CN100419422C - Method for determining nerve growth factor content - Google Patents
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- CN100419422C CN100419422C CNB2005101303483A CN200510130348A CN100419422C CN 100419422 C CN100419422 C CN 100419422C CN B2005101303483 A CNB2005101303483 A CN B2005101303483A CN 200510130348 A CN200510130348 A CN 200510130348A CN 100419422 C CN100419422 C CN 100419422C
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/48—Nerve growth factor [NGF]
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Abstract
A method for determining content of nerve growth factor includes chromatograph conditions as follows, gel chromatographic column in molecular weight detection scope of 10000-500000 dalton and with hole diameter of 200-300 angstrom gel column, column temperature in room temperature, detection wavelength of 205-220 nm, flow rate of 0.6-1.0ml/min, flowing phase in pH value of 6.5-7.2 and with salt solution concentration of 0.07-2.0 mol/L.
Description
Technical field
The present invention relates to the method for the nerve growth factor content in a kind of new high performance liquid chromatogram gel chromatography preparation.
Background technology
Nerve growth factor (NGF) is one of topmost active component of nervous system; it can keep the existence of sympathetic nerve and sensory neurone; influence the survival of axoneuron; promote the differentiation of neurocyte; normal function to the neuroprotective system has important effect, and China has ratified the nerve growth factor preparation and sold and use as medicine.
The content of NGF props up at 4～30 μ g/ in the existing nerve growth factor preparation, adopts human serum albumin as protective agent, because human serum albumin and NGF are protein, the content of human serum albumin in preparation is much higher than NGF, has influenced the quantitative measurement of NGF.For solving this difficult problem, people have attempted the multiple method of inspection, as Lowry method, ultraviolet absorption method, Bradford method and ELISA method etc., fail but all limit because of the sensitivity of the interference that can't get rid of human serum albumin or examined method itself.We once declared the content of analyzing NGF with exclusion chromatography, moving phase is 0.01～0.07M phosphate buffer, had solved the problem that NGF content can't quantitative test.We were through further a large amount of experiment discoveries afterwards, and the ionic strength of moving phase will influence the wash-out degree of NGF on chromatographic column to a great extent.In the patent of having declared, the ionic strength of moving phase is 0.01～0.07M phosphate buffer, lowest detection is limited to 10 μ g/ml, we are with the ionic strength of moving phase more preferably during 0.15～0.5M phosphate buffer, lowest detection is limited to 2 μ g/ml, and sensitivity has improved 5 times, even under the situation that NGF content is very low in preparation, also can accurately quantitatively detect, improve the accuracy of analyzing.
Summary of the invention
The method that the purpose of this invention is to provide the nerve growth factor content in a kind of new mensuration preparation.
In the analysis of high performance liquid chromatogram gel chromatography, select for use salt solusion and damping fluid, control the purpose that ionic strength in the moving phase can reach accurate quantitative test simultaneously as moving phase.
For achieving the above object, the present invention is by the following technical solutions:
A kind of method of measuring nerve growth factor content in the preparation, this method are exclusion chromatography, use high performance liquid chromatograph to detect, and chromatographic condition is:
(1) gel chromatographic columns: the molecular weight detection scope is 10000～500000 dalton, and the aperture is the gel column of 200～300 dusts;
(2) column temperature: room temperature;
(3) detect wavelength: 205～220nm;
(4) flow velocity: 0.6～1.0ml/min;
(5) moving phase: the pH value is 6.5～7.2, and concentration is 0.07～2.0mol/L salt solusion or damping fluid.
Wherein moving phase preferably: biphosphate sodium water solution, ammonium acetate aqueous solution, sodium sulphate (potassium) aqueous solution, sodium citrate (potassium) salt buffer, ammonium formate aqueous solution, sodium chloride (potassium) aqueous solution, Tris-acetate buffer solution, sodium acetate (potassium) salt buffer or sodium phosphate (potassium) salt buffer; More preferably sodium phosphate (potassium) salt buffer.
