Separation and purification method for vancomycin hydrochloride of high purity

Abstract

Provided is a separation and purification method for vancomycin hydrochloride of high purity. The method comprises the following steps: (1) obtaining a vancomycin hydrochloride solution from a crude vancomycin product by ion exchange chromatography and obtaining a concentrate by nanofiltration desalination and concentration; (2) adjusting the concentrate with a hydrochloric acid solution and then performing a column chromatography using a reverse chromatography column for the adjusted concentrate; (3) collecting the chromatographic solution of vancomycin to obtain a mixed chromatographic solution; (4) adjusting the mixed chromatographic solution, and separating the solution and the salts by nanofiltration desalination and concentration to obtain a concentrate; and (5) obtaining a vancomycin dry powder with a chromatographic purity of up to 99% and a pure white appearance by dehydrating and drying the concentrate of step (4), or by solvent crystallization or salting-out crystallization.

Claims

1. A method of separation and purification for vancomycin hydrochloride with high purity, comprising the following steps: (1) obtaining a vancomycin hydrochloride solution from a crude vancomycin product by ion exchange chromatography and obtaining a first vancomycin hydrochloride concentrate by nanofiltration, desalination and concentration; wherein a filler of the ion exchange chromatography column is a cation exchange glucan gel or agarose gel, a mobile phase is salt-water; (2) adjusting the first vancomycin hydrochloride concentrate with a hydrochloric acid solution to pH=3.5-4.5, and then performing a column chromatography using a reverse phase chromatography column for the first vancomycin hydrochloride pH adjusted concentrate, wherein a stationary phase is polystyrene, and a mobile phase is methanol aqueous solution or ethanol aqueous solution, wherein a proportion of methanol or ethanol in the mobile phase is 10% (V/V); (3) collecting a chromatographic solution of content of vancomycin hydrochloride of more than 98.5% purity; (4) adjusting the chromatographic solution with hydrochloric acid to pH=2.5-3.5, and separating solvent and salt by nanofiltration desalination and concentration to obtain a second vancomycin hydrochloride concentrate; and (5) obtaining a vancomycin dry powder with a chromatographic purity of up to 99% and a pure white appearance by dehydrating and drying the second vancomycin hydrochloride concentrate of step (4).

2. The method according to the claim 1, wherein the vancomycin hydrochloride solution of step (1) obtained by ion exchange chromatography has a chromatographic purity of more than 95%.

3. The method according to the claim 1, wherein the concentration of the vancomycin hydrochloride solution of step (1) is 100 mg/ml-200 mg/ml.

4. The method according to the claim 1, wherein the particle size of polystyrene is 20-40 m.

5. The method according to the claim 1, wherein the mobile phase of step (2) is adjusted to pH=3.5-5.5 with hydrochloric acid or acetic acid.

6. The method according to the claim 1, wherein the nanofiltration membrane with molecular weight of 100-800 Da of step (4) is used for nanofiltration.

7. The method according to the claim 1, wherein the vancomycin hydrochloride powder of step (5) has absorbance of less than 0.02; whiteness of more than 88%; and chromatographic purity of more than 99% when measured at 10 wt. % concentration of vancomycin hydrochloride powder at a wavelength of 450 nm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows chromatogram of crude vancomycin of example 1, the content of vancomycin B is 90.2%;

(2) FIG. 2 shows chromatogram of vancomycin hydrochloride concentrate of example 1, the content of vancomycin B is 95.3%;

(3) FIG. 3 shows chromatogram of the product of vancomycin hydrochloride of example 2, the content of vancomycin B is 99.1%;

(4) FIG. 4 shows chromatogram of vancomycin hydrochloride concentrate of examples 4-6, the content of vancomycin B is 95.8%;

(5) FIG. 5 shows chromatogram of the product of vancomycin hydrochloride of example 4, the content of vancomycin B is 99.0%;

(6) FIG. 6 shows chromatogram of the product of vancomycin hydrochloride of example 5, the content of vancomycin B is 99.2%;

