrated using the 1.sup.st international standard for LMWH (using techniques as described herein). The refractive index was used to monitor the concentration of the eluate.

(37) The tables provided in respect of each of Examples 2a to 2c below show the times taken to achieve a particular average molecular weight under the respective reaction conditions. The times indicated in respect of Example 2a are also shown in the graph provided as FIG. 1.

Example 2a

(38) Example 2a was reacted in the depolymerisation reaction at a pH of 11 using Batch A of heparin.

(39) TABLE-US-00002 Sample Mw (kDa) Time (min) 1 12.6 0 2 8.5 15 3 7.7 30 4 7.3 45 5 7.0 60 6 6.8 73 7 6.7 84 8 6.2 127 9 6.1 152 10 5.9 210 11 5.8 248

Example 2b

(40) Example 2b was reacted in the depolymerisation reaction at a pH of 11.5 using Batch A of heparin.

(41) TABLE-US-00003 Sample Mw (kDa) Time (min) 1 13.0 0 2 7.3 15 3 6.7 30 4 6.4 45 5 6.2 60 6 6.1 73 7 6.0 84 8 5.7 127 9 5.6 152 10 5.4 210 11 5.3 248

Example 2c

(42) Example 2c was reacted in the depolymerisation reaction at a pH of 11 using Batch B of heparin.

(43) TABLE-US-00004 Sample Mw (kDa) Time (min) 1 10.4 0 2 8.5 15 3 7.6 30 4 6.6 45 5 6.8 59 6 6.7 69 7 6.5 83 8 5.9 131 9 6.0 152 10 5.7 210 11 5.6 248

Example 3

(44) The below table shows the results of .sup.1H NMR analysis of heparin derivatives obtained using the procedure set out in Example 2, following the European Directorate for the

(45) Quality of Medicines & Healthcare (EDQM), monograph 7, as set out in the European Pharmacopeia.

(46) TABLE-US-00005 Intensity (% ratio) compared to the 5.42 ppm signal of unfractionated heparin 6.15 ppm 5.95 ppm Sample % of ref. signal % of ref. signal Example 2a 2.0 2.5 Example 2c 2.5 2.0

Example 4

(47) The product obtained from processes according to any one of the examples provided herein can be formulated into a pharmaceutical composition by a conventional aseptic process.

(48) In particular, a pharmaceutical composition may be prepared by forming a solution comprising 150 mg/mL of active product and Na phosphate to 15 mM, having a pH of 6-8. The so obtained pharmaceutical composition is intended primarily for subcutaneous administration, but is also suitable for intravenous administration.