CN112649537B - 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 PDFInfo
- Publication number
- CN112649537B CN112649537B CN202011638347.0A CN202011638347A CN112649537B CN 112649537 B CN112649537 B CN 112649537B CN 202011638347 A CN202011638347 A CN 202011638347A CN 112649537 B CN112649537 B CN 112649537B
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- sample
- liquid chromatography
- chromatographic column
- elution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000004458 analytical method Methods 0.000 title claims abstract description 17
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 7
- 239000000203 mixture Substances 0.000 title abstract description 7
- 229920001184 polypeptide Polymers 0.000 title abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 title abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 6
- 238000010828 elution Methods 0.000 claims abstract description 34
- 108010072051 Glatiramer Acetate Proteins 0.000 claims abstract description 18
- FHEAIOHRHQGZPC-KIWGSFCNSA-N acetic acid;(2s)-2-amino-3-(4-hydroxyphenyl)propanoic acid;(2s)-2-aminopentanedioic acid;(2s)-2-aminopropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound CC(O)=O.C[C@H](N)C(O)=O.NCCCC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CCC(O)=O.OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 FHEAIOHRHQGZPC-KIWGSFCNSA-N 0.000 claims abstract description 18
- 229960003776 glatiramer acetate Drugs 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 7
- 239000013558 reference substance Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 62
- 239000002245 particle Substances 0.000 claims description 46
- 239000000945 filler Substances 0.000 claims description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- 238000002347 injection Methods 0.000 claims description 23
- 239000007924 injection Substances 0.000 claims description 23
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 10
- 230000005284 excitation Effects 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 230000003247 decreasing effect Effects 0.000 claims 1
- 229920001577 copolymer Polymers 0.000 abstract description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 57
- 238000012360 testing method Methods 0.000 description 53
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 26
- 238000004587 chromatography analysis Methods 0.000 description 17
- 239000007788 liquid Substances 0.000 description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 14
- 241000202964 Mycoplasma mobile Species 0.000 description 14
- 229920003053 polystyrene-divinylbenzene Polymers 0.000 description 13
- 239000011780 sodium chloride Substances 0.000 description 13
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium group Chemical group [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 238000005277 cation exchange chromatography Methods 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000005571 anion exchange chromatography Methods 0.000 description 3
- MWWQKLKFKPWECK-UHFFFAOYSA-N aminomethanetriol;hydrochloride Chemical compound Cl.NC(O)(O)O MWWQKLKFKPWECK-UHFFFAOYSA-N 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 125000000998 L-alanino group Chemical group [H]N([*])[C@](C([H])([H])[H])([H])C(=O)O[H] 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 229960004441 tyrosine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- 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
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Hospice & Palliative Care (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Psychiatry (AREA)
Abstract
本发明涉及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
本申请是申请号201580079194.1,申请日为2015年4月28日,发明名称为“多肽混合物高效液相色谱分析方法”的中国专利申请的分案申请。
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,商品名为于1996年获美国FDA核准用于治疗多发性硬化症,目前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)xxCH3COOH
(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
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
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
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
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
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
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
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
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:
/>
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
/>
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
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
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 (4)