(7) FIG. 7 shows chromatogram of the product of vancomycin hydrochloride of example 6, the content of vancomycin B is 99.0%;

(8) FIG. 8 shows chromatogram of the vancomycin hydrochloride concentrate of examples 7-9, the content of vancomycin B is 95.9%;

(9) FIG. 9 shows chromatogram of the product of vancomycin hydrochloride of example 7, the content of vancomycin B is 99.0%;

(10) FIG. 10 shows chromatogram of the product of vancomycin hydrochloride of example 8, the content of vancomycin B is 99.2%;

(11) FIG. 11 shows chromatogram of the product of vancomycin hydrochloride of example 9, the content of vancomycin B is 99.3%.

DETAILED DESCRIPTION OF THE PRESENT INVENTION AND PREFERRED EMBODIMENTS THEREOF

(12) Hereafter, the present invention will be further illustrated with reference to examples. The examples are given only for illustration of the technical solution of the present invention and should not be construed to limit the present invention.

(13) The present invention is further specifically illustrated by the following examples, but is not limited to the following examples and the extent of process parameters of examples.

Example 1: Preparation of Vancomycin Hydrochloride Solution with a Content of Vancomycin B of not Lower than 95%

(14) 250 g of crude vancomycin is dissolved to 2.0 L purified water in a beaker, and stirred fully, and filtered with a filter membrane of pore size of 0.2 m after being completely dissolved, and then diluted by water, to finally obtain 3.0 L of the dissolved solution containing crude vancomycin hydrochloride with the concentration of 43.6 mg/ml and chromatographic purity of 90.2%, as shown in FIG. 1.

(15) 3000 ml of the dissolved solution of crude vancomycin hydrochloride is applied to a 8 cm*60 cm glass chromatographic column containing glucan SEPHADEX CM-25, the capacity of the column is 4% of the chromatographic column volume. The column solution is mixed with of chromatographic filler to form a mixed solution, and then the mixed solution is directly applied to the glass chromatographic column, and then is washed by a purified water at a flow rate of 1.5 times column volumes per hour, after washing 6 times column volumes, and is prewashed by 0.3% (w/v) NH.sub.4HCO.sub.3 aqueous solution with one times column volume per hour. the prewashing volume is 1520 times column volumes. After finishing prewashing, the content of vancomycin B of the eluent is more than 90%, and then eluted by 5% (w/v) NH.sub.4HCO.sub.3 aqueous solution to obtain vancomycin, and the eluent is collected in fractions, and the fractions with the content of vancomycin B of more than 95% determined by HPLC are mixed, and then adjusted to pH=3.1 with 4N hydrochloric acid, to obtain an effective eluent of 5600 ml with the concentration of 18.6 mg/ml, a chromatographic purity of 96.5%, as shown in FIG. 2.

(16) Afterwards the effective eluent is desalted and concentrated by nanofiltration membrane with pore diameter of molecular weight of 400, the purified water is added for 5 times, to finally obtain a dialysate with the conductivity of 1255 s/cm, and then the feed liquid is concentrated to obtain vancomycin hydrochloride concentrate with the concentration of 156 mg/ml, pH=3.8, the chromatographic purity of 95.3% (refer to FIG. 2), the volume of 680 ml, and the concentrate is stored at 2-8 C. for use.

Example 2: Preparation of Vancomycin Hydrochloride with High Purity

(17) 680 ml of the concentrate of example 1 is passed through a column (15 cm*30 cm) having C18 silica gel filler with particle size of 30 m, adjusted to pH 4.0 by hydrochloric acid, pre-washed for 100 mim with an aqueous solution containing NH.sub.4Cl of 0.2% (W/V) and methanol aqueous solution of 8% (V/V) as a mobile phase at the flow rate of 5 BV/h, and then the methanol proportion of the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. At the on-line detection wavelength =280, the eluent is collected about 10 bottles with 2.5 L per bottle when the absorption value begins rising rapidly, and the content of vancomycin B per bottle is determined, then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain the mixed chromatographic solution of 12.5 L, with the concentration of 6.8 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(18) The above chromatographic solution is passed through a nanofiltration membrane with the pore diameter of molecular weight of 400 for desolvation, when no solvent is produced anymore, the mixture solution is concentrated to a concentrate with concentration of 240 mg/ml and volume of 338 ml, and then the concentrate is filtered by filter membrane of 0.45 m and is freeze-dried in the freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 68.5 g, with the yield of 64.6%, chromatographic purity of 99.1% (refer to FIG. 3), and the absorbance of solution of 10% at 450 nm is 0.012.

