CN101613396B - Non-crystalline form Lyphocin (Fujisawa) and its production and use and its pharmaceutical composition - Google Patents
Non-crystalline form Lyphocin (Fujisawa) and its production and use and its pharmaceutical composition Download PDFInfo
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Abstract
The invention provides a kind of high density non-crystalline form Lyphocin (Fujisawa) and preparation method thereof and this Lyphocin (Fujisawa) for the purposes of oral or injection administration and the pharmaceutical composition containing this Lyphocin (Fujisawa).Described Lyphocin (Fujisawa) is non-crystalline form, and it has the feature essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1.The Lyphocin (Fujisawa) active princlple obtained by the present invention is improved significantly, other impurity is substantially reduced, purity is the highest, chromatographic purity can reach more than 95%, can be used not only for injection, can be additionally used in the solid preparations such as capsule, product appearance color obtains remarkable improvement simultaneously, is suitable to oral or injection administration.
Description
Technical field
The present invention relates to a kind of Lyphocin (Fujisawa), specifically, relate to a kind of high density without knot
Crystal formation state Lyphocin (Fujisawa) and preparation method thereof and this Lyphocin (Fujisawa) are used for being administered orally or noting
Penetrate the purposes of administration and the pharmaceutical composition containing this Lyphocin (Fujisawa).
Background technology
Lyphocin (Fujisawa) is the one two that Amycolatopsis orientais produces under controlling fermentation condition
Property glycopeptide antibiotics, its chemical formula is C66H75C12N9O24HCl, molecular weight is Isosorbide-5-Nitrae 86.
By fermentative microorganism Amycolatopis orientalis (Nocardia orientalis in the past)
Prepare the method for vancomycin in United States Patent (USP) 3,067,099 (nineteen fifty-five application) and
More detailed description in WO91/08,300.The lyophilized powder of Lyphocin (Fujisawa) presents off-white
Color, its aqueous solution is clear solution, and pH is 2.5-4.5.
Clinically, Lyphocin (Fujisawa) begins with commercial distribution from late 1950s.?
Know that Lyphocin (Fujisawa) is combined with mucopeptide antecedent end D-Ala-D-Ala, suppress bacterial cell
The synthesis of wall.Additionally, Lyphocin (Fujisawa) can also change cell permeability of the membrane and RNA
Synthesis.
It is tight that Lyphocin (Fujisawa) is used in particular for being caused by the staphylococcus of anti-β-lactam antibiotic
Weight or the Primary treatment of severe infection, be also used for treating allergic to penicillin or use penicillin and
The ineffective patient of cephalosporin.The Lyphocin (Fujisawa) of commercial distribution have oral (solution and
Capsule/bullet type capsule) and injection (the sterilization intravenous solution of bottle) form.
Lyphocin (Fujisawa) is used alone or is used in combination can treat with other glucosaminide
The endocarditis caused by staphylococcus, streptococcus, enterococcus or diphtheria corynebacterium.
The enterocolitis only caused staphylococcus at injection Lyphocin (Fujisawa) is not imitated
In the case of Guo, just allow oral hydrochloride vancomycin.Only vancomycin hydrochloride for injection is fitted
For other all of indication.
At present Lyphocin (Fujisawa) the most clinically is all to be that mouth prepared by raw material with lyophilized powder
Clothes and injection type, in the capsule formulation of peroral dosage form, because the density of lyophilized powder is smaller,
Prepare capsule particle relatively big, be difficult to take.
Summary of the invention
The technical problem to be solved is to provide the salt of a kind of high density non-crystalline form
Acid vancomycin and preparation method thereof and application pharmaceutically, not only can also be used with injection
In oral, it is particularly applicable to capsule.
According to an aspect of the present invention, the invention provides a kind of non-crystalline form hydrochloric acid the most mould
Element, it is characterised in that described Lyphocin (Fujisawa) is non-crystalline form, and it has and accompanying drawing 1
The feature that shown x-ray powder crystal diffraction collection of illustrative plates is essentially identical.Described hydrochloric acid is the most mould
The X-ray diffracting spectrum of element does not contains any peak shape.The bulk density of described Lyphocin (Fujisawa)
Being 0.45～0.65g/ml, compactness is 0.65～0.85g/ml.
