WO2015062168A1 - 一种高纯度盐酸万古霉素的分离纯化方法 - Google Patents

一种高纯度盐酸万古霉素的分离纯化方法 Download PDF

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WO2015062168A1
WO2015062168A1 PCT/CN2014/000950 CN2014000950W WO2015062168A1 WO 2015062168 A1 WO2015062168 A1 WO 2015062168A1 CN 2014000950 W CN2014000950 W CN 2014000950W WO 2015062168 A1 WO2015062168 A1 WO 2015062168A1
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vancomycin
solution
concentration
vancomycin hydrochloride
hydrochloric acid
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PCT/CN2014/000950
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English (en)
French (fr)
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李恩民
庄逸云
王珏
孙新强
劳学军
江必旺
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浙江医药股份有限公司新昌制药厂
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Priority to US15/033,116 priority Critical patent/US10131689B2/en
Priority to ES14857463T priority patent/ES2752554T3/es
Priority to JP2016550912A priority patent/JP6438966B2/ja
Priority to EP14857463.5A priority patent/EP3064214B1/en
Priority to KR1020167014663A priority patent/KR101925695B1/ko
Publication of WO2015062168A1 publication Critical patent/WO2015062168A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
    • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin

Definitions

  • the invention relates to a method for separating and purifying high-purity vancomycin hydrochloride.
  • the method uses crude vancomycin with a chromatographic purity of about 90% as a raw material, and firstly collects vancomycin hydrochloride with a chromatographic purity greater than 95% by chromatography on a Sephadex column containing a salt-water mobile phase. Perform nanofiltration desalting and concentration to 100mg/ml-200mg/ml, and then elute through a reversed-phase C18 silica gel column or reversed-phase polymer column with ammonium hydroxide aqueous solution or ethanol as mobile phase to collect chromatographic purity.
  • vancomycin hydrochloride More than 98.5% vancomycin hydrochloride was removed by nanofiltration, and then concentrated to 150 mg/ml-250 mg/ml for lyophilization to obtain vancomycin hydrochloride having a chromatographic purity of 99% and a white appearance.
  • Vancomycin hydrochloride is an amphoteric glycopeptide antibiotic produced by the actinomycetes of the genus Amycolatopsis genus under controlled fermentation conditions. Its chemical formula is C 66 H 75 C 12 N 9 O 24 ⁇ HCl, and its molecular weight is 1.486. It is known that vancomycin hydrochloride binds to the propeptide terminal D-Ala-D-Ala of mucopeptide and inhibits the synthesis of bacterial cell walls. In addition, vancomycin hydrochloride can also change the permeability of cell membranes and the synthesis of RNA.
  • Vancomycin hydrochloride is particularly useful for the initial treatment of severe or severe infections caused by staphylococci against ⁇ -lactam antibiotics, as well as for treating patients who are allergic to penicillin or have no effect with penicillin and cephalosporins.
  • vancomycin hydrochloride has serious renal and ototoxicity, so it has not been widely used clinically, but recently, due to the extensive use of antibiotics, clinically methicillin-resistant Staphylococcus aureus (MASA) infections are increasingly The increase has led to an increase in the use of vancomycin hydrochloride.
  • the vancomycin molecule consists of two basic structures, namely the glycosyl moiety ⁇ -o-vancosamine- ⁇ -o-glucosyl and the peptidyl moiety central heptapeptide nucleus whose structure determines its instability, and the vancomycin molecule Degradation products are produced by acid or alkali or high temperature conditions, and the structure also has a plurality of free phenolic hydroxyl groups, which causes them to be easily oxidized into hydrazine. It has been reported in the literature that vancomycin will hydrolyze under acidic conditions and high temperature conditions, and it will be removed. A glycosyl or a disaccharide group produces a monosaccharide vancomycin or aglucovancomycin.
  • an amide group may also be degraded to deamino vancomycin, which has two Isomers. Based on these characteristics of vancomycin chemical structure, it has brought certain difficulties to the manufacture of high-purity vancomycin hydrochloride.
  • the vancomycin hydrochloride product has been developed for decades.
  • the early preparation process is generally carried out by using solvents such as methanol, ethanol, isopropanol, acetone, etc., and precipitation by salting out by adding ammonium chloride or sodium chloride.
  • the preparation of the finished product but due to the presence of more impurities in the vancomycin fermentation broth, especially vancomycin structural analogs, the purity is generally not high, to achieve a chromatographic purity greater than 93% of the European Pharmacopoeia standards have a certain Difficulty.
  • the example in the patent WO2006061166 uses a 5 ⁇ m diameter ozone silica gel (Octadecyl silica gel) as a chromatographic medium, a 3% methanol solution containing 5 mM ammonium acetate and a pH adjustment of 4.0 as a mobile phase, and 2% pentanol in the mobile phase.
  • a resolving agent collect vancomycin hydrochloride with a chromatographic purity greater than 97.5%, then concentrate the concentration of the collected liquid to 140 mg/ml by vacuum, add methanol, adjust the pH to 8.5-9.0 with ammonia water, and cool the temperature of the solution to 0 degree to separate the precipitate.
  • the invention provides a method for separating and purifying vancomycin hydrochloride which is suitable for commercial production with high purity (chromatographic purity of more than 99%), low impurity and high efficiency, and the separation and purification method comprises the following steps: (1) The vancomycin hydrochloride solution was prepared by ion chromatography exchange chromatography on the crude vancomycin, and the first vancomycin hydrochloride concentrate was obtained by desalting and concentration by nanofiltration; (2) the first vancomycin hydrochloride was adjusted with hydrochloric acid solution.
  • the vancomycin hydrochloride solution in the step (1) is a vancomycin hydrochloride solution having a vancomycin chromatographic purity greater than 95% obtained by ion exchange chromatography.
  • the above vancomycin hydrochloride solution having a vancomycin chromatographic purity of not less than 95% is prepared by the following prior art method. (1) First, according to the method described in Chinese patent CN01132048.6, the fermenting strain is Amycolatopsis Oriertalis SIPI43491, which is inoculated into the first-class seed tank through the inoculum culture and expanded by the secondary seed.
  • the culture is carried out into a fermenter, the temperature is controlled at 24-34 ° C, the culture pressure is 0.01-0.08 MPa, the dissolved oxygen and pH are controlled in the process, and the fermentation cycle is 4-6 days, and the vancomycin fermentation liquid is obtained.