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,并分析醋酸格拉替雷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)配制待测醋酸格拉替雷溶液;
步骤(2)以反相液色谱法,对待测样本进行梯度洗脱,
步骤(3)检测分析醋酸格拉替雷各组分对应峰面积,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5-50μL,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%;
其中的N为5≤N≤15;
柱温25-50℃。
2.根据权利要求1所述的方法,其特征在于,N为8≤N≤12。
3.根据权利要求1-2任一所述的方法,其特征在于,N=10。
4.根据权利要求1-2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。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011638347.0A CN112649537B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201580079194.1A CN107531750B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
CN202011638347.0A CN112649537B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
PCT/CN2015/077690 WO2016172855A1 (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201580079194.1A Division CN107531750B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112649537A CN112649537A (zh) | 2021-04-13 |
CN112649537B true CN112649537B (zh) | 2024-03-29 |
Family
ID=57198918
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011638347.0A Active CN112649537B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
CN202011643350.1A Active CN112649538B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
CN201580079194.1A Active CN107531750B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011643350.1A Active CN112649538B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
CN201580079194.1A Active CN107531750B (zh) | 2015-04-28 | 2015-04-28 | 多肽混合物高效液相色谱分析方法 |
Country Status (5)
Country | Link |
---|---|
US (1) | US10330650B2 (zh) |
EP (3) | EP3290432B1 (zh) |
JP (1) | JP6676072B2 (zh) |
CN (3) | CN112649537B (zh) |
WO (1) | WO2016172855A1 (zh) |
Families Citing this family (11)
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 |
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 |
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 |
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 |
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 (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000018794A1 (en) * | 1998-09-25 | 2000-04-06 | Yeda Research And Development Co., Ltd. | Copolymer 1 related polypeptides for use as molecular weight markers and for therapeutic use |
WO2012123959A2 (en) * | 2011-02-14 | 2012-09-20 | Usv Limited | Copolymer-1, process for preparation and analytical methods thereof |
CN104098655A (zh) * | 2013-04-09 | 2014-10-15 | 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 |
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 |
Family Cites Families (22)
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 |
ES2228052T3 (es) * | 1998-06-01 | 2005-04-01 | Genentech, Inc. | Separacion demonomeros de anticuerpos de sus multimeros utilizando cromatografia de intercambio de iones. |
IL141021A0 (en) * | 1998-07-23 | 2002-02-10 | Yeda Res & Dev | Treatment of autoimmune conditions with copolymer 1 and related copolymers |
EP3388452A3 (en) * | 2002-09-11 | 2019-02-20 | 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 |
EP2013628B1 (en) * | 2006-04-28 | 2016-01-06 | Momenta Pharmaceuticals, Inc. | Methods of evaluating peptide mixtures |
WO2007142578A1 (en) * | 2006-06-09 | 2007-12-13 | Ge Healthcare Bio-Sciences Ab | Method of separating monomeric protein (s) |
EP2277050B2 (en) | 2008-04-16 | 2022-09-28 | Momenta Pharmaceuticals, Inc. | Analysis of amino acid copolymer compositions |
BRPI1011434A2 (pt) * | 2009-05-08 | 2016-03-15 | Scinopharm Taiwan Ltd | metodos de analisar misturas de peptideo |
CA2766571A1 (en) * | 2009-07-09 | 2011-02-24 | Biocon Limited | A preparative non-linear gradient based chromatographic method and purified products thereof |
JPWO2011090188A1 (ja) * | 2010-01-25 | 2013-05-23 | 株式会社日立ハイテクノロジーズ | 液体クロマトグラフ、および液体クロマトグラフ用送液装置 |
JP5826194B2 (ja) * | 2010-03-03 | 2015-12-02 | アブリンクス ナームローゼ フェンノートシャップ | 二パラトープ性a−ベータ結合ポリペプチド |
US9109006B2 (en) * | 2010-07-29 | 2015-08-18 | Santhanakrishnan Srinivasan | Glatiramer acetate molecular weight markers |
KR102057356B1 (ko) * | 2012-02-27 | 2019-12-18 | 아뮤닉스 파마슈티컬스, 인크. | Xten 콘주게이트 조성물 및 그의 제조 방법 |
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 |
CN103335873B (zh) * | 2013-06-04 | 2017-04-19 | 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 |
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 |
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 |
-
2015
- 2015-04-28 CN CN202011638347.0A patent/CN112649537B/zh active Active
- 2015-04-28 JP JP2017556727A patent/JP6676072B2/ja active Active
- 2015-04-28 CN CN202011643350.1A patent/CN112649538B/zh active Active