According to a further aspect in the invention, present invention also offers a kind of pharmaceutical composition, described
Pharmaceutical composition includes the non-crystalline form Lyphocin (Fujisawa) and pharmaceutically of therapeutically effective amount
Acceptable carrier, excipient, diluent or adjuvant.Wherein, the controlling of this Lyphocin (Fujisawa)
Treating effective dose is 0.5～5g.
Wherein, this kind of pharmaceutical composition can be made into injection and be administered, it is possible to make oral formulations to
Medicine.Injection is made predominantly into freeze-dried powder injection, pharmaceutic adjuvant used include glucose,
The polyalcohols such as sucrose, lactose, fructose, mannitol, xylitol make excipient, also include 5
Diluent during clinical administration made by % glucose injection and 0.9% sodium chloride injection.Injection
Agent is mainly used in treating the infection that the staphylococcus to methicillin resistance causes, to penicillin mistake
Quick patient and other antibiotic can not be used to include penicillin, cephalosporins, or after using
The infected patient such as staphylococcus, enterococcus and the corynebacterium failed to respond to any medical treatment, diphtheroid genus,
Such as endocarditis, osteomyelitis, septicemia or soft tissue infection etc. it can also be used to preventing and treating blood is saturating
The plasma viscosity shunt infection caused by staphylococcus that analysis patient occurs.Oral formulations is main
Making tablet and capsule, pharmaceutic adjuvant used includes excipient and filler such as microcrystalline cellulose
Element, hydroxypropylcellulose, starch, lactose, dextrin etc., binding agent such as PVP K30, hydroxypropyl
Methylcellulose etc., disintegrating agent such as carboxymethylstach sodium, cross-linking sodium carboxymethyl cellulose, the poly-dimension of crosslinking
The such as micropowder silica gel, magnesium stearate of ketone etc., fluidizer or lubricant, stearic acid, calcium stearate etc..
It is invalid that this product is administered orally for treating the metronidazole treatment caused by long-term taking broad ectrum antibiotic
Difficulty distinguish the pseudomembranous colitis or staphylococcal enteritis that bacillus fusiformis causes.
In accordance with a further aspect of the present invention, present invention also offers the system of described Lyphocin (Fujisawa)
Preparation Method, it is characterised in that comprise the following steps:
(1) there is vancomycin chromatographic purity be not less than the vancomycin crude product of 80% and containing
NH4HCO3The chromatographic column of flowing phase carries out column chromatography, obtains chromatographic purity and be not less than 93%
Effective chromatographic solution or effect chromatographic solution；
(2) add carbonate to effective chromatographic solution or effect chromatographic solution, produce precipitation；
(3) use ethanol to carry out top to wash, be dried to obtain Lyphocin (Fujisawa).
Preferably, described effective chromatographic solution can get the dense of effective chromatographic solution after ultrafiltration, nanofiltration
Contracting liquid, to produce precipitation by carbonate.Wherein, the reflux temperature of described ultrafiltration controls
10～20 DEG C, and wash filtering residue by purified water, 10000Da～50000Da selected by ultrafilter membrane.
Preferably, the concentrated solution of described effective chromatographic solution or effective chromatographic solution by 0.45 μm and
Precipitate again after the filter element filtering of 0.2 μm.
The temperature of described nanofiltration solution controls at 10～20 DEG C, and substep add purified water and
The ethylenediaminetetraacetic acid (W/bons) of 0.04%～0.06%, NF membrane selects 200Da～600Da.
Wherein, described carbonate includes (NH4)2CO3, NH4HCO3, Na2CO3With
NaHCO3.Preferably, in the concentrated solution of effective chromatographic solution or effective chromatographic solution, carbonate is added
Obtained carbonate concentration is 3%～7% (W/V).
Wherein, after adding carbonate, regulate pH=2.0～3.5, be stirred continuously 30～90 minutes,
Mixeding liquid temperature controls at 5～25 DEG C, and stands 24～48 hours.