  • the vancomycin fermentation broth is passed through a macroporous adsorption resin, vancomycin is eluted with an acidic aqueous solution containing ethanol, and then decolorized by adding activated carbon to the eluate. Ammonium bicarbonate is added to the decoloring solution, and the pH is adjusted to 7.5-8.5 with ammonia water.
  • the crude vancomycin having a chromatographic purity of not less than 80% is isolated (the crude vancomycin refers to the eternal in the step (1). Crudemycin).
  • Crudemycin (3)
  • the crude vancomycin was dissolved in purified water and filtered through a ceramic membrane having a pore size of 0.01 to 0.5 ⁇ m to obtain a clear vancomycin filtrate.
  • the vancomycin filtrate was clarified and purified by ion exchange chromatography to obtain an effective chromatographic solution containing vancomycin B in an amount of 95% or more (that is, vancomycin hydrochloride having a vancomycin chromatographic purity of not less than 95%).
  • the ion exchange chromatography column used is a cation exchange gel (Sephadex) or a Sepharose (Sepharose).
  • the vancomycin filtrate on the upper column needs to be chromatographed under acidic conditions, and alkaline or ammonium salt chromatography is added under alkaline conditions.
  • alkaline or ammonium salt chromatography is added under alkaline conditions.
  • NH 4 + salt and Na + salt such as NaCl, NH 4 . HCO 3 , (NH 4 ) 2 CO 3 , etc.
  • the fraction of vancomycin B collected above 93% during chromatography can make the content of vancomycin B in the mixed effective chromatography solution above 95%.
  • the effective chromatographic solution having the vancomycin chromatographic purity of not less than 95% is subjected to nanofiltration to obtain a 10-20% concentrated solution containing vancomycin hydrochloride (ie, the concentration is 100 mg/ml-200 mg/ml), and the concentrated solution is preferably Store at 2-8 °C.
  • Sodium filtration was carried out using a nanofiltration membrane having a molecular weight of 100-800 Da. Wherein the concentration temperature is ⁇ 20 °C.
  • a reverse phase chromatography column preferably C18 silica gel or a reverse phase polymer filler
  • the silica gel preferably has a particle size of 5 ⁇ m to 60 ⁇ m
  • the polymer such as polystyrene preferably has a particle size of 20 ⁇ m. 40 ⁇ m.
  • ammonium salt is mainly ammonium chloride or ammonium acetate for isocratic gradient elution.
  • concentration of the ammonium salt in the aqueous solution of methanol or ethanol containing an ammonium salt is from 0.1% to 1% by weight.
  • the fraction of vancomycin B content above 98.5% was collected, and the content of vancomycin B component in the mixed eluate was above 99%.
  • concentration temperature is ⁇ 20 °C.
  • the method of the refined dehydration belongs to the conventional prior art.
  • the chromatographic purity of the vancomycin hydrochloride powder obtained by drying is greater than 99%, and the vancomycin hydrochloride powder has a absorbance of less than 0.02 and a whiteness greater than 10% at a wavelength of 450 nm. 88%.
  • the preparation method of the invention has the following advantages: the purity of the final vancomycin hydrochloride is greater than 99% by ion exchange chromatography and reversed phase silica gel chromatography, and the appearance of the product color is greatly improved;
  • the method can achieve the target of vancomycin content greater than 99% only by ion exchange and reversed phase chromatography.
  • the method is simple; the method uses ammonium salt, ethanol or methanol as the mobile phase of reversed phase chromatography, compared with previous patents. It is easier to carry out subsequent treatment and solvent recovery, and can be easily concentrated by nanofiltration membrane, which is convenient to operate; therefore, the process not only improves product quality, but is also suitable for expanded commercial production.
  • Figure 1 shows the crude chromatogram of vancomycin in Example 1, vancomycin B content of 90.2%;
  • Figure 2 is a chromatogram of vancomycin hydrochloride concentrate in Example 1, vancomycin B content of 95.3%;
  • Figure 3 shows the chromatogram of vancomycin hydrochloride in Example 2, vancomycin B content of 99.1%;
  • Figure 4 shows a chromatogram of Example 4-6 vancomycin hydrochloride concentrate, vancomycin B content of 95.8%;
  • Figure 5 is a chromatogram of the finished vancomycin hydrochloride in Example 4, the vancomycin B content is 99.0%;
  • Figure 6 shows the chromatogram of vancomycin hydrochloride in Example 5, vancomycin B content of 99.2%;
  • Figure 7 is a chromatogram of the finished vancomycin hydrochloride in Example 6, the vancomycin B content is 99.0%;
  • Figure 8 shows a chromatogram of Example 7-9 vancomycin hydrochloride concentrate, vancomycin B content of 95.9%;
  • Figure 9 shows a chromatogram of the finished product vancomycin hydrochloride of Example 7, vancomycin B content of 99.0%;
  • Figure 10 shows the chromatogram of the finished vancomycin hydrochloride of Example 8, vancomycin B content of 99.2%;
  • Fig. 11 is a view showing the chromatogram of the finished vancomycin hydrochloride of Example 9, and the vancomycin B content was 99.3%.
  • Example 1 Preparation of vancomycin hydrochloride solution having a vancomycin B content of not less than 95%
  • the NH 4 HCO 3 aqueous solution is pre-washed, the pre-washing flow rate is 1 column volume/hour, the pre-washing volume is 15-20 column volumes, and the vancomycin B content in the eluate is more than 90% at the end of the pre-washing, and then Vancomycin was eluted with 5% (w/v) NH 4 HCO 3 aqueous solution, collected in stages, and the fraction of vancomycin B content greater than 95% was determined by HPLC to be mixed, and then pH was adjusted with 4N hydrochloric acid. 3.1, 5600 ml of effective eluent was obtained at a concentration of 18.6 mg/ml, and the chromatographic purity was 96.5%, see Figure 2.
  • the above effective eluate was desalted and concentrated through a nanofiltration membrane having a molecular weight of 400, wherein 5 times of purified water was added, and finally the permeate conductivity was 1255 ⁇ s/cm, and then the solution was concentrated to obtain vancomycin hydrochloride.
  • a mixed chromatography solution of 12.5 L was obtained at a concentration of 6.8 mg/ml, and pH was adjusted
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 240 mg/ml, the volume was 338 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • Example 3 Concentration effect of different pore size nanofiltration membranes
  • Nanofiltration membrane tubes with different pore sizes were selected, the pore diameters were 100Da, 200Da, 400Da and 800Da, respectively.
