WO2016172855A1 - 多肽混合物高效液相色谱分析方法 - Google Patents

多肽混合物高效液相色谱分析方法 Download PDF

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WO2016172855A1
WO2016172855A1 PCT/CN2015/077690 CN2015077690W WO2016172855A1 WO 2016172855 A1 WO2016172855 A1 WO 2016172855A1 CN 2015077690 W CN2015077690 W CN 2015077690W WO 2016172855 A1 WO2016172855 A1 WO 2016172855A1
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mobile phase
liquid chromatography
elution
gradient
ratio
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French (fr)
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唐洋明
何刚
李国弢
陶安进
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Hybio Pharmaceutical Co Ltd
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Hybio Pharmaceutical Co Ltd
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Priority to US15/559,428 priority Critical patent/US10330650B2/en
Priority to EP19198009.3A priority patent/EP3603764B1/en
Priority to PCT/CN2015/077690 priority patent/WO2016172855A1/zh
Priority to CN202011643350.1A priority patent/CN112649538B/zh
Priority to CN201580079194.1A priority patent/CN107531750B/zh
Priority to JP2017556727A priority patent/JP6676072B2/ja
Priority to EP15890235.3A priority patent/EP3290432B1/en
Priority to EP19198022.6A priority patent/EP3613756B1/en
Priority to CN202011638347.0A priority patent/CN112649537B/zh
Publication of WO2016172855A1 publication Critical patent/WO2016172855A1/zh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/03—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a method for analyzing a mixture of polypeptides by high performance liquid chromatography.
  • Glatiramer acetate is a synthetic peptide mixture (relative molecular weight: 4700-11000 Daltons) from L-alanine, L-glutamic acid, L-tyrosine A random polymer consisting of four amino acids of L-lysine, 45-100 amino acids in length. The molar ratio of each amino acid is about 0.392-0.462, 0.129-0.153, 0.086-0.100, 0.300-0.374.
  • the drug was developed by the Israeli pharmaceutical company Teva Pharmaceuticals (TEVA) under the trade name Approved by the US FDA for the treatment of multiple sclerosis in 1996, currently There are two products, water for injection and lyophilized powder for injection, all for subcutaneous injection.
  • Glatiramer acetate is a highly continuous copolymer whose structure is as follows:
  • compositional differences between the imitation glatiramer acetate and the control formulation glatiramer acetate can only be determined based on some inherent properties of the drug.
  • the invention is based on the charge, polarity and non-polarity of the product in different buffer solutions, and adopts anion exchange chromatography, cation exchange chromatography and reversed phase chromatography to carry out stepwise gradient elution method for comparative analysis of the prototype sample and commercial products. The difference in the preparation (control).
  • glatiramer acetate is a highly continuous copolymer, it is difficult to clarify the individual components by a separation method.
  • the existing analytical methods are based on the difference in molecular weight between components by the size exclusion method for simple separation analysis (single peak is divided into several components and then analyzed for its components), which shows that the literature method is more cumbersome. It is necessary to develop an analytical method that can effectively separate the components of glatiramer acetate.
  • the invention first relates to a method for performing linear or stepwise gradient elution of glatiramer acetate by high performance liquid chromatography and analyzing whether the content of each component of the copolymer is acceptable.
  • the method includes the following steps,
  • Step (1) configuring a glatiramer acetate solution to be tested
  • Step (3) detects and analyzes the corresponding peak area of each component of the copolymer, and compares with the reference product to determine whether the content range of each component of the sample to be tested is qualified.
  • step (2) The elution gradient described in step (2) is
  • the mobile phase A is contained in a solution containing 10-50 mM of trishydroxyammonium hydrochloride as the mobile phase A, and the pH is adjusted to 10-12 with a NaOH solution;
  • the mobile phase B is contained with 10-50 mM trishydroxyammonium hydrochloride, 0.5-1.5 M sodium chloride solution, and the mobile phase B is adjusted to pH 8-10 with a hydrochloric acid solution;
  • the injection volume is 5-50uL, and the sample concentration is 1-20mg/ml.
  • the flow rate is 0.5-1.5 mL/min, and the elution time is 50-250 min;
  • the elution gradient is:
  • the elution gradient is:
  • the total elution time is divided into N (2 ⁇ N ⁇ 20) steps, and the gradient elution is performed in sequence.
  • the proportion of mobile phase A in the previous N-1 step gradient is gradually reduced from 100% to 50%, and the ratio of mobile phase B is 0.
  • the injection volume is 5-50uL, and the sample concentration is 1-20mg/ml.
  • the flow rate is 0.5-1.5 mL/min, and the elution time is 50-250 min;
  • the elution gradient is:
  • the elution gradient is:
  • the total elution time is divided into N (2 ⁇ N ⁇ 20) steps, and the gradient elution is performed in sequence.
  • the proportion of mobile phase A in the previous N-1 step gradient is gradually reduced from 100% to 0%, and the ratio of mobile phase B is 0. Gradually increased to 100%, the mobile phase A ratio was 90% at the Nth step, and the mobile phase B was 10%;
  • the injection volume is 5-50uL, and the sample concentration is 1-20mg/ml.
  • the flow rate is 0.5-1.5 mL/min, and the elution time is 50-250 min;
  • the elution gradient is:
  • the elution gradient is:
  • the total elution time is divided into N (2 ⁇ N ⁇ 20) steps, and the gradient elution is performed in sequence.
  • the proportion of mobile phase A in the previous N-1 step gradient is gradually increased from 5% to 40%, and the ratio of mobile phase B is 95. % gradually decreased to 60%, the mobile phase A ratio was 5% at the Nth step, and the mobile phase B was 95%;
  • the detection analysis described in the step (3) uses a UV detector to detect the wavelength of 260-280 nm;
  • the excitation wavelength is 230 nm and the emission wavelength is 300 nm.
  • Figure 1-1 Example 1 chromatographic analysis of the test sample.
  • Figure 1-2 shows a chromatographic analysis of the sample of Example 2.
  • Example 7 is a chromatogram of the test sample.
  • Example 8 is a chromatogram of the test sample.
  • Example 9 is a chromatogram of the test sample.
  • Example 12 is a chromatogram of the test sample.
  • Example 13 shows a chromatographic analysis of the sample.
  • Example 14 is a chromatogram of the test sample.
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 150 mm long, 4.6 mm diameter
  • the column packing is polystyrene. - Divinylbenzene particles, 3 ⁇ m particle size.
  • Mobile phase A is 20 mM trishydroxyammonium hydrochloride, adjusted to pH 11.2 with NaOH solution
  • mobile phase B is 20 mM trishydroxyammonium hydrochloride, containing 1 M NaCl, adjusted to pH 9.8 with hydrochloric acid solution
  • the ratio of mobile phase A and B was set according to Table 1 below.
  • glatiramer acetate hereinafter referred to as reference
  • sample preparation of glatiramer acetate hereinafter referred to as prototype
  • the mobile phase was dissolved and made into a solution containing about 20 mg per 1 mL for testing.
  • the injection volume was 25 ⁇ L
  • the flow rate was set to 0.8 mL/min
  • the column temperature was 30 °C.
  • the proportion of each component is shown in Table 2, and the chromatogram of a sample is shown in Figure 1-1.
  • Table 1 is the ratio of mobile phase A to B in Example 1.
  • Table 2 is the comparison of the composition analysis of the anion exchange between the 3 batches of the control and the 3 batches of the sample.
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 150 mm long, 4.6 mm diameter
  • the column packing is polystyrene.
  • Mobile phase A is 10 mM trishydroxyammonium hydrochloride, adjusted to pH 10 with NaOH solution
  • mobile phase B is 10 mM trishydroxyammonium hydrochloride, containing 1.5 M NaCl, adjusted to pH with hydrochloric acid solution 8.
  • the ratio of mobile phase A and B is set according to Table 1.
  • the Waters 2695 high performance liquid chromatograph 2489 UV multi-wavelength detector was used with a wavelength of 275 nm, a column size of 150 mm long, and a diameter of 4.6 mm.
  • the column packing was polystyrene-divinylbenzene particles and a particle size of 10 ⁇ m.
  • Mobile phase A is 50 mM trishydroxyammonium hydrochloride, adjusted to pH 12 with NaOH solution; mobile phase B is 50 mM trishydroxyammonium hydrochloride, containing 0.5 M NaCl, adjusted to pH with hydrochloric acid solution 10.
  • the ratio of mobile phase A and B is set according to Table 1.
  • the Waters 2695 high performance liquid chromatograph 2489 UV multi-wavelength detector was used with a wavelength of 275 nm, a column size of 150 mm long, and a diameter of 4.6 mm.
  • the column packing was polystyrene-divinylbenzene particles and a particle size of 3 ⁇ m.
  • Mobile phase A is 30 mM trishydroxyammonium hydrochloride, adjusted to pH 10 with NaOH solution; mobile phase B is 30 mM trishydroxyammonium hydrochloride, containing 1 M NaCl, with salt
  • the pH of the acid solution was adjusted to 8, and the ratio of mobile phases A and B was set according to Table 3 below.
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 150 mm long, 4.6 mm diameter
  • the column packing is polystyrene. - Divinylbenzene particles, 3 ⁇ m particle size.
  • Mobile phase A is 20 mM trishydroxyammonium hydrochloride, adjusted to pH 11.2 with NaOH solution
  • mobile phase B is 20 mM trishydroxyammonium hydrochloride, containing 1 M NaCl, adjusted to pH 9.8 with hydrochloric acid solution
  • the ratio of mobile phase A and B was set according to Table 4 below.
  • the Waters 2695 high performance liquid chromatograph 2489 UV multi-wavelength detector was used with a wavelength of 275 nm, a column size of 150 mm long, and a diameter of 4.6 mm.
  • the column packing was polystyrene-divinylbenzene particles and a particle size of 3 ⁇ m.
  • Mobile phase A is 20 mM trishydroxyammonium hydrochloride, adjusted to pH 11.2 with NaOH solution; mobile phase B is 20 mM trishydroxyammonium hydrochloride, containing 1 M NaCl,
  • the hydrochloric acid solution was adjusted to pH 9.8, and the ratio of mobile phase A and B was set according to Table 4 below.
  • Table 5 is the ratio of mobile phase A to B in Example 6.
  • Table 6 is the comparison result of each component analysis of anion exchange between 3 batches of control and 3 batches of prototypes.
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 250 mm long, 4.6 mm diameter
  • the column packing is a tertiary ammonium group.
  • the base-bonded polystyrene-divinylbenzene particles were 5 ⁇ m in particle size.
  • Mobile phase A was 20 mM 2-(N-morpholino)ethanesulfonic acid containing 1 mM EDTA, adjusted to pH 5.2 with NaOH solution; mobile phase B was 20 mM 2-(N-morpholino)ethanesulfonic acid Containing 2 mM EDTA, containing 2 M NaCl, adjusting the pH to 5.8 with NaOH solution, and the ratio of mobile phase A and B was set according to Table 7 below. Take the control or the prototype and add the mobile phase to dissolve and make a solution containing about 20 mg per 1 mL for testing. The injection volume was 25 ⁇ L, the flow rate was set to 1 mL/min, and the column temperature was 30 °C. A chromatogram of a sample is shown in Figure 2-1.
  • Table 7 is the ratio of mobile phase A to B in Example 7.
  • Table 8 is the comparison result of each component analysis of cation exchange between 3 batches of control materials and 3 batches of prototype samples.
  • Mobile phase A is 10 mM 2-(N-morpholino)ethanesulfonic acid containing 0.5 mM EDTA, adjusted to pH 4 with NaOH solution;
  • mobile phase B is 10 mM 2-(N-morpholino)ethanesulfonate
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 250 mm long, 4.6 mm diameter
  • the column packing is a tertiary ammonium group.
  • the base-bonded polystyrene-divinylbenzene particles were 5 ⁇ m in particle size.
  • Mobile phase A is 50 mM 2-(N-morpholino)ethanesulfonic acid containing 1 mM EDTA, adjusted to pH 6 with NaOH solution; mobile phase B is 50 mM 2-(N-morpholino)ethanesulfonic acid
  • the pH was adjusted to 5 with 2 mM EDTA, 2M NaCl, and the ratio of mobile phase A and B was set according to Table 7 below.