Preferably, using the ethanol water of 90% repeatedly to push up and wash, top is washed till electrical conductivity
Stop adding the ethanol water of 90% during≤1000 μ s/cm.
Preferably, the chromatography media of employing is polydextran gel Sephadex CM-25, agarose
SP Sepharose or agarose CM Sepharose, flowing is 4～6% (w/v) mutually
NH4HCO3.Wherein, flowing is 0.5～1.5 column volumes/hour carry out eluting with flow.
Preferably, before being additionally included in column chromatography, vancomycin crude product is dissolved in pure water, stirring,
Adjust to pH=9～10 by 2～6mol/L NaOH solution, filter until completely dissolved, collect
Filtrate, with 2～6mol/L HCl solution adjustment filtrates to pH=4.5～6.0.
Preferably, it is additionally included in containing NH4HCO3Before flowing phase eluting, first with pure water and low
The NH of concentration4HCO3Solution carries out prewashing.
According to last aspect of the present invention, the invention provides a kind of Lyphocin (Fujisawa) for
The purposes of oral or injection administration.
Technological process provided by the present invention is described in detail below.
Vancomycin fermentation liquid filters in the basic conditions, and the clear filtrate obtained is inhaled by macropore
Attached resin, vancomycin is attracted to resin, water-soluble by the acidity containing solvent after resin washing
Liquid eluting, typical eluent is the aqueous hydrochloric acid solution containing ethanol.Vancomycin is eluted,
Eluent containing vancomycin adds activated carbon decolorizing, and the filtrate being filtrated to get is concentrated to give salt
Acid vancomycin aqueous solution, the concentration of aqueous solution is about at 100mg/mL.After concentration terminates,
Then precipitation operation is carried out.
NH is added in the aqueous solution containing Lyphocin (Fujisawa)4HCO3(quantity is concentrated solution
The 6% of volume～10% (W/V)), then with ammonia regulation vancomycin concentrated solution pH extremely
7.5～8.5, stirring 45～60min makes its mix homogeneously, and substantial amounts of vancomycin is heavy from solution
Form sediment out.The temperature of precipitation process must control at 10～20 DEG C, after standing 16 ± 2 hours,
Mixed liquor solid-liquid separation, making vancomycin precipitation separate from solution, to obtain solid the most mould
Element alkali, then washes with ethanol top, and debris in solid is gone on top, obtains vancomycin thick after separation
Product.In the Vancomycin obtained, vancomycin chromatographic purity is not less than 80% (HPLC), and one
As more than 85%.
Vancomycin crude product filters after dissolving in aqueous solution, and upper chromatography column chromatography purification obtains ten thousand
Effective chromatographic solution of ancient mycin chromatographic purity more than 93%.Effectively chromatographic solution is through ultrafiltration, nanofiltration
After obtain Lyphocin (Fujisawa) concentrated solution.Lyphocin (Fujisawa) concentrated solution by 0.45 μm and
Precipitate after the membrane filtration of 0.2 μm.
Carbonate, such as NaHCO is added in the concentrated solution containing Lyphocin (Fujisawa)3、
(NH4)2CO3、NH4HCO3, or Na2CO3, make carbonate [NaHCO in concentrated solution3Or
(NH4)2CO3Or NH4HCO3Or Na2CO3] concentration be 3%～7% (W/V), the denseest
Contracting liquid there is bubble to produce, then in solution, adds appropriate HCl regulation pH value to 2.0～3.5,
And continuously stirred 60 ± 30 minutes, control mixeding liquid temperature, at 5～25 DEG C, stands 24～48
Solid-liquid separation after hour.Lyphocin (Fujisawa) filter cake first carries out first with the ethanol water of 90%
Secondary top is washed, and top stops adding the ethanol water of 90% when being washed till electrical conductivity≤1000 μ s/cm.
Carrying out pushing up for the second time washing with the dehydrated alcohol after 0.45 μm membrane filtration again, top i.e. obtains salt after washing
Acid vancomycin precipitation wet product (ethanol water be by 0.45 μm membrane filtration after dehydrated alcohol
Formulated with purified water).Precipitation wet product removes residual ethanol and moisture by being dried, it is thus achieved that
Lyphocin (Fujisawa) finished product.
The Lyphocin (Fujisawa) active princlple obtained by the present invention is improved significantly, and other is miscellaneous
Matter is substantially reduced, and purity is the highest, and chromatographic purity can reach more than 95%, this Lyphocin (Fujisawa)
Bulk density be 0.45～0.65g/ml, compactness is 0.65～0.85g/ml.This density is than routine
Lyophilizing technique wants height, can be used not only for injection, it may also be used for the solid preparations such as capsule.With
Time product appearance color obtain remarkable improvement, be suitable to oral or injection administration.
Accompanying drawing explanation
Fig. 1 is the x-ray powder of the Lyphocin (Fujisawa) of high density non-crystalline form of the present invention
Diffraction spectrogram.
Detailed description of the invention
By following example, the present invention is further described in detail, it is not limited to
Process parameters range in lower embodiment and embodiment.
Embodiment 1: the preparation of vancomycin crude product
Vancomycin fermentation liquid filters in the basic conditions, and the clear filtrate obtained is inhaled by macropore
Attached resin XAD-1600, vancomycin is attracted to resin, uses 40% ethanol after resin washing
Acidic aqueous solution eluting, vancomycin is eluted, and the eluent containing vancomycin adds
Entering activated carbon decolorizing, the filtrate being filtrated to get is concentrated to give Lyphocin (Fujisawa) aqueous solution, then
Add NH4HCO3Precipitate, obtain vancomycin crude product.In vancomycin crude product through the ages
Mycin B content is 86.8%, and titer is 599u/mg.
Take this crude product 2500g, add purified water 20.0L, add NaOH solution regulation pH to 10.0,
Use 0.2M2The ceramic membrane filter in 0.5 μm aperture obtain 25.0L vancomycin filtrate, solution
Titer is 51224IU/ml, and vancomycin B content is 86.5%, and filtrate pH is 9.72, filter
Liquid Column chromatography after HCl/water solution regulation pH to 4.82, chromatographic column filler is that agarose coagulates
Glue.Upper prop terminates rear chromatographic column purification washing post 6BV, then with 0.1mol/l's
NH4HCO3Aqueous solution washes post 10BV, then with the NH of 0.6mol/l4HCO3Aqueous layer
Analysis, more than 93% and titer is in the part of 2000IU/ml to collect wherein vancomycin content,
Obtain effective chromatographic solution.Effectively chromatographic solution adds HCl, regulate pH to 2.76, then use
The ultrafilter membrane ultrafiltration of 50000Da obtains filtrate, then concentrates de-by the NF membrane in 200Da aperture
Salt, finally obtains the Lyphocin (Fujisawa) aqueous solution volume 4500mL that concentration is 200mg/mL,
Being 95.6% containing vancomycin chromatographic purity, pH value of solution is 2.85.
Embodiment 2: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g NaHCO is added while stirring after filter3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 2.80, continue stirring 60MIN so that it is mix homogeneously,
Stopping stirring and stand 38 hours, process temperature controls at 14～15 DEG C.Mixture divides by filtering
From and first wash with 200mL90% ethanol top, top wash end be top washing liquid electrical conductivity be
385us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
99.7g, wet product is by obtaining intermediate 72.5g after being dried removal residual ethanol, then passes through Gao Zhen
Sky obtains Lyphocin (Fujisawa) finished product 63.2g after drying, and moisture content of finished products is 3.4%, and hydrochloric acid is through the ages
Its bulk density of mycin is 0.62g/ml, and compactness is 0.81g/ml.This non-crystalline form hydrochloric acid ten thousand
Ancient mycin, it has essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Feature.Refer to Fig. 1.
Embodiment 3: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
15g NaHCO is added while stirring after filter3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 3.50, continue stirring 30MIN so that it is mix homogeneously,
Stopping stirring and stand 48 hours, process temperature controls at 5～6 DEG C.Mixture is isolated by filtration
And first wash with 200mL 90% ethanol top, it is that top washing liquid electrical conductivity is that end is washed on top
293us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
92.3g, wet product is by obtaining intermediate 68.5g after being dried removal residual ethanol, then passes through Gao Zhen
Sky obtains Lyphocin (Fujisawa) finished product 58.6g after drying, and moisture content of finished products is 3.1%, and hydrochloric acid is through the ages
Its bulk density of mycin is 0.45g/ml, and compactness is 0.69g/ml.This non-crystalline form hydrochloric acid ten thousand
Ancient mycin, it has essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Feature.Refer to Fig. 1.
Embodiment 4: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
35g NaHCO is added while stirring after filter3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 2.01, continue stirring 30MIN so that it is mix homogeneously,
Stopping stirring and stand 48 hours, process temperature controls at 24～25 DEG C.Mixture divides by filtering
From and first wash with 200mL90% ethanol top, top wash end be top washing liquid electrical conductivity be
396us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
102.1g, wet product is by obtaining intermediate 81.3g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 64.1g after vacuum drying, moisture content of finished products is 3.5%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.63g/ml, and compactness is 0.82g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 5: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g NaHCO is added while stirring after filter3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 3.00, continue stirring 90MIN so that it is mix homogeneously,
Stopping stirring and stand 24 hours, process temperature controls at 15～16 DEG C.Mixture divides by filtering
From and first wash with 200mL90% ethanol top, top wash end be top washing liquid electrical conductivity be
351us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
98.1g, wet product is by obtaining intermediate 72.2g after being dried removal residual ethanol, then passes through Gao Zhen
Sky obtains Lyphocin (Fujisawa) finished product 63.3g after drying, and moisture content of finished products is 3.2%, and hydrochloric acid is through the ages
Its bulk density of mycin is 0.59g/ml, and compactness is 0.79g/ml.This non-crystalline form hydrochloric acid ten thousand
Ancient mycin, it has essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Feature.Refer to Fig. 1.
Embodiment 6: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g Na is added while stirring after filter2CO3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 2.82, continue stirring 60MIN so that it is mix homogeneously,
Stopping stirring and stand 36 hours, process temperature controls at 14～15 DEG C.Mixture divides by filtering
From and first wash with 200mL90% ethanol top, top wash end be top washing liquid electrical conductivity be
405us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
101.5g, wet product is by obtaining intermediate 73.0g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 62.4g after vacuum drying, moisture content of finished products is 2.8%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.64g/ml, and compactness is 0.85g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 7: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
15g Na is added while stirring after filter2CO3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 3.50, continue stirring 60MIN so that it is mix homogeneously,
Stopping stirring and stand 48 hours, process temperature controls at 5～6 DEG C.Mixture is isolated by filtration
And first wash with 200mL90% ethanol top, it is that top washing liquid electrical conductivity is that end is washed on top
325us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
91.3g, wet product is by obtaining intermediate 62.5g after being dried removal residual ethanol, then passes through Gao Zhen
Sky obtains Lyphocin (Fujisawa) finished product 50.8g after drying, and moisture content of finished products is 2.9%, and hydrochloric acid is through the ages
Its bulk density of mycin is 0.46g/ml, and compactness is 0.65g/ml.This non-crystalline form hydrochloric acid ten thousand
Ancient mycin, it has essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Feature.Refer to Fig. 1.