- 2015-04-28 US US15/559,428 patent/US10330650B2/en active Active
- 2015-04-28 WO PCT/CN2015/077690 patent/WO2016172855A1/zh active Application Filing
- 2015-04-28 EP EP15890235.3A patent/EP3290432B1/en active Active
- 2015-04-28 EP EP19198022.6A patent/EP3613756B1/en active Active
- 2015-04-28 CN CN201580079194.1A patent/CN107531750B/zh active Active
- 2015-04-28 EP EP19198009.3A patent/EP3603764B1/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000018794A1 (en) * | 1998-09-25 | 2000-04-06 | Yeda Research And Development Co., Ltd. | Copolymer 1 related polypeptides for use as molecular weight markers and for therapeutic use |
WO2012123959A2 (en) * | 2011-02-14 | 2012-09-20 | Usv Limited | Copolymer-1, process for preparation and analytical methods thereof |
CN104098655A (zh) * | 2013-04-09 | 2014-10-15 | 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 |
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 |
Non-Patent Citations (1)
Title |
---|
RP-HPLC METHOD FOR QUANTITATIVE ESTIMATION OF GLATIRAMER ACETATE FOR INJECTION IN PHARMACEUTICAL DOSAGE FORMS;ANUSHA DEVI.V等;《The Experiment》;20121031;第33卷(第3期);第193-198页 * |
Also Published As
Publication number | Publication date |
---|---|
CN112649538A (zh) | 2021-04-13 |
CN112649537A (zh) | 2021-04-13 |
EP3613756B1 (en) | 2024-02-21 |
CN107531750B (zh) | 2021-03-16 |
US20180080909A1 (en) | 2018-03-22 |
CN112649538B (zh) | 2024-03-29 |
EP3290432B1 (en) | 2022-03-30 |
CN107531750A (zh) | 2018-01-02 |
US10330650B2 (en) | 2019-06-25 |
EP3290432A4 (en) | 2019-03-27 |
JP6676072B2 (ja) | 2020-04-08 |
EP3603764B1 (en) | 2022-03-30 |
EP3290432A1 (en) | 2018-03-07 |
EP3613756A1 (en) | 2020-02-26 |
EP3603764A1 (en) | 2020-02-05 |
JP2018515766A (ja) | 2018-06-14 |
WO2016172855A1 (zh) | 2016-11-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112649537B (zh) | 多肽混合物高效液相色谱分析方法 | |
Murphy et al. | Endogenous pyrogens made by rabbit peritoneal exudate cells are identical with lymphocyte-activating factors made by rabbit alveolar macrophages. | |
MX2012014838A (es) | Comparacion de las muestras de proteina. | |
Nice et al. | Synergies between micropreparative high-performance liquid chromatography and an instrumental optical biosensor | |
Maidment et al. | Measurement of extracellular neuropeptides in the brain: microdialysis linked to solid-phase radioimmunoassays with sub-femtomole limits of detection | |
CN103134892A (zh) | 复方氨基酸(15)双肽(2)注射液的分析方法 | |
Rea et al. | Capillary ion-exchange chromatography with nanogram sensitivity for the analysis of monoclonal antibodies | |
CN104897832B (zh) | 转移因子溶液及其制剂中游离氨基酸含量的高效液相色谱检测方法 | |
JP2016027326A (ja) | 測定方法、測定装置および溶離液 | |
Stuting et al. | Determination of pituitary and recombinant human growth hormone molecular weights by modern high-performance liquid chromatography with low angle laser light scattering detection | |
Yaron et al. | Lysine oligopeptides. Preparation by ion‐exchange chromatography | |
EP3170836B1 (en) | Rp-hplc analysis of complex polypeptide mixtures | |
Souto et al. | Stability-indicating capillary zone electrophoresis method for the assessment of recombinant human interleukin-11 and its correlation with reversed-phase liquid chromatography and biossay | |
Scanlon et al. | Evaluation of the precision of high-performance liquid chromatography for wheat cultivar identification | |
Abed et al. | Evaluation of the combined effects of pegylation and glycosylation on the stability of erythropoietin using a stability-indicating SE-HPLC | |
Hamidi et al. | A reversed‐phase high‐performance liquid chromatography method for bovine serum albumin assay in pharmaceutical dosage forms and protein/antigen delivery systems | |
CN105223281B (zh) | 一种用于检测卡帕松制剂中醋酸格拉替雷浓度的色谱方法 | |
Jadaun et al. | Comparison of two pharmacopoeia methods for determining higher molecular weight proteins in insulin glargine | |
US11867671B2 (en) | Method for amino acid analysis and system for amino acid analysis | |
Souto et al. | Assessment of rhIL-11 by Validated SE-LC Method and its Correlation with RP-LC and CZE Methods | |
Saadane et al. | Inhibition of liver RNA breakdown during acute inflammation in the rat | |
CN105218646B (zh) | 一种用于检测醋酸格拉替雷样本的uplc方法 | |
Lemieux et al. | Separation of a casein hydrolyzate by HPSEC with a new mobile phase and characterization of peptides by FABMS | |
Glajch et al. | Molecular Weight Analysis of Glatiramer Acetate and Related Compounds by Size-Exclusion Chromatography with Multi-Angle Light Scattering Detection (SEC-MALS) | |
CN114252519A (zh) | 一种测定神经生长因子纯度的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |