CN105277606A - Determination method for exenatide related substance - Google Patents
Determination method for exenatide related substance Download PDFInfo
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- CN105277606A CN105277606A CN201410321588.0A CN201410321588A CN105277606A CN 105277606 A CN105277606 A CN 105277606A CN 201410321588 A CN201410321588 A CN 201410321588A CN 105277606 A CN105277606 A CN 105277606A
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Abstract
The invention provides a determination method for an exenatide related substance. Compared with traditional HPLC detection methods, the determination method provided by the invention has the advantages of simpler operation, faster analysis speed, better separation effect and low cost.
Description
Technical field
The present invention relates to the detection method of Exenatide related substance.
Background technology
Glucagon-like-peptide-1 (GLP-1) analog Exenatide (exenatide) be develop in diabetes B treatment field in recent years with one of two Hormone Deficiency newly-developed hypoglycemic agent that is target.Exenatide is secretin's analog, it is glucagon-like-peptide-1 (GLP-1) receptor stimulating agent, can the effect of class GLP-1 be played with GLP-1 receptors bind thus reduce blood sugar, there is promotion insulin secretion, increase insulin sensitivity and improve the effect of islet cell function, having the advantage that traditional treatment diabetes medicament is incomparable.Exenatide is the 1st the GLP-1 analog gone on the market by U.S. food Drug Administration (FDA) approval in 2005, in 2009 in the granted listing of China.
Polypeptide drug is combined in a certain order by different seed amino acid, and need the synthesis step of process many compared to small-molecule drug, consequent related substance is comparatively complicated.The related substance of improvement on synthesis can be divided into four classes, the process contaminants being mainly 1, bringing in building-up process: disappearance peptide, and fracture peptide, inserts peptide, the peptide class related substanceses such as non-deprotection peptide; 2, degradation impurity: polypeptide deacylated tRNA amine, oxidation, reduction, hydrolysis, disulfide bond mispairing, the labile factors such as β elimination and the catabolite that produces; 3, polymkeric substance: dimer and polymer; 4, optics impurity: racemization, diastereo-isomerism impurity.Polypeptide drug preparation is parenteral solution and injection powder pin mainly, in production and storage process, degraded change more easily occurs, produces degradation impurity.Because impurity is numerous in improvement on synthesis, composition is comparatively complicated, and character is different, and what have is very similar to polypeptide drugs, and some difference are comparatively large, and some impurity also may produce larger toxicity under low concentration.According to relevant research, one or two amino acid order is had to change in polypeptide, just likely activator is become antagonist, produce opposite effect, Drug safety is not only also relevant with the toxicity of impurity with the toxicity of main active, detecting these impurity so set up suitable method and carry out assay, is the key of carrying out polypeptide drug quality control.The related substance limit of polypeptide drug is limited in European Pharmacopoeia 7.0 annex " Substancesforpharmaceuticaluse ", formulate reporting limit (0.1%) respectively, limit of identification (0.5%) and Quality Control limit (1.0%).As can be seen here, the control research of polypeptide drugs related substance needs deeply.For different types of impurity, corresponding detection method must be adopted.
Exenatide is 39 peptides of solid phase technique synthesis, wherein impurity be included in that produce in building-up process with assorted peptide that is target polypeptides similar (as diastereo-isomerism, lack peptide, rupture peptide etc.), the catabolite (as disulfide exchange, oxidation product etc.) produced because polypeptide is unstable and polymkeric substance etc.These impurity have a strong impact on the quality and safety of product, therefore need to select the related substance of reliable analytical approach to Exenatide to check.
Polypeptide drug is commonly used preparative high performance liquid chromatography and is carried out purifying, and some impurity may elute with target peptide simultaneously, if use the HPLC method of same principle, purity testing the possibility of result is untrue.Simultaneously HPLC method exists that separating effect is not good enough, analysis time long, high in cost of production limitation.Therefore be necessary to develop the related substance that additive method detects Exenatide.
Summary of the invention
The invention provides a kind of detection method of Exenatide related substance, adopt capillary electrophoresis method, wherein the pH of damping fluid is 5.0 ~ 7.0, is preferably 5.5 ~ 6.5.Said damping fluid can be selected from phosphate buffer, ammonium acetate buffer system, HEPES buffer system, MES buffer system, is preferably phosphate buffer, ammonium acetate buffer system, especially phosphate buffer.
The concentration of damping fluid is 150 ~ 250mmol/L, is preferably 200 ~ 250mmol/L, is more preferably 200mmol/L.Can add acetonitrile in described damping fluid, in damping fluid, the concentration of volume percent of acetonitrile is 13 ~ 20%, is preferably 15%.Kapillary column temperature is 20 ~ 30 DEG C, is preferably 25 DEG C.Working voltage is 15 ~ 25kV, is preferably 20kV.
Capillary column can select non-coating fused quartz kapillary, saves cost.
Determined wavelength is: 214nm
The detection method of Exenatide related substance provided by the invention, relative to traditional HPLC detection method, its operation is easier, analysis speed is faster, separating effect is better and with low cost.