Embodiment 8: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
35g Na is added while stirring after filter2CO3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 2.02, continue stirring 30MIN so that it is mix homogeneously,
Stopping stirring and stand 48 hours, process temperature controls at 24～25 DEG C.Mixture divides by filtering
From and first wash with 200mL90% ethanol top, top wash end be top washing liquid electrical conductivity be
413us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
103.8g, wet product is by obtaining intermediate 73.9g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 63.6g after vacuum drying, moisture content of finished products is 3.0%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.63g/ml, and compactness is 0.83g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 9: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g Na is added while stirring after filter2CO3, vancomycin therefrom separates out, and defines slurry,
Add HCl/water solution regulation pH to 3.10, continue stirring 90MIN so that it is mix homogeneously,
Stopping stirring and stand 24 hours, process temperature controls at 14～15 DEG C.Mixture divides by filtering
From and first wash with 200mL90% ethanol top, top wash end be top washing liquid electrical conductivity be
394us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
99.7g, wet product is by obtaining intermediate 72.8g after being dried removal residual ethanol, then passes through Gao Zhen
Sky obtains Lyphocin (Fujisawa) finished product 62.8g after drying, and moisture content of finished products is 3.0%, and hydrochloric acid is through the ages
Its bulk density of mycin is 0.61g/ml, and compactness is 0.84g/ml.This non-crystalline form hydrochloric acid ten thousand
Ancient mycin, it has essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Feature.Refer to Fig. 1.
Embodiment 10: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g NH is added while stirring after filter4HCO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 2.78, continues stirring 60MIN so that it is mixing is all
Even, stop stirring and stand 30 hours, process temperature controls at 11～12 DEG C.Mixture passed through
Filter separates and first washes with 200mL90% ethanol top, and it is that top washing liquid electrical conductivity is that end is washed on top
524us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
103.7g, wet product is by obtaining intermediate 74.5g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 64.5g after vacuum drying, moisture content of finished products is 3.2%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.64g/ml, and compactness is 0.82g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 11: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
35g NH is added while stirring after filter4HCO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 3.48, continues stirring 60MIN so that it is mixing is all
Even, stop stirring and stand 48 hours, process temperature controls at 5～6 DEG C.Mixture is by filtering
Separating and first wash with 200mL90% ethanol top, it is that top washing liquid electrical conductivity is that end is washed on top
531us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
104.3g, wet product is by obtaining intermediate 75.8g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 66.9g after vacuum drying, moisture content of finished products is 3.1%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.65g/ml, and compactness is 0.85g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 12: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
15g NH is added while stirring after filter4HCO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 2.04, continues stirring 30MIN so that it is mixing is all
Even, stop stirring and stand 24 hours, process temperature controls at 24～25 DEG C.Mixture passed through
Filter separates and first washes with 200mL90% ethanol top, and it is that top washing liquid electrical conductivity is that end is washed on top
524us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
88.2g, wet product is by obtaining intermediate 61.7g after being dried removal residual ethanol, then passes through Gao Zhen
Sky obtains Lyphocin (Fujisawa) finished product 49.8g after drying, and moisture content of finished products is 2.8%, and hydrochloric acid is through the ages
Its bulk density of mycin is 0.45g/ml, and compactness is 0.66g/ml.This non-crystalline form hydrochloric acid ten thousand
Ancient mycin, it has essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Feature.Refer to Fig. 1.
Embodiment 13: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g NH is added while stirring after filter4HCO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 3.02, continues stirring 90MIN so that it is mixing is all
Even, stop stirring and stand 48 hours, process temperature controls at 12～13 DEG C.Mixture passed through
Filter separates and first washes with 200mL90% ethanol top, and it is that top washing liquid electrical conductivity is that end is washed on top
385us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
101.8g, wet product is by obtaining intermediate 72.9g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 62.8g after vacuum drying, moisture content of finished products is 2.9%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.59g/ml, and compactness is 0.78g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 14: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g (NH is added while stirring after filter4)2CO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 2.82, continues stirring 60MIN so that it is mixing is all
Even, stop stirring and stand 40 hours, process temperature controls at 8～9 DEG C.Mixture is by filtering
Separating and first wash with 200mL90% ethanol top, it is that top washing liquid electrical conductivity is that end is washed on top
352us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
104.2g, wet product is by obtaining intermediate 75.1g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 68.3g after vacuum drying, moisture content of finished products is 3.3%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.65g/ml, and compactness is 0.84g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 15: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
15g (NH is added while stirring after filter4)2CO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 3.47, continues stirring 30MIN so that it is mixing is all
Even, stop stirring and stand 48 hours, process temperature controls at 5～6 DEG C.Mixture is by filtering
Separating and first wash with 200mL90% ethanol top, it is that top washing liquid electrical conductivity is that end is washed on top
318us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
94.5g, wet product is by obtaining intermediate 69.2g after being dried removal residual ethanol, then passes through Gao Zhen
Sky obtains Lyphocin (Fujisawa) finished product 58.0g after drying, and moisture content of finished products is 3.2%, and hydrochloric acid is through the ages
Its bulk density of mycin is 0.51g/ml, and compactness is 0.69g/ml.This non-crystalline form hydrochloric acid ten thousand
Ancient mycin, it has essentially identical with the x-ray powder crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Feature.Refer to Fig. 1.
Embodiment 16: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
35g (NH is added while stirring after filter4)2CO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 2.00, continues stirring 30MIN so that it is mixing is all
Even, stop stirring and stand 48 hours, process temperature controls at 24～25 DEG C.Mixture passed through
Filter separates and first washes with 200mL90% ethanol top, and it is that top washing liquid electrical conductivity is that end is washed on top
502us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
108.2g, wet product is by obtaining intermediate 78.4g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 69.0g after vacuum drying, moisture content of finished products is 2.9%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.62g/ml, and compactness is 0.80g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
Embodiment 17: the precipitation of Lyphocin (Fujisawa)
Concentrated solution 500mL in Example 1, by the filter membrane mistake of 0.45 μm and 0.2 μm
25g (NH is added while stirring after filter4)2CO3, vancomycin therefrom separates out, and defines pulpous state
Thing, adds HCl/water solution regulation pH to 3.05, continues stirring 90MIN so that it is mixing is all
Even, stop stirring and stand 24 hours, process temperature controls at 20～21 DEG C.Mixture passed through
Filter separates and first washes with 200mL90% ethanol top, and it is that top washing liquid electrical conductivity is that end is washed on top
401us/cm, then washes with 200mL dehydrated alcohol top, has obtained Lyphocin (Fujisawa) wet product
101.9g, wet product is by obtaining intermediate 73.2g after being dried removal residual ethanol, then passes through height
Obtaining Lyphocin (Fujisawa) finished product 66.5g after vacuum drying, moisture content of finished products is 2.7%, hydrochloric acid ten thousand
Ancient its bulk density of mycin is 0.61g/ml, and compactness is 0.79g/ml.This non-crystalline form hydrochloric acid
Vancomycin, it has and the x-ray powder basic phase of crystal diffraction collection of illustrative plates shown in accompanying drawing 1
Same feature.Refer to Fig. 1.
The present invention is illustrated by above embodiment, it is understood, however, that this
Bright it is not limited to particular example as described herein and embodiment.Here comprise these special
The purpose of example and embodiment is to help those of skill in the art to put into practice the present invention.Appoint
What those of skill in the art is easy to without departing from the spirit and scope of the present invention
Being further improved and perfect, therefore the present invention is only by the content of the claims in the present invention
With the restriction of scope, it is intended to contain all these that limited by appendix claim of being included in
Alternative in bright spirit and scope and equivalent.
Claims (11)
1. the preparation method of a non-crystalline form Lyphocin (Fujisawa), it is characterised in that bag
Include following steps:
(1) there is vancomycin chromatographic purity be not less than the vancomycin crude product of 80% and containing
NH4HCO3The chromatographic column of flowing phase carries out column chromatography, obtains chromatographic purity and be not less than 93%
Effective chromatographic solution or the concentrated solution of effective chromatographic solution；Wherein, vancomycin fermentation liquid is in alkalescence
Under the conditions of filter, the clear filtrate obtained pass through macroporous adsorbent resin XAD-1600, the most mould
Element is attracted to resin, with the acidic aqueous solution eluting of 40% ethanol after resin washing, the most mould
Element is eluted, and the eluent containing vancomycin adds activated carbon decolorizing, is filtrated to get
Filtrate is concentrated to give Lyphocin (Fujisawa) aqueous solution, is subsequently added into NH4HCO3Precipitate,
Obtain vancomycin crude product；The column chromatography medium used is polydextran gel Sephadex
CM-25, agarose SP Sepharose or agarose CM Sepharose, flowing is 4～6% mutually
(w/v)NH4HCO3；Flowing with flow is 0.5～1.5 column volumes/hour carry out eluting；
(2) concentrated solution to effective chromatographic solution or effective chromatographic solution adds carbonate, and it is heavy to produce
Form sediment；
(3) use ethanol to carry out top to wash, be dried to obtain Lyphocin (Fujisawa).