  • the injection volume was 50 ⁇ L, the flow rate was set to 1 mL/min, and the column temperature was 30 °C.
  • a chromatographic analysis of a sample is shown in Figure 2-3.
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 250 mm long, 4.6 mm diameter
  • the column packing is a tertiary ammonium group.
  • the base-bonded polystyrene-divinylbenzene particles were 5 ⁇ m in particle size.
  • Mobile phase A was 20 mM 2-(N-morpholino)ethanesulfonic acid containing 1 mM EDTA, adjusted to pH 5.2 with NaOH solution; mobile phase B was 20 mM 2-(N-morpholino)ethanesulfonic acid The pH was adjusted to 5.8 with 2 mM EDTA, 2M NaCl, and the ratio of mobile phase A and B was set according to Table 9 below.
  • the injection volume was 25 ⁇ L, the flow rate was set to 1 mL/min, and the column temperature was 30 °C.
  • a chromatogram of a sample is shown in Figures 2-4. It can be seen that under this elution condition, the components of the sample cannot be effectively separated.
  • Mobile phase A was 30 mM 2-(N-morpholino)ethanesulfonic acid containing 3 mM EDTA, adjusted to pH 5 with NaOH solution; mobile phase B was 10 mM 2-(N-morpholino)ethanesulfonic acid 2% EDTA, containing 2M NaCl, adjusted to pH 6 with NaOH solution, the ratio of mobile phase A and B was set according to Table 10 below.
  • Table 10 is the ratio of mobile phase A to B in Example 11.
  • Table 11 shows the comparison results of the cation exchange of the three batches of the control and the three batches of the sample.
  • the Agilent 1260 High Performance Liquid Chromatograph UV multi-wavelength detector was used with a detection wavelength of 275 nm, a column size of 250 mm long, and a diameter of 4.6 mm.
  • the column packing was a silica matrix bonded C18 ligand particle with a 3 ⁇ m particle size.
  • Mobile phase A was acetonitrile; mobile phase B was a 50 mM ammonium sulfate solution, the pH was adjusted to 2.5 with a phosphoric acid solution, and the ratio of mobile phase A to B was set as shown in Table 12 below.
  • the control and the sample were separately dissolved in the mobile phase and made into a solution containing about 20 mg per 1 mL for testing.
  • the injection volume was 25 ⁇ L, the flow rate was set to 1 mL/min, and the column temperature was 30 °C.
  • the comparison analysis of the components of the reference product and the prototype sample is shown in Table 13 below, and the chromatogram analysis of a sample is shown in Figure 3-1.
  • Table 12 is the ratio of mobile phase A to B of Example 12:
  • Table 13 is the comparison of the components of the three batches of the control and the three batches of the sample.
  • the detection wavelength was set to 275 nm, the column size was 250 mm long, and the diameter was 4.6 mm.
  • the column packing was silica gel matrix bonded C8 ligand particles, 10 ⁇ m particle size.
  • Mobile phase A was acetonitrile; mobile phase B was 0.1% trifluoroacetic acid solution, and the ratio of mobile phase A to B was set as shown in Table 12.
  • the control and the sample were separately dissolved in the mobile phase and made into a solution containing about 10 mg per 1 mL for testing.
  • the injection volume was 50 ⁇ L, the flow rate was set to 1 mL/min, and the column temperature was 50 °C.
  • a chromatogram of a sample is shown in Figure 3-2.
  • the Agilent 1260 High Performance Liquid Chromatograph UV multi-wavelength detector was used with a detection wavelength set at 275 nm, a column size of 250 mm long, and a diameter of 4.6 mm.
  • the column packing was a silica matrix bonded C4 ligand particle with a 3 ⁇ m particle size.
  • Mobile phase A was acetonitrile; mobile phase B was a 30 mM ammonium sulfate solution, the pH was adjusted to 2 with a phosphoric acid solution, and the ratio of mobile phase A to B was set as shown in Table 12.
  • the injection volume was 15 ⁇ L, the flow rate was set to 0.5 mL/min, and the column temperature was 25 °C.
  • a chromatogram of a sample is shown in Figure 3-3.
  • the Agilent 1260 High Performance Liquid Chromatograph UV multi-wavelength detector was used with a detection wavelength set at 275 nm, a column size of 250 mm long, and a diameter of 4.6 mm.
  • the column packing was a silica matrix bonded C4 ligand particle with a 3 ⁇ m particle size.
  • Mobile phase A was acetonitrile; mobile phase B was 80 mM ammonium sulfate solution, pH was adjusted to 3 with phosphoric acid solution, and the ratio of mobile phase A to B was set as shown in Table 12.
  • the injection volume was 25 ⁇ L, the flow rate was set to 0.5 mL/min, and the column temperature was 50 °C.
  • a chromatographic analysis of a sample is shown in Figure 3-4.
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 250 mm long, 4.6 mm diameter
  • the column packing is a silica matrix bond.
  • Mobile phase A was acetonitrile
  • mobile phase B was 0.1% trifluoroacetic acid solution
  • the ratio of mobile phase A to B was set as shown in Table 14.
  • the injection volume was 50 ⁇ L, the flow rate was set to 1 mL/min, and the column temperature was 30 °C.
  • a chromatogram of a sample is shown in Figures 3-5. It can be seen that the conventional method cannot completely separate the components in the prototype.
  • the excitation wavelength (Ex) is 230 nm
  • the emission wavelength (Em) is 300 nm
  • the column size is 250 mm long, 4.6 mm diameter
  • the column packing is a silica matrix bond.
  • Mobile phase A was acetonitrile
  • mobile phase B was 0.1% trifluoroacetic acid solution
  • the ratio of mobile phase A to B was set as shown in Table 15 below.
  • Table 15 is the ratio of mobile phase A to B in Example 17.
  • Table 16 is the comparison of the components of the three batches of the control and the three batches of the sample.
  • the method for analyzing the content of each component in the glatiramer acetate sample is effective and feasible, and can be used for the comparative analysis and quality consistency study of glatiramer acetate.

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Abstract

提供一种多肽混合物高效液相色谱分析方法，具体而言，该方法包括如下步骤，步骤(1)配置待测醋酸格拉替雷溶液；步骤(2)以阴离子交换液相色谱法、阳离子交换液相色谱法或反相液色谱法，对待测样本进行梯度洗脱，步骤(3)检测分析共聚物各组分对应峰面积，通过与对照品比对，确定待测样本各组分含量范围是否合格。

Description

多肽混合物高效液相色谱分析方法 技术领域
本发明属于生物医药领域，具体而言，涉及一种多肽混合物高效液相色谱分析方法。
背景技术
醋酸格拉替雷(Glatiramer acetate，缩写为GA)是一种人工合成多肽类混合物(相对分子量：4700-11000道尔顿)，由L-丙氨酸、L-谷氨酸、L-酪氨酸和L-赖氨酸四个氨基酸所组成的随机聚合物，长为45-100个氨基酸。各氨基酸摩尔比大约为0.392-0.462，0.129-0.153，0.086-0.100，0.300-0.374。该药物由以色列药厂梯瓦制药(TEVA)所研发制造，商品名为
Figure PCTCN2015077690-appb-000001
于1996年获美国FDA核准用于治疗多发性硬化症，目前
Figure PCTCN2015077690-appb-000002
有注射用水针和注射用冻干粉针两种产品，均用于皮下注射。
醋酸格拉替雷是一个连续性很强的共聚物，其结构如下式所示：
(Glu，Ala，Lys，Tyr)x.xCH3COOH
(C5H9NO4.C3H7NO2.C6H14N2O2.C9H11NO3)x.
xC2H4O2
CAS-147245-92-9
对于仿制该药厂商，只能基于该药的一些固有性质来考察仿制醋酸格拉替雷与对照制剂醋酸格拉替雷的组分差异。
本发明基于本品在不同缓冲溶液中所带电荷、极性、非极情况，采用阴离子交换色谱、阳离子交换色谱、反相色谱，进行阶梯式梯度洗脱的方式对比分析试制样品与市售商品制剂(对照品)的差异。
基于醋酸格拉替雷是一个连续性很强的共聚物，很难用一种分离方法弄清各个组分。现有的分析方法基于组分间分子量差异用体积排阻法进行简单分离分析(将单一峰分为若干组分收集后对其组分再进行分析)，可见文献方法较为繁琐。开发能有效分离醋酸格拉替雷各组分的分析方法是非常有必要的。
发明内容
本发明首先涉及一种采用高效液相色谱法对醋酸格拉替雷进行线性或阶梯式梯度洗脱，并分析该共聚物各组分含量是否合格的方法。
该方法包括如下步骤，
步骤(1)配置待测醋酸格拉替雷溶液；
步骤(2)以阴离子交换液相色谱法、阳离子交换液相色谱法或反相液色谱法，对待测样本进行梯度洗脱，
步骤(3)检测分析共聚物各组分对应峰面积，通过与对照品比对，确定待测样本各组分含量范围是否合格。
步骤(2)所述的洗脱梯度为
步骤(2)所述采用阴离子交换液相色谱法的色谱条件为：
采用羧基键合聚苯乙烯-二乙烯苯颗粒为填料的色谱柱，所述的色谱柱填料颗粒粒径为1.7-10μm；
以含10-50mM的三羟基氨基甲烷盐酸盐溶液为流动相A，所述流动相A用NaOH溶液调节pH值至10-12；
以含10-50mM三羟基氨基甲烷盐酸盐、0.5-1.5M氯化钠溶液为流动相B，所述流动相B用盐酸溶液调节pH值至8-10；
进样量5-50uL，样品浓度1-20mg/ml，
流速0.5-1.5mL/min，洗脱时间50-250min；
洗脱梯度为：
采用不同形式的洗脱梯度进行洗脱，
柱温25-50℃；
所述的洗脱梯度为：
总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱，前N-1步梯度中流动相A的比例由100％渐次降至50％，流动相B的比例由0渐次升至50％，第N步时流动相A比例为100％，流动相B为0；
优选的5≤N≤15，更优选的8≤N≤12，最优选的，N＝10。
步骤(2)所述采用阳离子交换液相色谱法的色谱条件为：
采用叔铵基键合聚苯乙烯-二乙烯苯颗粒为填料的色谱柱，所述的色谱柱填料颗粒粒径为1.7-10μm；
以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA溶液为流动相A，用NaOH溶液调pH值至4-6；
以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA、1-2.5M的NaCl的溶液为流动相B，用NaOH溶液调pH值至5-7；
进样量5-50uL，样品浓度1-20mg/ml，
流速0.5-1.5mL/min，洗脱时间50-250min；
洗脱梯度为：
采用不同形式的洗脱梯度进行洗脱，
柱温25-50℃；
所述的洗脱梯度为：
总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱，前N-1步梯度中流动相A的比例由100％渐次降至0％，流动相B的比例由0渐次升至100％，第N步时流动相A比例为90％，流动相B为10％；
优选的5≤N≤15，更优选的8≤N≤12，最优选的，N＝10。
步骤(2)所述采用反相液色谱法的色谱条件为：
采用C18、C12、C8、C4键合硅胶颗粒为填料的色谱柱，所述的色谱柱填料颗粒粒径为1.7-10μm；
以乙腈为流动相A；
以含30-80mM的硫酸铵的溶液为流动相B，用磷酸溶液调pH值至2-3；
或以0.1％三氟乙酸溶液作为流动相B；
进样量5-50uL，样品浓度1-20mg/ml，
流速0.5-1.5mL/min，洗脱时间50-250min；
洗脱梯度为：
采用不同形式的洗脱梯度进行洗脱，
柱温25-50℃；
所述的洗脱梯度为：
总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱，前N-1步梯度中流动相A的比例由5％渐次升至40％，流动相B的比例由95％渐次降至60％，第N步时流动相A比例为5％，流动相B为95％；
优选的5≤N≤15，更优选的8≤N≤12，最优选的，N＝10。
步骤(3)所述的检测分析采用紫外检测器检测波长260-280nm；
采用荧光检测器时，其激发波长为230nm，发射波长为300nm。
附图说明
图1-1，实施例1检测样本色谱分析图。
图1-2，实施例2检测样本色谱分析图。
图1-3，实施例3检测样本色谱分析图。
图1-4，实施例4检测样本色谱分析图。
图1-5，实施例5检测样本色谱分析图。
图2-1，实施例7检测样本色谱分析图。
图2-2，实施例8检测样本色谱分析图。
图2-3，实施例9检测样本色谱分析图。
图2-4，实施例10检测样本色谱分析图。
图3-1，实施例12检测样本色谱分析图。
图3-2，实施例13检测样本色谱分析图。
图3-3，实施例14检测样本色谱分析图。
图3-4，实施例15检测样本色谱分析图。
图3-5，实施例16检测样本色谱分析图。
具体实施方式
实施例1
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸150mm长，4.6mm直径，色谱柱填料为聚苯乙烯-二乙烯苯颗粒，3μm粒径。流动相A是20mM的三羟基氨基甲烷盐酸盐，用NaOH溶液调pH值至11.2；流动相B是20mM的三羟基氨基甲烷盐酸盐，含1M的NaCl，用盐酸溶液调pH值至9.8，流动相A和B的比例依据下表1进行设置。分别取市售醋酸格拉替雷(下称对照品)、试制样品醋酸格拉替雷(下称试制样品)，加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为0.8mL/min，柱温采用30℃。其各组分比例见表2，某一样品色谱分析图见附图1-1。
表1是实施例1流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
15 100 0
15.1 97.5 2.5
30 97.5 2.5
30.1 95 5
45 95 5
45.1 90 10
60 90 10
60.1 87.5 12.5
75 87.5 12.5
75.1 85 15
90 85 15
90.1 80 20
105 80 20
105.1 70 30
120 70 30
120.1 50 50
135 50 50
135.1 100 0
140 100 0
表2是3批对照品与3批试制样品阴离子交换各组成分析比对结果
Figure PCTCN2015077690-appb-000003
实施例2
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸150mm长，4.6mm直径，色谱柱填料为聚苯乙烯-二乙烯苯颗粒，5μm粒径。流动相A是10mM的三羟基氨基甲烷盐酸盐，用NaOH溶液调pH值至10；流动相B是10mM的三羟基氨基甲烷盐酸盐，含1.5M的NaCl，用盐酸溶液调pH值至8，流动相A和B的比例依据表1进行设置。分别取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量15μL，流速设为0.5mL/min，柱温采用25℃。某一样品色谱分析图见附图1-2。
实施例3
使用沃特世2695高效液相色谱仪2489紫外多波长检测器，波长为275nm，色谱柱尺寸150mm长，4.6mm直径，色谱柱填料为聚苯乙烯-二乙烯苯颗粒，10μm粒径。流动相A是50mM的三羟基氨基甲烷盐酸盐，用NaOH溶液调pH值至12；流动相B是50mM的三羟基氨基甲烷盐酸盐，含0.5M的NaCl，用盐酸溶液调pH值至10，流动相A和B的比例依据表1进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约10mg的溶液，供测试。进样量50μL，流速设为1.5mL/min，柱温采用50℃。某一样品色谱分析图见附图1-3
实施例4
使用沃特世2695高效液相色谱仪2489紫外多波长检测器，波长为275nm，色谱柱尺寸150mm长，4.6mm直径，色谱柱填料为聚苯乙烯-二乙烯苯颗粒，3μm粒径。流动相A是30mM的三羟基氨基甲烷盐酸盐，用NaOH溶液调pH值至10；流动相B是30mM的三羟基氨基甲烷盐酸盐，含1M的NaCl，用盐 酸溶液调pH值至8，流动相A和B的比例依据下表3进行设置。分别取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为0.8mL/min，柱温采用40℃。某一样品色谱分析图见附图1-4。
表3实施例4流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
15 95 5
30 92.5 7.5
45 90 10
60 87.5 12.5
75 85 15
90 80 20
105 70 30
120 50 50
135 100 0
150 100 0
实施例5
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸150mm长，4.6mm直径，色谱柱填料为聚苯乙烯-二乙烯苯颗粒，3μm粒径。流动相A是20mM的三羟基氨基甲烷盐酸盐，用NaOH溶液调pH值至11.2；流动相B是20mM的三羟基氨基甲烷盐酸盐，含1M的NaCl，用盐酸溶液调pH值至9.8，流动相A和B的比例依据下表4进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为1mL/min，柱温采用30℃。某一样品色谱分析图见附图1-5，可见，在该洗脱条件下，不能对样品的组分进行有效分离。
表4.实施例5流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
10 100 0
30 0 100
40 0 100
41 100 0
55 100 0
实施例6
使用沃特世2695高效液相色谱仪2489紫外多波长检测器，波长为275nm，色谱柱尺寸150mm长，4.6mm直径，色谱柱填料为聚苯乙烯-二乙烯苯颗粒，3μm粒径。流动相A是20mM的三羟基氨基甲烷盐酸盐，用NaOH溶液调pH值至11.2；流动相B是20mM的三羟基氨基甲烷盐酸盐，含1M的NaCl，用 盐酸溶液调pH值至9.8，流动相A和B的比例依据下表4进行设置。分别取对照品、试制样品，加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为0.8mL/min，柱温采用30℃。其各组分比例见表6。
表5是实施例6流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
10 100 0
10.1 97 3
15 97 3
15.1 93 7
20 93 7
20.1 90 10
30 90 10
30.1 85 15
40 85 15
40.1 80 20
50 80 20
50.1 75 25
60 75 25
60.1 70 30
75 70 30
75.1 65 35
85 65 35
85.1 60 40
95 60 40
95.1 55 45
105 55 45
105.1 50 50
120 50 50
120.1 100 0
140 100 0
表6是3批对照品与3批试制样品阴离子交换各组成分析比对结果
Figure PCTCN2015077690-appb-000004
阳离子交换液相色谱(实施例7-11)
实施例7
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒，5μm粒径。流动相A是20mM的2-(N-吗啉代)乙磺酸，含1mM EDTA，用NaOH溶液调pH值至5.2；流动相B是20mM的2-(N-吗啉代)乙磺酸，含2mM EDTA，含2M的NaCl，用NaOH溶液调pH值至5.8，流动相A和B的比例依据下表7进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为1mL/min，柱温采用30℃。某一样品色谱分析图见附图2-1。
表7是实施例7流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 90 10
15 90 10
15.1 80 20
30 80 20
30.1 70 30
45 70 30
45.1 60 40
60 60 40
60.1 50 50
75 50 50
75.1 40 60
90 40 60
90.1 30 70
105 30 70
105.1 20 80
120 20 80
120.1 0 100
135 0 100
135.1 90 10
150 90 10
表8是3批对照品与3批试制样品阳离子交换各组成分析比对结果
Figure PCTCN2015077690-appb-000005
实施例8
使用沃特世2695高效液相色谱仪2489紫外多波长检测器，波长为275nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒，10μm粒径。流动相A是10mM的2-(N-吗啉代)乙磺酸，含0.5mM EDTA，用NaOH溶液调pH值至4；流动相B是10mM的2-(N-吗啉代)乙磺酸，含5mM EDTA，含2M的NaCl，用NaOH溶液调pH值至7，流动相A和B的比例依据表7进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量15μL，流速设为1mL/min，柱温采用25℃。某一样品色谱分析图见附图2-2
实施例9
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒，5μm粒径。流动相A是50mM的2-(N-吗啉代)乙磺酸，含1mM EDTA，用NaOH溶液调pH值至6；流动相B是50mM的2-(N-吗啉代)乙磺酸，含2mM EDTA，2M的NaCl，用NaOH溶液调pH值至5，流动相A和B的比例依据下表7进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约10mg的溶液，供测试。进样量50μL，流速设为1mL/min，柱温采用30℃。某一样品色谱分析图见附图2-3。
实施例10
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒，5μm粒径。流动相A是20mM的2-(N-吗啉代)乙磺酸，含1mM EDTA，用NaOH溶液调pH值至5.2；流动相B是20mM的2-(N-吗啉代)乙磺酸，含2mM EDTA，2M的NaCl，用NaOH溶液调pH值至5.8，流动相A和B的比例依据下表9进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为1mL/min，柱温采用30℃。某一样品色谱分析图见附图2-4，可见，在该洗脱条件下，不能对样品的组分进行有效分离。
表9实施例10流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
20 100 0
55 0 100
70 0 100
71 100 0
90 100 0
实施例11
使用沃特世2695高效液相色谱仪2489紫外多波长检测器，波长为275nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒，5μm粒径。流动相A是30mM的2-(N-吗啉代)乙磺酸，含3mM EDTA，用NaOH溶液调pH值至5；流动相B是10mM的2-(N-吗啉代)乙磺酸，含2mM EDTA，含2M的NaCl，用NaOH溶液调pH值至6，流动相A和B的比例依据下表10进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为1mL/min，柱温采用35℃。其各组分比例见表11。
表10是实施例11流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
10 100 0
10.1 97.5 2.5
15 97.5 2.5
15.1 93 7
20 93 7
20.1 90 10
30 90 10
30.1 87.5 12.5
40 87.5 12.5
40.1 83 17
50 83 17
50.1 80 20
60 80 20
60.1 75 25
75 75 25
75.1 70 30
85 70 30
85.1 65 35
95 65 35
95.1 60 40
105 60 40
105.1 55 45
115 55 45
115.1 50 50
125 50 50
125.1 100 0
140 100 0
表11是3批对照品与3批试制样品阳离子交换各组成分析比对结果
Figure PCTCN2015077690-appb-000006
反相液相色谱案例(实施例12-17)
实施例12
使用安捷伦1260高效液相色谱仪紫外多波长检测器，检测波长设为275nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为硅胶基体键合C18配基颗粒，3μm粒径。流动相A是乙腈；流动相B是50mM硫酸铵溶液，用磷酸溶液调pH值至2.5，流动相A与B的比例按下表12进行设置。分别取对照品和试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为1mL/min，柱温采用30℃。其对照品与试制样品个组分比对分析见下表13，某一样品色谱分析图见附图3-1。
表12是实施例12流动相A与B的比例：
时间(min) 流动相A(％) 流动相B(％)
0 5 95
5 5 95
5.1 12.5 87.5
20 12.5 87.5
20.1 15 85
35 15 85
35.1 17.5 82.5
50 17.5 82.5
50.1 20 80
65 20 80
65.1 22.5 78.5
80 22.5 78.5
80.1 25 75
95 25 75
95.1 30 70
110 30 70
110.1 40 60
125 40 60
125.1 5 95
135 5 95
表13是3批对照品与3批试制样品反相各组分分析比对结果
Figure PCTCN2015077690-appb-000007
实施例13
使用安捷伦1260高效液相色谱仪紫外多波长检测器，检测波长设为275nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为硅胶基体键合C8配基颗粒，10μm粒径。流动相A是乙腈；流动相B是0.1％三氟乙酸溶液，动相A与B的比例按表12进行设置。分别取对照品和试制样品加流动相溶解并制成每1mL含约10mg的溶液，供测试。进样量50μL，流速设为1mL/min，柱温采用50℃。其某一样品色谱分析图见附图3-2。
实施例14
使用安捷伦1260高效液相色谱仪紫外多波长检测器，检测波长设为275nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为硅胶基体键合C4配基颗粒，3μm粒径。流动相A是乙腈；流动相B是30mM硫酸铵溶液，用磷酸溶液调pH值至2，流动相A与B的比例按表12进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量15μL，流速设为0.5mL/min，柱温采用25℃。某一样品色谱分析图见附图3-3。
实施例15
使用安捷伦1260高效液相色谱仪紫外多波长检测器，检测波长设为275nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为硅胶基体键合C4配基颗粒，3μm粒径。流动相A是乙腈；流动相B是80mM硫酸铵溶液，用磷酸溶液调pH值至3，流动相A与B的比例按表12进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量25μL，流速设为0.5mL/min，柱温采用50℃。某一样品色谱分析图见附图3-4。
实施案16
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为硅胶基体键合C18配基颗粒，3μm粒径。流动相A是乙腈；流动相B是0.1％三氟乙酸溶液，动相A与B的比例按下表14进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量50μL，流速设为1mL/min，柱温采用30℃。某一样品色谱分析图见附图3-5，可见，常规方法完全无法分离试制样本中的各个成分。
表14实施例16流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
15 100 0
35 0 100
45 0 100
46 100 0
55 100 0
实施案17
使用沃特世2695高效液相色谱仪2475荧光多波长检测器，激发波长(Ex)为230nm，发射波长(Em)为300nm，色谱柱尺寸250mm长，4.6mm直径，色谱柱填料为硅胶基体键合C18配基颗粒，3μm粒径。流动相A是乙腈；流动相B是0.1％三氟乙酸溶液，动相A与B的比例按下表15进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液，供测试。进样量50μL，流速设为1.5mL/min，柱温采用30℃。其各组分比例见表16。
表15是实施例17流动相A与B的比例
时间(min) 流动相A(％) 流动相B(％)
0 100 0
15 100 0
15.1 90 10
30 90 10
30.1 80 20
45 80 20
45.1 70 30
60 70 30
60.1 60 40
75 60 40
75.1 55 45
85 55 45
85.1 50 50
100 50 50
100.1 100 0
120 100 0
表16是3批对照品与3批试制样品反相各组分分析比对结果
Figure PCTCN2015077690-appb-000008
由以上实施例可知，本发明用于分析醋酸格拉替雷样本中各个组分的含量的方法是有效的，可行的，可以用于醋酸格拉替雷的对比分析与质量一致性研究。
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，而不用做对本发明保护范围的限制。

Claims (5)

  1. 一种采用高效液相色谱法对醋酸格拉替雷进行线性或阶梯式梯度洗脱，并分析该共聚物各组分含量是否合格的方法，其特征在于，该方法包括如下步骤，
    步骤(1)配置待测醋酸格拉替雷溶液；
    步骤(2)以阴离子交换液相色谱法、阳离子交换液相色谱法或反相液色谱法，对待测样本进行梯度洗脱，
    步骤(3)检测分析共聚物各组分对应峰面积，通过与对照品比对，确定待测样本各组分含量范围是否合格。
  2. 根据权利要求1所述的方法，其特征在于，
    步骤(2)所述采用阴离子交换液相色谱法的色谱条件为：
    采用羧基键合聚苯乙烯-二乙烯苯颗粒为填料的色谱柱，所述的色谱柱填料颗粒粒径为1.7-10μm；
    以含10-50mM的三羟基氨基甲烷盐酸盐溶液为流动相A，所述流动相A用NaOH溶液调节pH值至10-12；
    以含10-50mM三羟基氨基甲烷盐酸盐、0.5-1.5M氯化钠溶液为流动相B，所述流动相B用盐酸溶液调节pH值至8-10；
    进样量5-50uL，样品浓度1-20mg/ml，
    流速0.5-1.5mL/min，洗脱时间50-250min；
    洗脱梯度为：
    总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱，前N-1步梯度中流动相A的比例由100％渐次降至50％，流动相B的比例由0渐次升至50％，第N步时流动相A比例为100％，流动相B为0；
    优选的，5≤N≤15，更优选的，8≤N≤12，最优选的，N＝10；
    柱温25-50℃。
  3. 根据权利要求1所述的方法，其特征在于，
    步骤(2)所述采用阳离子交换液相色谱法的色谱条件为：
    采用叔铵基键合聚苯乙烯-二乙烯苯颗粒为填料的色谱柱，所述的色谱柱填料颗粒粒径为1.7-10μm；
    以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA溶液为流动相A，用NaOH溶液调pH值至4-6；
    以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA、1-2.5M的NaCl的溶液为流动相B，用NaOH溶液调pH至5-7；
    进样量5-50uL，样品浓度1-20mg/ml，
    流速0.5-1.5mL/min，洗脱时间50-250min；
    洗脱梯度为：
    总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱，前N-1步梯度中流动相A的比例由100％渐次降至0％，流动相B的比例由0渐次升至100％，第N步时流动相A比例为90％，流动相B为10％；
    优选的，5≤N≤15，更优选的，8≤N≤12，最优选的，N＝10；
    柱温25-50℃。
  4. 根据权利要求1所述的方法，其特征在于，
    步骤(2)所述采用反相液色谱法的色谱条件为：
    采用C18、C12、C8、C4键合硅胶颗粒为填料的色谱柱，所述的色谱柱填料颗粒粒径为1.7-10μm；
    以乙腈为流动相A；
    以含30-80mM的硫酸铵的溶液为流动相B，用磷酸溶液调pH值至2-3；
    或以0.1％三氟乙酸溶液作为流动相B；
    进样量5-50uL，样品浓度1-20mg/ml，
    流速0.5-1.5mL/min，洗脱时间50-250min；
    洗脱梯度为：
    总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱，前N-1步梯度中流动相A的比例由5％渐次升至40％，流动相B的比例由95％渐次降至60％，第N步时流动相A比例为5％，流动相B为95％；
    优选的5≤N≤15，更优选的8≤N≤12，最优选的，N＝10；
    柱温25-50℃。
  5. 根据权利要求1-4任一所述的方法，其特征在于，
    步骤(3)所述的检测分析采用紫外检测器检测波长260-280nm；
    采用荧光检测器时，其激发波长为230nm，发射波长为300nm。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114384194A (zh) * 2021-12-28 2022-04-22 湖南中晟全肽生化有限公司 一种高通量检测多肽的方法
CN115586288A (zh) * 2022-10-08 2023-01-10 广州朗圣药业有限公司 一种含szfz13-a的组合物中szfz13-a含量的高效液相色谱检测方法

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111060635A (zh) * 2019-11-27 2020-04-24 吉尔生化（上海）有限公司 一种卡贝缩宫素的分析检测方法
CN111138516B (zh) * 2020-01-10 2023-04-25 大理大学 一种大胡蜂肽及其制备方法和应用
CN112858522B (zh) * 2021-02-20 2022-09-20 重庆工贸职业技术学院 一种高效液相色谱分离测定高纯缬氨酸中杂氨基酸的方法
CN113009014B (zh) * 2021-02-24 2023-04-07 上海旭东海普药业有限公司 一种2-甲氧基-5-氟尿嘧啶杂质的高效液相检测方法
CN113720941B (zh) * 2021-09-18 2023-03-24 广东丸美生物技术股份有限公司 护肤品原料或者护肤品中多肽的检测方法及多肽的高效液相色谱检测方法
WO2023065013A1 (en) * 2021-10-22 2023-04-27 Barrelwise Technologies Ltd. System and method for rapid determination of free sulfur dioxide concentration in a liquid
CN114280179B (zh) * 2021-12-22 2024-03-15 北京美福润医药科技股份有限公司 艾塞那肽的前处理及其得到的His氨基酸洗脱液中异构体的检测方法
CN114609286A (zh) * 2022-03-18 2022-06-10 浙江理工大学 一种检测涤纶织物中间苯二甲酸含量的方法
KR102742623B1 (ko) * 2022-04-07 2024-12-16 에쓰대시오일 주식회사 임팩트 폴리프로필렌 내 에틸렌-프로필렌 고무 함량 측정 방법 및 측정 장치
CN115078592B (zh) * 2022-07-15 2024-01-23 广东广纳安疗科技有限公司 丙烯酰胺类单体的检测方法及其应用
CN115656391B (zh) * 2022-12-12 2023-04-07 哈尔滨吉象隆生物技术有限公司 一种特立帕肽含有的杂质的检测方法
CN116429950B (zh) * 2023-06-07 2023-08-11 成都佩德生物医药有限公司 多肽pd-dp-005的有关物质分析方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090263347A1 (en) * 2008-04-16 2009-10-22 Momenta Pharmaceutical, Inc. Analysis of amino acid copolymer compositions
WO2012123959A2 (en) * 2011-02-14 2012-09-20 Usv Limited Copolymer-1, process for preparation and analytical methods thereof
CN103335873A (zh) * 2013-06-04 2013-10-02 深圳翰宇药业股份有限公司 测量氨基酸-n-甲酸酐中氯离子含量的方法

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL119989A0 (en) * 1997-01-10 1997-04-15 Yeda Res & Dev Pharmaceutical compositions for oral treatment of multiple sclerosis
JPH11271294A (ja) * 1998-03-23 1999-10-05 Mitsubishi Chemical Corp 球状多孔質架橋重合体粒子およびその製造方法
EP1500661A1 (en) * 1998-06-01 2005-01-26 Genetech, Inc. Separation of protein monomers from aggregates by use of ion-exchange chromatography
IL141021A0 (en) * 1998-07-23 2002-02-10 Yeda Res & Dev Treatment of autoimmune conditions with copolymer 1 and related copolymers
DK2239269T3 (da) * 1998-09-25 2013-04-29 Yeda Res & Dev Anvendelse af peptider afledt af copolymer-1 som molekylvægtsmarkører
HUE033623T2 (en) * 2002-09-11 2017-12-28 Genentech Inc protein purification
CN1591005A (zh) * 2003-09-02 2005-03-09 浙江大学 键合型阳离子色谱柱及其制备方法
WO2005078125A2 (en) * 2004-02-18 2005-08-25 Guy's And St. Thomas' Nhs Foundation Trust Itpase gene polymorphisms associated with adverse drug reactions to azathioprine therapy
ES2565035T3 (es) * 2006-04-28 2016-03-30 Momenta Pharmaceuticals, Inc. Métodos para evaluar mezclas de péptidos
US20090264630A1 (en) * 2006-06-09 2009-10-22 Ge Healthcare Bio-Sciences Ab Method of separating monomeric protein(s)
KR101696948B1 (ko) * 2009-05-08 2017-01-16 시노팜 타이완 리미티드 펩타이드 혼합물을 분석하는 방법
MX2012000087A (es) * 2009-07-09 2012-02-28 Biocon Ltd Metodo cromatografico basado en gradiente no lineal preparativo y productos purificados del mismo.
CN102713599A (zh) * 2010-01-25 2012-10-03 株式会社日立高新技术 液相色谱仪以及液相色谱仪用送液装置
NZ601117A (en) * 2010-03-03 2014-06-27 Boehringer Ingelheim Int Biparatopic abeta binding polypeptides
US9109006B2 (en) * 2010-07-29 2015-08-18 Santhanakrishnan Srinivasan Glatiramer acetate molecular weight markers
JP6355563B2 (ja) * 2012-02-27 2018-07-11 アムニクス オペレーティング インコーポレイテッド Ｘｔｅｎ共役組成物およびそれを製造する方法
CN104098655B (zh) * 2013-04-09 2018-01-30 深圳翰宇药业股份有限公司 用于合成醋酸格拉替雷的质谱内标的多肽
CN103265624B (zh) * 2013-05-27 2015-04-22 成都圣诺生物制药有限公司 格拉替雷的制备方法
WO2015008845A1 (ja) * 2013-07-17 2015-01-22 積水メディカル株式会社 試料分析装置におけるグラジエント送液装置
CN104371012A (zh) * 2013-08-12 2015-02-25 深圳翰宇药业股份有限公司 一种合成醋酸格拉替雷的方法
CN104297404B (zh) * 2014-09-26 2016-08-24 深圳翰宇药业股份有限公司 一种用于测定醋酸格拉替雷样品中哌啶杂质含量的方法
US20180164275A1 (en) * 2015-04-30 2018-06-14 Teva Pharmaceutical Industries Ltd. Method of determining the molecular weight distribution of glatiramer acetate using multi-angle laser light scattering (malls)
EP3170836B1 (en) * 2015-11-23 2018-10-24 Chemi SPA Rp-hplc analysis of complex polypeptide mixtures

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090263347A1 (en) * 2008-04-16 2009-10-22 Momenta Pharmaceutical, Inc. Analysis of amino acid copolymer compositions
WO2012123959A2 (en) * 2011-02-14 2012-09-20 Usv Limited Copolymer-1, process for preparation and analytical methods thereof
CN103335873A (zh) * 2013-06-04 2013-10-02 深圳翰宇药业股份有限公司 测量氨基酸-n-甲酸酐中氯离子含量的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3290432A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114384194A (zh) * 2021-12-28 2022-04-22 湖南中晟全肽生化有限公司 一种高通量检测多肽的方法
CN114384194B (zh) * 2021-12-28 2023-09-22 湖南中晟全肽生化有限公司 一种高通量检测多肽的方法
CN115586288A (zh) * 2022-10-08 2023-01-10 广州朗圣药业有限公司 一种含szfz13-a的组合物中szfz13-a含量的高效液相色谱检测方法

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