Example 3: Comparison of Concentration Effects by Nanofiltration Membrane with Different Pore Diameter

(19) Nanofiltration membrane tubes with different pore diameter such as 100 Da, 200 Da, 400 Da and 800 Da and with the same filter area of 0.32 m.sup.2 are selected respectively, to install in turn on a small nanofiltration membrane equipment for lab (type LNG-NF-101). 8000 ml of concentrate containing 10% vancomycin hydrochloride is divided into 4 portions, and one of them is concentrated by nanofiltration, with the pressure of 10 bar controlled by circulating pump. Potency of the dialysis sample is detected at the beginning of the concentration, and record the flow rate of dialysis, when the circulating fluid is concentrated to volume of 1000 ml, the potency detection of dialysis sample is finished, and the flow rate of dislysis is recorded. The equipment is washed after using per time, and the nanofiltration membrane tube is replaced by next one, use another concentrate for experiment. The assay results are shown as follows:

(20) TABLE-US-00001 Pore diameter Testing item 100 Da 200 Da 400 Da 800 Da Initiate flow rate 2.2 4 4.2 4.2 (L/H) Initiate potency 5 6 5 8 (/ml) End flow rate 1.6 3.8 3.8 4.0 (L/H) End potency 6 7 7 8 (/ml)

(21) As shown in above Table, nanofiltration membranes with different pore diameter have little impact on potency of dialysate. So all of them are suitable for concentrating vancomycin hydrochloride. But membrane tubes with different pore diameters have effects on flow rates, and the membrane tubes with pore diameters of greater than 200 Da is more appropriate to the flow rate.

Example 4: Comparison of C18 Silica Gels with Different Particle Sizes

(22) 330 ml of the concentrate with the concentration of 150 mg/ml, chromatographic purity of 95.8% (refer to FIG. 4) is adjusted to pH=4.0 with hydrochloric acid or sodium hydroxide solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with the particle size of 5 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.1% NH.sub.4Cl(W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 8 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 10 L, with the concentration of 3.8 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(23) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 248 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 30.8 g, with the yield of 62.2%, the chromatographic purity of 99.0% (refer to FIG. 5), and the absorbance A of solution of 10% at 450 nm is 0.014.

Example 5: Comparison of C18 Silica Gels with Different Particle Sizes

(24) 330 ml of the concentrate with the concentration of 150 mg/ml, chromatographic purity of 95.8% (refer to FIG. 4) is adjusted to pH=4.0 with hydrochloric acid or sodium hydroxide solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.1% NH.sub.4Cl(W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 8 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 10 L, with the concentration of 4.1 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(25) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 265 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 32.5 g, with the yield of 65.6%, the chromatographic purity of 99.2% (refer to FIG. 6), and the absorbance A of solution of 10% at 450 nm is 0.015.

Example 6: Comparison of C18 Silica Gels with Different Particle Sizes

(26) 330 ml of the concentrate with the concentration of 150 mg/ml, chromatographic purity of 95.8% is adjusted to pH=4.0 with hydrochloric acid or sodium hydroxide solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 60 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.1% NH.sub.4Cl(W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 8 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 10 L, with the concentration of 3.8 mg/ml mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(27) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 245 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 29.8 g, with the yield of 60.2%, the chromatographic purity of 99.0% (refer to FIG. 7), and the absorbance A of solution of 10% at 450 nm is 0.012.

(28) It can be found from comparison of examples 4, 5 and 6 that the reverse chromatography containing C18 silica gel filler with different particle size has little impact on the chromatographic purity, yield and absorbance. But, as for operating pressures, the smaller the particle size is, the greater the pressure is. So it is most suitable for mass production to choose C18 silica gel with particle size of 30 m-60 m.

Example 7: Comparison of CH3COONH4 and NH4Cl in the Mobile Phase

(29) 330 ml of the concentrate with the concentration of 152 mg/ml, chromatographic purity of 95.9% (refer to FIG. 8) is adjusted to pH=4.0 with hydrochloric acid or sodium hydroxide solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.0 with acetic acid, and pre-washed for 100 min with an aqueous solution containing 0.1% CH.sub.3COONH.sub.4 (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 8 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 10 L, with the concentration of 4.0 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(30) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 252 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 31.1 g, the chromatographic purity of 99.0% (refer to FIG. 9), and the absorbance A of solution of 10% at 450 nm is 0.017.

(31) It can be found from comparison of example 5 that the components of the mobile phase containing NH4Cl and its yields is little difference from that of CH3COONH4.

Example 8: Comparison of Different Proportions of NH4Cl in Mobile Phase

(32) 330 ml of the concentrate with the concentration of 152 mg/ml, chromatographic purity of 95.9% is adjusted to pH=4.0 with hydrochloric acid solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.5% NH.sub.4Cl (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 9 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12.5 L, with the concentration of 3.2 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(33) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 260 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 32.1 g, the yield of 64.0%, the chromatographic purity of 99.2% (refer to FIG. 10), and the absorbance A of solution of 10% at 450 nm is 0.016.

Example 9: Comparison of Different Proportions of NH4Cl in Mobile Phase

(34) 330 ml of the concentrate with the concentration of 152 mg/ml, chromatographic purity of 95.9% is adjusted to pH=4.0 with hydrochloric acid solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 1.0% NH.sub.4Cl (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 9 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12.5 L, with the concentration of 3.1 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(35) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 258 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 32.0 g, the yield of 63.8%, the chromatographic purity of 99.3% (refer to FIG. 10), and the absorbance A of solution of 10% at 450 nm is 0.014.

(36) It can be found from comparison of examples 5, 8 and 9 that the aqueous solution containing different contents of NH.sub.4Cl (W/V) and 8% (V/V) methanol aqueous solution as the mobile phases has little impact on the yields, chromatographic components and absorbancy, but has impacts on the volume collected.

Example 10: Comparison of Different pH of the Column Solution and the Mobile Phase

(37) 670 ml of the concentrate with the concentration of 146 mg/ml, chromatographic purity of 96.4% is adjusted to pH=3.5 with hydrochloric acid solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=3.5 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.5% NH.sub.4Cl (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h, the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluent with the chromatographic purity of more than 98.5% are mixed to obtain the mixed chromatographic solution of 12.5 L, with the concentration of 6.3 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(38) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 380 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 63.1 g, the yield of 64.5%, the chromatographic purity of 99.2%, and the absorbance A of solution of 10% at 450 nm is 0.015.

Example 11: Comparison of Different pH of the Column Solution and the Mobile Phase

(39) 670 ml of the concentrate with the concentration of 146 mg/ml, chromatographic purity of 96.4% is adjusted to pH=4.5 with hydrochloric acid solution, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.5 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.5% NH.sub.4Cl (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then a proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h, the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluent with the chromatographic purity of more than 98.5% are mixed to obtain the mixed chromatographic solution of 15 L, with the concentration of 5.1 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(40) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 360 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 58.2 g, the yield of 59.5%, the chromatographic purity of 99.0%, and the absorbance A of solution of 10% is 0.018.

(41) It can be found from comparison of examples 7, 10 and 11 that pH=3.5 and pH=4.0 of the column solution and the mobile phase have little effect on the product yield and components, and pH=4.5 of the column solution would result in increasing of the collected volume, and the yield and components are affected slightly.

Example 12: Comparison of Different Concentration CH3COONH4 in the Mobile Phase

(42) 330 ml of the concentrate with the concentration of 154 mg/ml, chromatographic purity of 95.9% is adjusted to pH=4.0 with hydrochloric acid or sodium hydroxide, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.5% CH.sub.3COONH.sub.4 (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 8 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 10 L, with the concentration of 4.1 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(43) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 260 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 32.5 g, the yield of 64.0%, the chromatographic purity of 99.1%, and the absorbance A of solution of 10% is 0.013.

Example 13: Comparison of Different Concentration CH3COONH4 in the Mobile Phase

(44) 330 ml of the concentrate with the concentration of 154 mg/ml is adjusted to pH=4.0 with hydrochloric acid or sodium hydroxide, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 1% CH.sub.3COONH.sub.4 (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 9 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12.5 L, with the concentration of 3.2 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(45) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 150 mg/ml and volume of 265 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 33.0 g, the yield of 65.0%, the chromatographic purity of 99.0%, and the absorbance A of solution of 10% is 0.015.

(46) It can be found from comparison of examples 7, 12 and 13 that the aqueous solution containing different concentration CH.sub.3COONH.sub.4 (W/V) and 8% (V/V) methanol aqueous solution as the mobile phase has little impact on the product component, yield and absorbancy, however high concentration CH.sub.3COONH.sub.4 has impact on the collected volume.

Example 14: Comparison of Different pH in CH3COONH4 System as Mobile Phase

(47) 670 ml of the concentrate with the concentration of 145 mg/ml, chromatographic purity of 96.6% is adjusted to pH=3.5 with hydrochloric acid or sodium hydroxide, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=3.5 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.5% CH.sub.3COONH.sub.4 (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12.5 L, with the concentration of 6.2 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(48) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 370 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 60.6 g, the yield of 62.4%, the chromatographic purity of 99.2%, and the absorbance A of solution of 10% is 0.016.

Example 15: Comparison of Different pH in the CH3COONH4 Systems as Mobile Phase

(49) 650 ml of the concentrate with the concentration of 156 mg/ml, chromatographic purity of 95.7% is adjusted to pH=4.0 with hydrochloric acid or sodium hydroxide, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 0.5% CH.sub.3COONH.sub.4 (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12.5 L, with the concentration of 6.4 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(50) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 385 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 63.9 g, the yield of 63.0%, the chromatographic purity of 99.0%, and the absorbance A of solution of 10% is 0.014.

Example 16: Comparison of Different pH in the CH3COONH4 System as Mobile Phase

(51) 670 ml of the concentrate with the concentration of 145 mg/ml, chromatographic purity of 96.6% is adjusted to pH=4.5 with sodium hydroxide, is filled into a preparative column (15 cm*30 cm) containing C18 silica gel filler with particle size of 30 m and is adjusted to pH=4.5 with acetic acid, and pre-washed for 100 min with an aqueous solution containing 0.5% CH.sub.3COONH.sub.4 (W/V) and 8% (V/V) methanol aqueous solution as mobile phase, at the flow rate of 5 BV/h, then the proportion of methanol in the mobile phase is increased to 12% to elute at the flow rate of 5 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2.5 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12.5 L, with the concentration of 6.1 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(52) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 370 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 61.0 g, the yield of 62.8%, the chromatographic purity of 99.1%, and the absorbance A of solution of 10% is 0.018.

(53) It can be found from comparison of examples 14, 15 and 16 that the pH is controlled between 3.54.5 in the column solution and the mobile phase, little impact is on the yield and quality of the prepared product.

Example 17: Example of Reverse Phase Preparation with Polymer Filler PS Resin

(54) 680 ml of the concentrate with the concentration of 148 mg/ml, chromatographic purity of 95.9% is adjusted to pH=4.0 with sodium hydroxide, is filled into a preparative column (15 cm*30 cm) containing PS adsorption resin with particle size of 20 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 100 min with an aqueous solution containing 5% ethanol (V/V) as mobile phase, at the flow rate of 2 BV/h, then the proportion of ethanol in the mobile phase is increased to 10% to elute at the flow rate of 2 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12 L, with the concentration of 6.4 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(55) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 365 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 60.6 g, the yield of 60.2%, the chromatographic purity of 99.3%, and the absorbance A of solution of 10% is 0.016.

Example 18: Example of Reverse Phase Preparation with Polymer Filler PS Resin

(56) 680 ml of the concentrate with the concentration of 148 mg/ml, chromatographic purity of 95.9% is adjusted to pH=3.5 with hydrochloric acid, is filled into a preparative column (15 cm*30 cm) containing PS adsorption resin with particle size of 20 m and is adjusted to pH=3.5 with hydrochloric acid, and pre-washed for 60 min with an aqueous solution containing 5% ethanol (V/V) as mobile phase, at the flow rate of 2 BV/h, then the proportion of ethanol in the mobile phase is increased to 10% to elute at the flow rate of 2 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12 L, with the concentration of 6.2 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(57) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 370 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 61.2 g, the yield of 60.8%, the chromatographic purity of 99.0%, and the absorbance A of solution of 10% is 0.015.

Example 19: Example of Reverse Phase Preparation with Polymer Filler PS Resin

(58) 650 ml of the concentrate with the concentration of 158 mg/ml, chromatographic purity of 96.2% is adjusted to pH=3.5 with hydrochloric acid, is filled into a preparative column (15 cm*30 cm) containing PS adsorption resin with particle size of 40 m and is adjusted to pH=3.5 with hydrochloric acid, and pre-washed for 60 min with an aqueous solution containing 5% ethanol (V/V) as mobile phase, at the flow rate of 2 BV/h, then the proportion of ethanol in the mobile phase is increased to 10% to elute at the flow rate of 2 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12 L, with the concentration of 7.1 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(59) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 370 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 64.7 g, the yield of 63.0%, the chromatographic purity of 99.0%, and the absorbance A of solution of 10% is 0.012.

Example 20: Example of Reverse Phase Preparation with Polymer Filler PS Resin

(60) 650 ml of the concentrate with the concentration of 158 mg/ml, chromatographic purity of 96.2% is adjusted to pH=4.0 with hydrochloric acid, is filled into a preparative column (15 cm*30 cm) containing PS adsorption resin with particle size of 40 m and is adjusted to pH=4.0 with hydrochloric acid, and pre-washed for 60 min with an aqueous solution containing 5% ethanol (V/V) as mobile phase, at the flow rate of 2 BV/h, then the proportion of ethanol in the mobile phase is increased to 10% to elute at the flow rate of 2 BV/h. the on-line detection wavelength =280, the eluent is collected when the absorptive value begins with rising rapidly, to totally collect 10 bottles, one bottle per 2 L, and to determine the content of vancomycin B per bottle, and then the eluents with a chromatographic purity of more than 98.5% are mixed to obtain a mixed chromatographic solution of 12 L, with the concentration of 6.8 mg/ml, and the mixed chromatographic solution is adjusted to pH=2.8 with 4N hydrochloric acid.

(61) The above chromatographic solution is passed through a nanofiltration membrane with a pore diameter of molecular weight of 400 for desolvation. the mixture solution is concentrated to a concentration of 200 mg/ml and volume of 380 ml when no solvent is produced anymore, and then the concentrate is filtered by a filter membrane of 0.45 m, and is freeze-dried in a freeze dryer to obtain freeze-dried powders of vancomycin hydrochloride of 63.5 g, the yield of 61.8%, the chromatographic purity of 99.1%, and the absorbance A of solution of 10% is 0.018.

(62) In the examples 17-20, chromatography is prepared by using polymer reverse phase fillers PS with two specifications, and is prepared by using indifferent pH. It may be shown from results that the differences of the product component, yield and absorbance between them are little, and their effects are better.

(63) The present invention illustrates by the above examples, however, it is understood that, the present invention is not limited to special instance and implementation scheme described herein. Here the purpose including these special instances and implementation schemes is aimed at helping the persons skilled in the art to achieve this invention. It is easy for any persons skilled in the art to carry out further improvement and perfection not from the spirit and scope of the invention, so the present invention is just limited by the content and scope of claims of the present invention, its intention to cover all included all alternative solutions and equivalent solutions within the spirit and scope of the present invention limited by the appendix claims.