  • the filtration area was 0.32m 2 , which was installed on the laboratory small nanofiltration membrane equipment (Model LNG-NF-101).
  • 10% concentrated solution of 10% vancomycin hydrochloride divided into 4 parts, first take one of them for nanofiltration and concentration, the circulating pump pressure is controlled at 10 bar, and the dialysis sample is taken for titer detection at the beginning of concentration, and the flow rate is revealed.
  • the circulating fluid volume is 1000 ml
  • the dialysis sample is taken for titer detection, and the flow rate is revealed.
  • the device is cleaned after each use, and the next nanofiltration membrane tube is replaced, and another concentrated liquid is used for the test.
  • the test results are as follows:
  • nanofiltration membranes with different pore diameters have little effect on the titer of dialysate. Therefore, they are suitable for concentrated vancomycin hydrochloride.
  • membrane tubes with different pore sizes have an effect on the flow rate, and the membrane flow rate with pore diameter greater than 200 Da is suitable.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore diameter of 400, and the concentration of the mixture was concentrated to 150 mg/ml in a volume of 248 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • the membrane was filtered and placed in a freeze dryer for lyophilization to obtain 30.8 g of vancomycin hydrochloride freeze-dried powder, the yield was 62.2%, the chromatographic purity was 99.0% (see Figure 5), and the 10% solution absorbance A was 0.014.
  • the eluent When the absorption value starts to rise rapidly, the eluent is collected, and one bottle is collected every 2.5L, and about 8 bottles are collected to detect each bottle.
  • the content of vancomycin B was more than 98.5%, the mixed chromatogram was obtained, and the concentration was 4.1 mg/ml, and the pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 150 mg/ml in a volume of 265 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 150 mg/ml in a volume of 245 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • the membrane was filtered and placed in a freeze dryer for lyophilization to obtain 29.8 g of vancomycin hydrochloride freeze-dried powder.
  • Example 4 By comparing Example 4, Example 5 and Example 6, it can be found that the reversed-phase chromatography effect of different particle size C18 silica gel fillers has little effect on chromatographic purity, product yield and product absorbance, but for operating pressure, The smaller the particle size, the greater the pressure, so it is preferable to use C18 silica gel of 30 ⁇ m to 60 ⁇ m for mass production.
  • Example 7 Comparison of CH 3 COONH 4 and NH 4 Cl in a mobile phase
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 150 mg/ml, the volume was 252 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • Example 8 Comparison of different ratios of NH 4 Cl in the mobile phase
  • One bottle is collected every 2.5L, and about 9 bottles are collected to detect the vancomycin B content of each bottle.
  • a mixed chromatography solution of 12.5 L was obtained at a concentration of 3.2 mg/ml, and pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 150 mg/ml in a volume of 260 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • One bottle is collected every 2.5L, and about 9 bottles are collected to detect the vancomycin B content of each bottle.
  • a mixed chromatography solution of 12.5 L was obtained at a concentration of 3.1 mg/ml, and pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 150 mg/ml in a volume of 258 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • the membrane was filtered and placed in a freeze dryer for lyophilization to obtain 32.0 g of vancomycin hydrochloride freeze-dried powder, the yield was 63.8%, the chromatographic purity was 99.3% (see Figure 11), and the 10% solution absorbance A was 0.014.
  • Example 5 By comparing Example 5, Example 8 and Example 9, it can be found that different contents of NH 4 Cl (W / V) and 8% aqueous methanol (V / V) as flow relative to product yield, chromatographic components and Absorbance has little effect, but it has an effect on the volume collected.
  • Example 10 Comparison of different pH of upper column liquid and mobile phase
  • One bottle is collected every 2.5L, and about 10 bottles are collected to detect the vancomycin B content of each bottle.
  • a mixed chromatography solution of 12.5 L was obtained at a concentration of 6.3 mg/ml, and pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml in a volume of 380 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • Example 11 Comparison of different pH of upper column liquid and mobile phase
  • One bottle is collected every 2.5L, and about 10 bottles are collected to detect the vancomycin B content of each bottle.
  • a mixed chromatography solution of 15 L was obtained at a concentration of 5.1 mg/ml, and pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml in a volume of 360 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • Example 12 Comparison of different concentrations of CH 3 COONH 4 in the mobile phase
  • the eluent When the absorption value starts to rise rapidly, the eluent is collected, and one bottle is collected every 2.5L, and about 8 bottles are collected to detect each bottle.
  • the content of vancomycin B was more than 98.5%, the mixed chromatogram was obtained, and the concentration was 4.1 mg/ml, and the pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 150 mg/ml in a volume of 260 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • Example 13 Comparison of different concentrations of CH 3 COONH 4 in the mobile phase
  • the eluent When the absorption value starts to rise rapidly, the eluent is collected. One bottle is collected every 2.5L, and about 9 bottles are collected to detect the vancomycin B content of each bottle. In the case where the chromatographic purity was more than 98.5%, a mixed chromatography solution of 12.5 L was obtained at a concentration of 3.2 mg/ml, and pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 150 mg/ml in a volume of 265 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • Example 7 By comparing Example 7, Example 12 and Example 13, it was found that different concentrations of CH 3 COONH 4 (W/V) and 8% aqueous methanol (V/V) were used as flow relative to the finished product content, yield and absorbance. The effect is small, and high concentration CH 3 COONH 4 has an effect on the collection volume.
  • W/V CH 3 COONH 4
  • V/V aqueous methanol
  • Example 14 Comparison of different pH of mobile phase to CH 3 COONH 4 system
  • One bottle is collected every 2.5L, and about 10 bottles are collected to detect vancomycin B per bottle.
  • the content of the chromatographic purity was greater than 98.5%, and a total of 12.5 L of the mixed chromatography solution was obtained at a concentration of 6.2 mg/ml, and the pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution is desolvated through a nanofiltration membrane having a pore diameter of 400, and the permeate is not dissolved.
  • the concentration of the mixture is concentrated, the concentration of the mixture is concentrated to 200 mg/ml, and the volume is 370 ml.
  • Example 15 Comparison of different pH of mobile phase to CH 3 COONH 4 system
  • the eluent When the absorption value starts to rise rapidly, the eluent is collected, and one bottle is collected every 2.5L, and about 10 bottles are collected, and each bottle is tested.
  • the content of vancomycin B was mixed with a chromatographic purity of more than 98.5%, and a total of 12.5 L of a mixed chromatography solution was obtained at a concentration of 6.4 mg/ml, and pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml, the volume was 385 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • the membrane was filtered and placed in a freeze dryer for lyophilization to obtain 63.9 g of vancomycin hydrochloride freeze-dried powder, the yield was 63.0%, the chromatographic purity was 99.0%, and the 10% solution absorbance A was 0.014.
  • Example 16 Comparison of different pH of mobile phase to CH 3 COONH 4 system
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml, the volume was 370 ml, and the concentrate was filtered through 0.45 um.
  • the pH of the upper column and mobile phase can be found by comparing Example 14, Example 15 and Example 16. Control between 3.5 and 4.5 has little effect on the yield and quality of the prepared product.
  • the vancomycin B content of each bottle is detected, and the chromatographic purity is greater than 98.5% for mixing.
  • the chromatographic solution was 12 L at a concentration of 6.4 mg/ml, and pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml, the volume was 365 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • the vancomycin B content of each bottle is detected, and the chromatographic purity is greater than 98.5% for mixing.
  • the chromatographic solution was 12 L at a concentration of 6.2 mg/ml, and the pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml, the volume was 370 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • the membrane was filtered and placed in a freeze dryer for lyophilization to obtain 61.2 g of vancomycin hydrochloride freeze-dried powder, the yield was 60.8%, the chromatographic purity was 99.0%, and the 10% solution absorbance A was 0.0105.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml, the volume was 400 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • One bottle is collected every 2L, and about 10 bottles are collected.
  • the vancomycin B content of each bottle is detected, and the chromatographic purity is greater than 98.5% for mixing.
  • the chromatographic solution was 12 L at a concentration of 6.8 mg/ml, and the pH was adjusted to 2.8 with 4N hydrochloric acid.
  • the above chromatographic solution was desolvated through a nanofiltration membrane having a pore size of 400, and the concentration of the mixture was concentrated to 200 mg/ml in a volume of 380 ml, and the concentrate was filtered through 0.45 ⁇ m.
  • Examples 17-20 were prepared by preparative chromatography using two kinds of polymer reversed phase packings PS, and prepared by using different pH conditions respectively. The results showed that the prepared components, yield and absorbance did not differ greatly, and the effect was small. better.

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Abstract

本发明提供了一种高纯度盐酸万古霉素的分离纯化方法，所述方法包括下列步骤：（1）万古霉素粗品通过离子交换层析得到盐酸万古霉素溶液，并通过纳滤脱盐浓缩得到浓缩液；（2）先用盐酸溶液调节浓缩液，再将调节后的浓缩液通过反向色谱层析柱进行柱层析；（3）收集万古霉素的层析液以得到混合层析液；（4）用盐酸调节混合层析液，然后经纳滤脱盐浓缩脱除溶剂和盐，以得到浓缩液；（5）脱水干燥步骤（4）的浓缩液，或者通过溶剂结晶或盐析结晶以得到色谱纯度达99%，外观纯白的万古霉素干粉。

Description

一种高纯度盐酸万古霉素的分离纯化方法 技术领域
本发明涉及一种高纯度盐酸万古霉素的分离纯化方法。该方法以色谱纯度为90％左右的万古霉素粗品为原料，首先通过以含盐-水为流动相的葡聚糖凝胶柱进行层析，收集色谱纯度大于95％的盐酸万古霉素并进行纳滤脱盐浓缩至100mg/ml-200mg/ml，然后经过反相C18硅胶色谱柱或反相聚合物色谱柱，用含铵盐的甲醇水溶液或乙醇水溶液作为流动相进行洗脱，收集色谱纯度大于98.5％的盐酸万古霉素，再通过纳滤脱去溶剂，然后浓缩至150mg/ml-250mg/ml进行冷冻干燥，得到色谱纯度达到99％，外观纯白的盐酸万古霉素。
背景技术
盐酸万古霉素是放线菌属东方拟无枝酸菌在控制发酵条件下产生的一种两性糖肽类抗生素，其化学式为C66H75C12N9O24·HCl，分子量为1.486。已知盐酸万古霉素与黏肽的前质末端D-Ala-D-Ala结合，抑制细菌细胞壁的合成。此外，盐酸万古霉素还可以改变细胞膜的渗透性和RNA的合成。盐酸万古霉素特别用于由抗β-乳胺抗生素的葡萄球菌引起的严重或重症感染的初步治疗，也用于治疗对青霉素过敏或使用青霉素和头孢菌素没有效果的患者。早期有文献报道认为盐酸万古霉素有严重的肾、耳毒性，故在临床上未被广泛使用，但近来由于抗生素的大量使用，导致临床上耐甲氧西林金葡菌(MASA)感染病例日益增多，使得盐酸万古霉素的使用量逐年增长。因此，降低盐酸万古霉素的使用毒性，增加用药的安全性变得非常重要，而其中提高药用盐酸万古霉素的纯度是其中的一种有效的手段。近年来，随着科学技术的不断发展，技术人员不断的提高了盐酸万古霉素的纯度，过去认为盐酸万古霉素所具有的严重的肾、耳毒性在不断的减少。基于以上原因，开发一种更高纯度、简易可行、适于工业化生产的盐酸万古霉素精制工艺有着十分重要的意义。
万古霉素分子由两个基本结构组成，即糖基部分α-o-vancosamine-β-o-glucosyl和肽基部分中心七肽核，其结构决定了它的不稳定性，万古霉素分子在酸碱或高温条件下会降解产生降解产物，同时结构还带有多个游离酚羟基，导致其易氧化成醌式。已有文献报道了，万古霉素在酸性条件和高温条件下会发生水解，脱去 一个糖基或二个糖基生成单糖万古霉素或无糖万古霉素(desvancosaminyl vancomycin and aglucovancomycin)，在弱酸环境下，也可能脱去一个酰胺基降解成去氨基万古霉素，它有两个异构体。基于万古霉素化学结构的这些特点，给制造高纯度盐酸万古霉素带来了一定的困难。
盐酸万古霉素产品开发已有几十年的历史，早期的制备工艺普遍采用如甲醇、乙醇、异丙醇、丙酮等溶剂进行结晶，也有通过加入氯化铵或氯化钠进行盐析法沉淀的方式制备成品，但由于万古霉素发酵液中存在较多的杂质，尤其是万古霉素结构类似物较多，使得纯度普遍不高，要达到色谱纯度大于93％的欧洲药典标准有一定的难度。
近些年随着各种介质的色谱分离技术的发展，该技术在盐酸万古霉素分离纯化上得到了广泛的应用。CN200710187300.5采用离子交换填料葡聚糖凝胶Sephadex CM-25、琼脂糖SP Sepharose或琼脂糖CM Sepharose进行色谱层析，流动相为4-6％的碳酸氢铵，得到色谱纯度大于95％，小于98％的盐酸万古霉素，再通过加入氯化钠溶液进行盐析沉淀，分离并用乙醇顶洗，烘干得到盐酸万古霉素成品。该工艺能得到纯度较高的盐酸万古霉素霉素，回收率不高，另外离子交换色谱方法对于去除色素不太理想，因此该工艺产品在外观颜色和溶液吸光度上不是十分理想。
专利WO2006061166中的实例采用粒径5um的反相硅胶(Octadecyl silica gel)作为色谱介质，含5mM醋酸铵且pH调节为4.0的3％甲醇溶液作为流动相，在流动相中加入2％的戊醇作为解析剂，收集色谱纯度大于97.5％的盐酸万古霉素，然后通过真空浓缩收集液浓度至140mg/ml，加入甲醇，用氨水调节pH8.5-9.0，冷却料液温度至0度，分离沉淀物，并用甲醇顶洗，将分离物用水溶解并调节pH至3.2，然后用异丙醇进行结晶，真空烘干，最终得到纯度达到97-99.3％的盐酸万古霉素成品。该专利所涉及的工艺存在一些缺陷，尤其对于商业化扩大生产有一定的难度，首先，该方法中流动相和洗脱剂使用了不同溶剂，对于溶剂回收存在一定的困难；其次，由于该工艺通过反相纯化，色谱纯度并不能一次性达到99％以上，因此需要有两步溶剂结晶操作，最后通过真空干燥获得成品，而溶剂结晶存在的普遍问题是残留溶剂不能有效去除，达不到ICH对残留溶剂的要求；最后，该方法由于步骤较多，在产品收率上并不理想。
因此，从目前的文献资料看，由于存在种种的缺陷，市面上还没有一种制备高纯度(色谱纯度达到99％以上)的适用于商业化生产的盐酸万古霉素生产方法，而鉴于盐酸万古霉素用药安全性，需要一种制备高纯度盐酸万古霉素的方法。
发明内容
本发明提供了一种高纯度(色谱纯度达到99％以上)、低杂质、高效率的适用于商业化生产的盐酸万古霉素的分离纯化方法，所述分离纯化方法包括如下步骤：(1)万古霉素粗品通过离子色谱交换层析法制备得到盐酸万古霉素溶液，并通过纳滤脱盐浓缩，得到第一盐酸万古霉素浓缩液；(2)用盐酸溶液调节第一盐酸万古霉素浓缩液pH＝3.5-4.5，再将调节后的第一盐酸万古霉素浓缩液通过反相色谱层析柱进行柱层析，其中，固定相为C18硅胶或聚苯乙烯聚合物，流动相为含有铵盐的甲醇水溶液或乙醇水溶液；(3)收集万古霉素B含量大于98.5％的层析液；(4)用盐酸调节上述层析液pH＝2.5-3.5，再用纳滤浓缩脱除溶剂和盐，以得到第二盐酸万古霉素浓缩液；以及(5)将步骤(4)的第二盐酸万古霉素浓缩液精制脱水以得到色谱纯度达到99％，纯白的盐酸万古霉素粉。
优选地，步骤(1)中所述盐酸万古霉素溶液为通过离子交换色谱层析法获得的万古霉素色谱纯度大于95％的盐酸万古霉素溶液。上述制备万古霉素色谱纯度不低于95％的盐酸万古霉素溶液是通过如下现有技术方法制得。(1)首先，按照中国专利CN01132048.6所述的方法，采用发酵菌种为东方拟无枝酸(Amycolatopsis Oriertalis)SIPI43491，经过接种体的培养，接种至一级种子罐，经二级种子扩大培养后，进入发酵罐培养，控制温度24-34℃，培养压力0.01-0.08MPa，过程中控制溶解氧和pH，发酵周期为4-6天，得到万古霉素发酵液。(2)其次，按照中国专利CN200710198599.4所述，将万古霉素发酵液通过大孔吸附树脂，用含乙醇的酸性水溶液将万古霉素洗脱，然后在洗脱液中加入活性炭脱色，在脱色液中加入碳酸氢铵，并用氨水调节pH至7.5-8.5搅拌静置后分离得到色谱纯度不低于80％的万古霉素粗品(该万古霉素粗品指的是步骤(1)中的万古霉素粗品)。(3)万古霉素粗品在纯化水里溶解后用0.01～0.5μm孔径的陶瓷膜过滤，获得澄清的万古霉素滤液。澄清万古霉素滤液经过离子交换层析柱层析纯化得到万古霉素B含量95％以上的有效层析液(即，万古霉素色谱纯度不低于95％的盐酸万古霉素溶液)。这里，所说的离子交换层析柱所用填料阳离子交换葡聚糖凝胶(Sephadex)或琼脂糖凝胶(Sepharose)。上柱的万古霉素滤液需在酸性条件下上层析柱，在碱性条件下加入碱性金属盐或铵盐层析，一般我们采用NH4 +盐和Na+盐，如NaCl、NH4HCO3、(NH4)2CO3等；层析时收集万古霉素B含量在93％以上的部分可以使混合有效层析液中万古霉素B含量在95％以上。
该万古霉素色谱纯度不低于95％的有效层析液经过纳滤后得到含盐酸万古霉素10-20％浓缩液(即，浓度为100mg/ml-200mg/ml)，该浓缩液优选在2-8℃下保存。采用分子量为100-800Da的纳滤膜进行钠滤。其中，所述浓缩的温度为≤20℃。
将上述浓缩液用4N盐酸溶液调节pH＝3.5-4.5经过反相色谱柱优选C18硅胶或反相聚合物填料，硅胶粒径优选为5μm-60μm，聚合物如聚苯乙烯粒径优选为20μm-40μm。以甲醇或乙醇作为流动相并加入铵盐作为调节pH的缓冲剂，优选用盐酸或醋酸将调节pH＝3.5-5.5，这里优选的铵盐主要为氯化铵或醋酸铵进行等度梯度洗脱，其中，在含有铵盐的甲醇水溶液或乙醇水溶液中所述铵盐的浓度在0.1％-1wt％。收集万古霉素B含量在98.5％以上的部分，并使得混合洗脱液中万古霉素B组分含量在99％以上。
将混合洗脱液用4N的盐酸进行酸化调节pH＝2.5-3.5，然后经过纳滤脱除溶剂和盐，优选地，采用分子量为100-800Da的纳滤膜进行钠滤。其中，所述浓缩的温度为≤20℃。再浓缩得到含盐酸万古霉素15-25％浓缩液，放入冷冻干燥机或喷雾干燥机进行精制脱水(其中，精制脱水的方法为冷冻干燥、或喷雾干燥法、盐析结晶法或溶剂结晶法，所述精制脱水的方法属于常规现有技术)干燥得到盐酸万古霉素粉的色谱纯度大于99％，该盐酸万古霉素粉在10％浓度，波长450nm下，吸光度小于0.02，白度大于88％。
本发明的制备方法相比以往的专利有如下优势：通过离子交换层析和反相硅胶色谱层析，使得最终的盐酸万古霉素纯度大于99％，产品颜色外观得到了很大的提升；本方法仅通过离子交换和反相两部层析即可达到万古霉素含量大于99％的目标，方法简易；本方法采用铵盐、乙醇或甲醇作为反相色谱的流动相，与以往专利相比，更易于后续的处理和溶剂的回收，可以通过纳滤膜简单的进行浓缩处理，操作方便；因此该工艺不仅提高了产品品质，而且适合于扩大的商业化生产。
附图说明
图1表示实施例1中万古霉素粗品色谱图，万古霉素B含量90.2％；
图2表示实施例1中盐酸万古霉素浓缩液色谱图，万古霉素B含量95.3％；
图3表示实施例2中盐酸万古霉素成品色谱图，万古霉素B含量99.1％；
图4表示实施例4-6盐酸万古霉素浓缩液色谱图，万古霉素B含量95.8％；
图5表示实施例4中盐酸万古霉素成品色谱图，万古霉素B含量99.0％；
图6表示实施例5中盐酸万古霉素成品色谱图，万古霉素B含量99.2％；
图7表示实施例6中盐酸万古霉素成品色谱图，万古霉素B含量99.0％；
图8表示实施例7-9盐酸万古霉素浓缩液色谱图，万古霉素B含量95.9％；
图9表示实施例7盐酸万古霉素成品色谱图，万古霉素B含量99.0％；
图10表示实施例8盐酸万古霉素成品色谱图，万古霉素B含量99.2％；
图11表示实施例9盐酸万古霉素成品色谱图，万古霉素B含量99.3％。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
通过以下实施例来对本发明作进一步具体说明，但并不仅限于以下实施例和实施例中的工艺参数范围。
实施例1：制备万古霉素B含量不低于95％的盐酸万古霉素溶液
在烧杯中将万古霉素粗品250g溶于2.0L的纯化水中，搅拌充分，待完全溶解后用孔径为0.2um的滤膜过滤，然后加水稀释，最后得到盐酸万古霉素粗品溶解液3.0L，浓度为43.6mg/ml，色谱纯度为90.2％，参见图1。
取上述盐酸万古霉素粗品溶解液3000ml，通过装有葡聚糖Sephadex CM-25的8cm*60cm的玻璃层析柱，上柱量为层析柱体积的4％左右，吸附方法为将上柱液和1/5的层析填料混合，然后将混合液直接上柱，再用纯化水以1.5倍柱体积/小时的流速冲洗，洗涤6倍柱体积后，用0.3％(w/v)的NH4HCO3水溶液进行预洗，预洗流速为1倍柱体积/小时，预洗体积为15-20倍柱体积，预洗结束时洗脱液中万古霉素B含量大于90％以上，然后用5％(w/v)NH4HCO3水溶液将万古霉素洗脱下来，分段收集，用HPLC检测万古霉素B的含量大于95％的部分进行混合，然后用4N的盐酸调节pH＝3.1，得到有效洗脱液5600ml，浓度为18.6mg/ml，色谱纯度为96.5％，参见图2。
然后将上述有效洗脱液通过孔径为400分子量的纳滤膜进行脱盐和浓缩，其中加入5次纯化水，最后透出液电导率为1255μs/cm,然后将料液浓缩以得到盐酸万古霉素浓缩液的浓度156mg/ml，pH＝3.8，色谱纯度为95.3％(见图2)，体积680ml，所述浓缩液在2-8℃下保存以备用。
实施例2：制备高纯度盐酸万古霉素成品
取例1中制备的浓缩液680ml，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用盐酸调节pH＝4.0，含0.2％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约10瓶，检测每瓶的万古霉 素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为6.8mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至240mg/ml，体积338ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品68.5g，收率为64.6％，色谱纯度99.1％(见图3)，10％溶液吸光度A450nm＝0.012。
实施例3：不同孔径纳滤膜浓缩效果对比
选取不同孔径的纳滤膜管，孔径分别为100Da、200Da、400Da和800Da，过滤面积均为0.32m2，轮流安装在实验室小型纳滤膜设备上(型号LNG-NF-101)，取含10％盐酸万古霉素的浓缩液8000ml，分成4份，先取其中一份进行纳滤浓缩，循环泵压力控制在10bar，浓缩开始时取透析样进行效价检测，并记录透出流速，待浓缩至循环液体积为1000ml时结束取透析样进行效价检测，并记录透出流速，每次使用完后清洗设备，并更换下一根纳滤膜管，使用另一份浓缩液进行试验。试验结果如下：
Figure PCTCN2014000950-appb-000001
如上表，孔径不同的纳滤膜对于透析液效价影响不大，因此对于浓缩盐酸万古霉素都适用，但不同孔径的膜管对于流速有影响，孔径大于200Da的膜管流速较为合适。
实施例4：不同粒径C18硅胶对比
取浓缩液330ml，浓度150mg/ml，色谱纯度95.8％(见图4)，用盐酸或氢氧化钠溶液调节pH＝4.0，通过装有粒径为5μm的C18硅胶填料的制备柱 (15cm*30cm)，用盐酸调节pH＝4.0，含0.1％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约8瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液10L，浓度为3.8mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积248ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品30.8g，收率为62.2％，色谱纯度99.0％(见图5)，10％溶液吸光度A＝0.014。
实施例5：不同粒径C18硅胶对比
取浓缩液330ml，浓度150mg/ml，色谱纯度95.8％，用盐酸或氢氧化钠溶液调节pH＝4.0，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用盐酸调节pH＝4.0，含0.1％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约8瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液10L，浓度为4.1mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积265ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品32.5g，色谱纯度99.2％(见图6)，收率65.6％，10％溶液吸光度A＝0.015。
实施例6：不同粒径C18硅胶对比
取浓缩液330ml，浓度150mg/ml，色谱纯度95.8％，用盐酸或氢氧化钠溶液调节pH＝4.0，通过装有粒径为60μm的C18硅胶填料的制备柱(15cm*30cm)，用盐酸调节pH＝4.0，含0.1％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液， 每2.5L收集一瓶，共收集约8瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液10L，浓度为3.8mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积245ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品29.8g，色谱纯度99.0％(见图7)，收率为60.2％，10％溶液吸光度A＝0.012。
通过对比实施例4、实施例5和实施例6可以发现，不同粒径的C18硅胶填料反相层析效果在色谱纯度、产品收率及产品吸光度方面影响不大，但对于操作压力来说，粒径越小压力越大，因此适用于大规模生产最好选用30μm-60μm的C18硅胶。
实施例7：流动相中CH3COONH4和NH4Cl的对比
取浓缩液330ml，浓度152mg/ml，色谱纯度95.9％(见图8)，用盐酸调节pH＝4.0，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用醋酸调节pH＝4.0，含0.1％的CH3COONH4(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约8瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液10L，浓度为4.0mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积252ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品31.1g，色谱纯度99.0％(见图9)，10％溶液吸光度A＝0.017。
通过和实施例5对比可以发现，用含有NH4Cl的流动相组分和收率与用含有CH3COONH4的流动相区别不大。
实施例8：流动相中不同比例NH4Cl对比
取浓缩液330ml，浓度152mg/ml，色谱纯度95.9％，用盐酸调节pH＝4.0，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用盐酸调节pH＝4.0，含0.5％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim， 流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约9瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为3.2mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积260ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品32.1g，收率为64.0％，色谱纯度99.2％(见图10)，10％溶液吸光度A＝0.016。
实施例9：流动相中不同比例NH4Cl对比
取浓缩液330ml，浓度152mg/ml，色谱纯度95.9％，用盐酸调节pH＝4.0，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用盐酸调节pH＝4.0，含1％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约9瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为3.1mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积258ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品32.0g，收率为63.8％，色谱纯度99.3％(见图11)，10％溶液吸光度A＝0.014。
通过对比实施例5、实施例8和实施例9可以发现，不同含量的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相对于产品收率、色谱组分及吸光度影响不大，但对于收集的体积会有影响。
实施例10：上柱液和流动相不同pH对比
取浓缩液670ml，浓度146mg/ml，色谱纯度96.4％，用盐酸调节pH＝3.5，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用盐酸调节pH＝3.5，含0.5％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一 瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为6.3mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积380ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品63.1g，收率为64.5％，色谱纯度99.2％，10％溶液吸光度A＝0.015。
实施例11：上柱液和流动相不同pH对比
取浓缩液670ml，浓度146mg/ml，色谱纯度96.4％，用盐酸调节pH＝4.5，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用盐酸调节pH＝4.5，含0.5％的NH4Cl(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液15L，浓度为5.1mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积360ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品58.2g，收率为59.5％，色谱纯度99.0％，10％溶液吸光度A＝0.018。
通过对比实施例7、实施例10和实施例11可以发现，上柱液和流动相pH＝3.5和pH＝4.0的产品收率和组分影响不大，上柱液pH＝4.5会导致收集的体积增大，收率和组分略有影响。
实施例12：流动相中不同浓度的CH3COONH4对比
取浓缩液330ml，浓度154mg/ml，色谱纯度95.9％，用盐酸或氢氧化钠调节pH＝4.0，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用醋酸调节pH＝4.0，含0.5％的CH3COONH4(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约8瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液10L，浓度为4.1mg/ml，用4N的盐酸调 节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积260ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品32.5g，收率为64.0％，色谱纯度99.1％，10％溶液吸光度A＝0.013。
实施例13：流动相中不同浓度的CH3COONH4对比
取浓缩液330ml，浓度154mg/ml，用盐酸或氢氧化钠调节pH＝4.0，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用醋酸调节pH＝4.0，含1％的CH3COONH4(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约9瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为3.2mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至150mg/ml，体积265ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品33.0g，收率65.0％，色谱纯度99.0％，10％溶液吸光度A＝0.015。
通过对比实施例7、实施例12和实施例13可以发现，不同浓度的CH3COONH4(W/V)和8％的甲醇水溶液(V/V)作为流动相对于成品含量、收率及吸光度影响不大，高浓度CH3COONH4对于收集体积有影响。
实施例14：流动相为CH3COONH4体系下不同pH的对比
取浓缩液670ml，浓度145mg/ml，色谱纯度96.6％，用盐酸调节pH＝3.5，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用醋酸调节pH＝3.5，含0.5％的CH3COONH4(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为6.2mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶 剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积370ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品60.6g，收率为62.4％，色谱纯度99.2％，10％溶液吸光度A＝0.016。
实施例15：流动相为CH3COONH4体系下不同pH的对比
取浓缩液650ml，浓度156mg/ml，色谱纯度95.7％，用盐酸或氢氧化钠调节pH＝4.0，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用醋酸调节pH＝4.0，含0.5％的CH3COONH4(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为6.4mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积385ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品63.9g，收率为63.0％，色谱纯度99.0％，10％溶液吸光度A＝0.014。
实施例16：流动相为CH3COONH4体系下不同pH的对比
取浓缩液670ml，浓度145mg/ml，色谱纯度96.6％，用氢氧化钠调节pH＝4.5，通过装有粒径为30μm的C18硅胶填料的制备柱(15cm*30cm)，用醋酸调节pH＝4.5，含0.5％的CH3COONH4(W/V)和8％的甲醇水溶液(V/V)作为流动相进行预洗100mim，流速为5BV/h，然后将流动相中甲醇比例提高至12％进行洗脱，流速为5BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2.5L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12.5L，浓度为6.1mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积370ml，然后将浓缩液经过0.45um滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品61.0g，收率为62.8％，色谱纯度99.1％，10％溶液吸光度A＝0.018。
通过对比实施例14、实施例15和实施例16可以发现，上柱液和流动相的pH 控制在3.5-4.5之间，对于制备产品的收率和质量影响不大。
实施例17：聚合物填料PS树脂反相制备实施例
取浓缩液680ml，浓度148mg/ml，色谱纯度95.9％，用盐酸调节pH＝4.0，通过装有粒径为20μm的PS吸附树脂的制备柱(15cm*30cm)，用盐酸调节pH＝4.0，5％的乙醇水溶液(V/V)作为流动相进行预洗60mim，流速为2BV/h，然后将流动相中乙醇比例提高至10％进行洗脱，流速为2BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12L，浓度为6.4mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积365ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品60.6g，收率为60.2％，色谱纯度99.3％，10％溶液吸光度A＝0.016。
实施例18：聚合物填料PS树脂反相制备实施例
取浓缩液680ml，浓度148mg/ml，色谱纯度95.9％，用盐酸调节pH＝3.5，通过装有粒径为20μm的PS吸附树脂的制备柱(15cm*30cm)，用盐酸调节pH＝3.5，5％的乙醇水溶液(V/V)作为流动相进行预洗60mim，流速为2BV/h，然后将流动相中乙醇比例提高至10％进行洗脱，流速为2BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12L，浓度为6.2mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积370ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品61.2g，收率为60.8％，色谱纯度99.0％，10％溶液吸光度A＝0.015。
实施例19：聚合物填料PS树脂反相制备实施例
取浓缩液650ml，浓度158mg/ml，色谱纯度96.2％，用盐酸调节pH＝3.5，通过装有粒径为40μm的PS吸附树脂的制备柱(15cm*30cm)，用盐酸调节pH＝3.5， 5％的乙醇水溶液(V/V)作为流动相进行预洗60mim，流速为2BV/h，然后将流动相中乙醇比例提高至10％进行洗脱，流速为2BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12L，浓度为7.1mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积400ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品64.7g，收率63.0％，色谱纯度99.0％，10％溶液吸光度A＝0.012。
实施例20：聚合物填料PS树脂反相制备实施例
取浓缩液650ml，浓度158mg/ml，色谱纯度96.2％，用盐酸调节pH＝4.0，通过装有粒径为40μm的PS吸附树脂的制备柱(15cm*30cm)，用盐酸调节pH＝4.0，5％的乙醇水溶液(V/V)作为流动相进行预洗60mim，流速为2BV/h，然后将流动相中乙醇比例提高至10％进行洗脱，流速为2BV/h，在线检测波长λ＝280，待吸收值开始快速上升时，开始收集洗脱液，每2L收集一瓶，共收集约10瓶，检测每瓶的万古霉素B含量情况，色谱纯度大于98.5％的进行混合，共得到混合层析液12L，浓度为6.8mg/ml，用4N的盐酸调节pH＝2.8。
将上述层析液通过孔径为400分子量的纳滤膜进行脱溶剂，待透出液没有溶剂产生则开始浓缩，将混合液浓度浓缩至200mg/ml，体积380ml，然后将浓缩液经过0.45μm滤膜进行过滤，放入冷冻干燥机进行冻干，得到盐酸万古霉素冻干粉成品63.5g，收率61.8％，色谱纯度99.1％，10％溶液吸光度A＝0.018。
实施例17-20，使用两种规格的聚合物反相填料PS进行了制备层析，并分别利用不同的pH条件进行制备，结果显示制备的成品组分、收率及吸光度差异不大，效果较好。
需要说明的是，上述发明内容及具体实施方式意在证明本发明所提供技术方案的实际应用，不应解释为对本发明保护范围的限定。本领域技术人员在本发明的精神和原理内，当可作各种修改、等同替换、或改进。本发明的保护范围以所附权利要求书为准。

Claims (10)

  1. 一种高纯度盐酸万古霉素的分离纯化方法，所述方法包括下列步骤：
    (1)万古霉素粗品通过离子交换色谱层析法制备得到盐酸万古霉素溶液，并通过纳滤脱盐浓缩，得到第一盐酸万古霉素浓缩液；
    (2)用盐酸溶液调节第一盐酸万古霉素浓缩液pH＝3.5-4.5，再将调节后的第一盐酸万古霉素浓缩液通过反相色谱层析柱进行柱层析，其中，固定相为C18硅胶或聚苯乙烯聚合物，流动相为含有铵盐的甲醇水溶液或乙醇水溶液；
    (3)收集万古霉素B含量大于98.5％的层析液；
    (4)用盐酸调节上述层析液pH＝2.5-3.5，再用纳滤浓缩脱除溶剂和盐，以得到第二盐酸万古霉素浓缩液；以及
    (5)将步骤(4)的第二盐酸万古霉素浓缩液精制脱水以得到色谱纯度达到99％，纯白的盐酸万古霉素粉。
  2. 如权利要求1所述的分离纯化方法，其中，步骤(1)中所述盐酸万古霉素溶液为通过离子交换色谱层析法获得的万古霉素色谱纯度大于95％的盐酸万古霉素溶液。
  3. 如权利要求1所述的分离纯化方法，其中，步骤(1)中盐酸万古霉素浓缩液的浓度为100mg/ml-200mg/ml。
  4. 如权利要求1所述的分离纯化方法，其中，步骤(2)中所述C18硅胶的粒径为5-60tm，聚苯乙烯聚合物的粒径为20-40μm。
  5. 如权利要求1所述的分离纯化方法，其中，步骤(2)中所用的溶剂为甲醇或乙醇。
  6. 如权利要求1所述的分离纯化方法，其中，步骤(2)中所述铵盐为氯化铵或醋酸铵。
  7. 如权利要求1所述的分离纯化方法，其中，在含有铵盐的甲醇水溶液或乙醇水溶液中所述铵盐的浓度在0.1％-1wt.％。
  8. 如权利要求1所述的分离纯化方法，其中，步骤(2)中所述流动相采用盐酸或醋酸调节pH至3.5-5.5。
  9. 如权利要求1所述的分离纯化方法，其中，步骤(4)中采用分子量为100-800Da的纳滤膜进行钠滤。
  10. 如权利要求1所述的分离纯化方法，其中，步骤(5)中制备的盐酸万古霉素粉在10wt.％浓度，波长450nm下，吸光度小于0.02，白度大于88％，制备的盐酸万古霉素粉的色谱纯度达到99％以上。
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CN114989262A (zh) * 2022-07-06 2022-09-02 丽珠集团福州福兴医药有限公司 一种万古霉素的提纯方法
CN116754534A (zh) * 2023-08-09 2023-09-15 长春中医药大学 一种快速检测金属离子锡以及碱性物质的方法和应用
CN116754534B (zh) * 2023-08-09 2023-10-27 长春中医药大学 一种快速检测金属离子锡以及碱性物质的方法和应用

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CN104610434B (zh) 2019-06-07
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CN104610434A (zh) 2015-05-13
US20160264620A1 (en) 2016-09-15
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KR20160123284A (ko) 2016-10-25
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