2. preparation method as claimed in claim 1, it is characterised in that described effective chromatography
Liquid is the concentrated solution of available effective chromatographic solution after ultrafiltration, nanofiltration, to be produced by carbonate
Precipitation.
3. preparation method as claimed in claim 2, it is characterised in that described effective chromatography
The concentrated solution of liquid or effectively chromatographic solution is carried out after the filter element filtering by 0.45 μm and 0.2 μm again
Precipitation.
4. preparation method as claimed in claim 1, it is characterised in that described carbonate bag
Include (NH4)2CO3, NH4HCO3, Na2CO3And NaHCO3。
5. preparation method as claimed in claim 4, it is characterised in that to effective chromatographic solution
Or to add the concentration of carbonate obtained by carbonate in the concentrated solution of effective chromatographic solution be 3%～7%
(W/V)。
6. preparation method as claimed in claim 1, it is characterised in that after adding carbonate,
Regulation pH=2.0～3.5, is stirred continuously 30～90 minutes, and mixeding liquid temperature controls at 5～25 DEG C,
And stand 24～48 hours.
7. preparation method as claimed in claim 1, it is characterised in that also include using 90%
Ethanol water repeatedly push up and wash, top stops adding when being washed till electrical conductivity≤1000 μ s/cm
The ethanol water of 90%.
8. preparation method as claimed in claim 2, it is characterised in that returning of described ultrafiltration
Stream temperature controls at 10～20 DEG C, and washs filtering residue by purified water, and ultrafilter membrane is selected
10000Da～50000Da.
9. preparation method as claimed in claim 2, it is characterised in that described nanofiltration solution
Temperature control at 10～20 DEG C, and substep adds purified water and the ethylenediamine of 0.04%～0.06%
Tetraacethyl (W/bou), NF membrane selects 200Da～600Da.
10. preparation method as claimed in claim 1, it is characterised in that be additionally included in post layer
Before analysis, being dissolved in pure water by vancomycin crude product, stirring, by 2～6mol/L NaOH solution
Adjust to pH=9～10, filter until completely dissolved, collect filtrate, with 2～6mol/L HCl
Solution adjusts filtrate to pH=4.5～6.0.
11. preparation methoies as claimed in claim 1, are additionally included in containing NH4HCO3
Before flowing phase eluting, first with pure water and the NH of low concentration4HCO3Solution carries out prewashing.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033997A (en) * | 1987-12-28 | 1989-07-19 | 伊莱利利公司 | Improved vancomycin precipitation process |
| CN1616651A (en) * | 2004-10-15 | 2005-05-18 | 上海医药工业研究院 | Vancomycine producing fungus and its use |
| CN1626542A (en) * | 2003-12-09 | 2005-06-15 | 华北制药集团有限责任公司 | Method for preparing vancomycin of norhydrochloric acid |
| CN1858061A (en) * | 2006-01-06 | 2006-11-08 | 上海医药工业研究院 | Vacomycin column chromatography purifying method |
-
2008
- 2008-06-26 CN CN200810126436.XA patent/CN101613396B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033997A (en) * | 1987-12-28 | 1989-07-19 | 伊莱利利公司 | Improved vancomycin precipitation process |
| CN1626542A (en) * | 2003-12-09 | 2005-06-15 | 华北制药集团有限责任公司 | Method for preparing vancomycin of norhydrochloric acid |
| CN1616651A (en) * | 2004-10-15 | 2005-05-18 | 上海医药工业研究院 | Vancomycine producing fungus and its use |
| CN1858061A (en) * | 2006-01-06 | 2006-11-08 | 上海医药工业研究院 | Vacomycin column chromatography purifying method |
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