Accompanying drawing explanation
Fig. 1: embodiment 1 Exenatide Capillary Electrophoresis collection of illustrative plates
Fig. 2: embodiment 3 pH of buffer-degree of separation figure
Fig. 3: embodiment 4 buffer concentrations-degree of separation figure
Fig. 4: embodiment 5 column temperatures-degree of separation figure
Fig. 5: embodiment 6 Exenatide HPLC analyzes collection of illustrative plates
Embodiment
Further illustrate the present invention with test example by the following examples, but do not limit the present invention in any way.
The detection of embodiment 1 Exenatide bulk drug related substance
The preparation of sample: get Exenatide bulk drug, uses water-soluble solution, and compound concentration is the solution of 2mg/mL
Capillary Electrophoresis condition:
Running buffer is 200mmol/L sodium dihydrogen phosphate-disodium phosphate soln (pH6.2, containing 15%v/v acetonitrile); Working voltage 20kV, temperature 25 DEG C; Hydrodynamic injection 5kPa × 10s; Determined wavelength 214nm
Non-capillary column having coated layer (48.5cm × 50 μm, effective length 40cm)
Test apparatus: Agilent7100 type capillary electrophoresis apparatus, joins diode array detector
Test method: kapillary 1mol/L sodium hydroxide solution, 0.1mol/L sodium hydroxide solution, water, running buffer respectively rinse 10min under 100kPa.Respectively rinse 2min with 0.1mol/L sodium hydroxide solution, 0.1mol/L hydrochloric acid solution, water between two pins, running buffer rinses 5min.After flushing completes, analyze according to deposition condition.At the end of with 0.1molL
-1sodium hydroxide solution rinses 10min under 300kPa pressure, 0.1molL
-1hydrochloric acid solution rinses 10min, and water rinses 10min, and kapillary two ends are placed in water and spend the night.
Calculate by area normalization method, impurity content is 1.7%.Test findings is shown in Fig. 1.In Fig. 1, peak 1 is Exenatide; Peak 2-10 is impurity (wherein 5 is Acetyl-Exenatide).
The contrast test of the different pH damping fluid of embodiment 2
According to the method for embodiment 1, adopt different pH buffer system, measure Exenatide related substance, the degree of separation of main peak and Acetyl-Exenatide, and impurity number is in table 1.
The different pH damping fluid of table 1 detects degree of separation and the impurity number of Exenatide related substance
Result shows: as the pH<5 of damping fluid, does not substantially have removing foreign matter; As pH>7, separating effect weakens, and impurity A cetyl-Exenatide does not separate with main peak; When pH5 ~ 7, separating effect is relatively better, at least can isolate 6 impurity.
The contrast test of embodiment 3 variable concentrations damping fluid
Damping fluid: select 100 respectively, 150,200,250mmol/L sodium dihydrogen phosphate-disodium phosphate soln, other test conditions and test method are with embodiment 1, with main peak in Fig. 11 and other impurities peak 5, the degree of separation of the impurity peaks 6 and 7 that content is relatively large is inspection target, and test findings is in table 2 and Fig. 2.
Table 2 variable concentrations damping fluid detects the degree of separation of Exenatide related substance
Result shows: along with the increase of buffer concentration, R (1,5) increase gradually, R (6,7) first increases and then decreases, when buffer concentration is 150 ~ 250mmol/L, separating effect is better, but when concentration is 200mmol/L, not only good separating effect, and current value is relatively little, reproducible.
Embodiment 4 adds the contrast test of variable concentrations acetonitrile
According to the method for embodiment 1, add the acetonitrile of variable concentrations in damping fluid, with main peak in Fig. 11 and other impurities peak 5, the degree of separation of the impurity peaks 6 and 7 that content is relatively large is inspection target, and test findings is in table 3 and Fig. 3.
Table 3 variable concentrations acetonitrile detects the degree of separation of Exenatide related substance
Result shows: along with acetonitrile concentration increases, R (1,5) increases gradually, and R (6,7) reduces gradually; Acetonitrile concentration reaches good separating effect in 13 ~ 20% scopes.
The contrast test of the different kapillary column temperature of embodiment 5
According to the method for embodiment 1, adopt different column temperature (15,20,25,30 DEG C) to investigate, with main peak in Fig. 11 and other impurities peak 5, the degree of separation of the impurity peaks 6 and 7 that content is relatively large is inspection target, and test findings is in table 4 and Fig. 4.
The different capillary column temperature detector of table 4 surveys the degree of separation of Exenatide related substance
Result shows: along with the rising of column temperature, and R (1,5) and R (6,7) first rise and decline afterwards, and have good degree of separation at 20 ~ 30 DEG C, when 25 DEG C, degree of separation reaches optimal value.
Embodiment 6 capillary electrophoresis and HPLC method contrast test
Capillary electrophoresis: according to the method for embodiment 1
HPLC method:
Chromatographic column: YMC-PackProC18 (150mm × 4.6mm, 3 μm); With 0.1% trifluoroacetic acid-water for mobile phase A, 0.1% trifluoroacetic acid-acetonitrile is Mobile phase B, adopts gradient elution: (0 ~ 30min, 20% → 50%B; 30 ~ 30.1min, 50% → 20%B; 30.1 ~ 40min, 20% → 30%B; 40.1 ~ 50min, 20%B); Flow velocity 0.9ml/min; Column temperature 40 DEG C; Determined wavelength 214nm; Sample size 50 μ L.
Investigate degree of separation and the analysis time of two kinds of method main peaks and adjacent peaks respectively, test findings is in table 5, Fig. 1 and Fig. 5.
Table 5 Capillary Electrophoresis and HPLC detect the degree of separation of Exenatide related substance
Result shows: the impurity peaks separating effect that HPLC method is closed on main peak is not good enough, and the degree of separation at main peak and Acetyl-Exenatide peak (peak 7) is 1.09; The separating effect of capillary electrophoresis is better, and the degree of separation at main peak and Acetyl-Exenatide peak (peak 5) is 1.62.HPLC method main peak retention time is 21.703min, and last impurity peaks retention time is 24.190min; The main peak transit time of capillary electrophoresis is 12.053min, and last impurity peaks transit time is 14.646min, and analysis time is shorter, more saves time.
Claims (9)
1. a detection method for Exenatide related substance, it is characterized in that adopting capillary electrophoresis method, the pH of damping fluid is 5.0 ~ 7.0.
2. want the detection method described in 1 according to right, it is characterized in that the pH of damping fluid is 5.5 ~ 6.5.
3. want the detection method described in 1 according to right, it is characterized in that damping fluid is phosphate buffer, ammonium acetate buffer system, HEPES buffer system, MES buffer system, be preferably phosphate buffer, ammonium acetate buffer system.
4. want the detection method described in 2 according to right, it is characterized in that damping fluid is sodium dihydrogen phosphate-disodium phosphate soln.
5. want the arbitrary described detection method of 1-4 according to right, it is characterized in that the concentration of damping fluid is 150 ~ 250mmol/L, be preferably 200 ~ 250mmol/L, be more preferably 200mmol/L.
6. want the arbitrary described detection method of 1-4 according to right, it is characterized in that adding acetonitrile in damping fluid.
7. want the detection method described in 6 according to right, it is characterized in that the concentration of volume percent of acetonitrile in damping fluid is 13 ~ 20%.
8. want the arbitrary described detection method of 1-4 according to right, it is characterized in that kapillary column temperature is 20 ~ 30 DEG C, be preferably 25 DEG C.
9. want the arbitrary described detection method of 1-4 according to right, it is characterized in that working voltage is 15 ~ 25kV, be preferably 20kV.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107515269A (en) * | 2017-08-15 | 2017-12-26 | 浙江美华鼎昌医药科技有限公司 | A kind of ultra performance liquid chromatography analysis method of Exenatide |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1916602A (en) * | 2005-08-19 | 2007-02-21 | 上海市公安局刑事侦查总队 | Capillary electrophoresis method for detecting medications and poisons in blood and urine at the same time |
| EP2013628A2 (en) * | 2006-04-28 | 2009-01-14 | Momenta Pharmaceuticals, Inc. | Methods of evaluating peptide mixtures |
| CN102323320A (en) * | 2011-06-07 | 2012-01-18 | 首都师范大学 | Capillary electrophoresis method for identifying low molecular weight glutelin subunit of wheat and application thereof |
| CN102827270A (en) * | 2012-09-13 | 2012-12-19 | 无锡和邦生物科技有限公司 | Pegylated exenatide ramification and use thereof |
| CN103163200A (en) * | 2013-03-25 | 2013-06-19 | 江苏省原子医学研究所 | Method for measuring content of pyrroloquinoline quinone dimethyl ester by micellar electrokinetic chromatography |
-
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- 2014-07-08 CN CN201410321588.0A patent/CN105277606A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1916602A (en) * | 2005-08-19 | 2007-02-21 | 上海市公安局刑事侦查总队 | Capillary electrophoresis method for detecting medications and poisons in blood and urine at the same time |
| EP2013628A2 (en) * | 2006-04-28 | 2009-01-14 | Momenta Pharmaceuticals, Inc. | Methods of evaluating peptide mixtures |
| CN102323320A (en) * | 2011-06-07 | 2012-01-18 | 首都师范大学 | Capillary electrophoresis method for identifying low molecular weight glutelin subunit of wheat and application thereof |
| CN102827270A (en) * | 2012-09-13 | 2012-12-19 | 无锡和邦生物科技有限公司 | Pegylated exenatide ramification and use thereof |
| CN103163200A (en) * | 2013-03-25 | 2013-06-19 | 江苏省原子医学研究所 | Method for measuring content of pyrroloquinoline quinone dimethyl ester by micellar electrokinetic chromatography |
Non-Patent Citations (1)
| Title |
|---|
| 刘洁欣 等: "全柱成像毛细管等电聚焦电泳测定艾塞那肽等电点", 《生物技术通讯》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107515269A (en) * | 2017-08-15 | 2017-12-26 | 浙江美华鼎昌医药科技有限公司 | A kind of ultra performance liquid chromatography analysis method of Exenatide |
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