Use the exclusion chromatography in the high performance liquid chromatography that the content of NGF in the preparation is measured, its ultimate principle is by the difference of the molecular weight of the various materials of the chromatographic column of flowing through it to be separated, and molecular weight is big more, and retention time is short more.Because every kind of material has specific retention time under certain condition, therefore can know the position and the correlation parameter at purpose peak, and calculate the content of object by above-mentioned parameter by the retention time on the chromatogram.Selecting the cardinal rule of gel column for use is the molecular weight that its sensing range comprises material to be checked.The molecular weight of NGF monomer is 13.6kD, and its dimeric molecular weight is 26kD, thus the present invention selected for use the molecular weight detection scope at 10000～50000 daltonian gel columns as the analysis chromatographic column.The standard that wavelength is chosen is relevant with the optical absorption property of material to be checked, and to a kind of protein or polypeptide, its maximum absorption wavelength is fixed.By UV scanning, find that NGF all has light absorption at 205～220nm wavelength, can be used as the detection wavelength.
The preferred TSK G3000 of described gel chromatographic columns SW XL, 7.8 * 300,5 μ, (TOSOH, Japan), SHODEXProtein KW-804 (SHODEX, Japan) or BSA-7 (MN, Germany), Phenomenex K3 (Phenomenex, the U.S.).The molecular weight ranges of these pillars is all 10000～500000 dalton, and the aperture is 200～300 dusts.
By experiment, find out preferable embodiment of the present invention, wherein the pH value of moving phase is 7.0, and flow velocity is 0.6～1.0ml/min, and the detection wavelength is 214nm.
Above exclusion chromatography can use external standard method that preparation is measured.
Described external standard method comprises the steps:
(1) make typical curve: getting the pure product of the known NGF of content is standard items, and gradient dilution is a moving phase with the phosphate buffer, cross gel chromatographic columns, under the detection wavelength, detect, the record chromatogram, obtain parameters such as retention time, peak height, peak area, obtain the typical curve and the range of linearity;
(2) external standard method NGF content: select suitable concentration in the range of linearity, preparation contrast solution and sample solution are measured under selected chromatographic condition, and the record chromatogram is pressed external standard method with calculated by peak area, promptly.
Experiment shows:
(1) same duplicate samples solution, sample size 20 μ l, when moving phase was the 0.05M phosphate buffer, the NGF peak area was 752103, and peak height is 28955, and lowest detection is limited to 10 μ g/ml.When moving phase was the 0.25M phosphate buffer, the NGF peak area was 3215088, and peak height is 128628, and lowest detection is limited to 2 μ g/ml.Detectability is low more, and then sensitivity is high more, and quantitative test is accurate more.Can calculate the sensitivity of the inventive method comparison photograph method by lowest detectable limit and improve 5 times.See accompanying drawing 1 and accompanying drawing 2.
When (2) ionic strength was 0.07～2.0M, there be not auxiliary material peak interference problem in nerve growth factor main peak and albumin main peak degree of separation greater than 1.5, but the equal quantitative Analysis of nerve growth factor main peak area in the preparation.See embodiment 1, embodiment 2.
When (3) we were preferably 0.10M～0.75M with moving phase intermediate ion intensity, nerve growth factor main peak area response value increased in the preparation, equally can quantitative Analysis.See embodiment 3 and embodiment 4.
(4) we are with moving phase intermediate ion intensity more preferably during 0.15M～0.5M, nerve growth factor main peak area response value maximum in the preparation, and sensitivity is the highest.See embodiment 5 and embodiment 6.
Advantage of the present invention is: adopting ionic strength is that salt solusion or the damping fluid of 0.07～2.0M is moving phase, can improve the detectability of nerve growth factor in the preparation greatly, has got rid of the interference of auxiliary materials such as human serum albumin in the preparation simultaneously.
Description of drawings
Fig. 1 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is the 0.05M phosphate buffer, and NGF peak retention time: 13min, NGF peak area are 752103, and peak height is 28955;
Fig. 2 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is the 0.25M phosphate buffer, the NGF peak area is 3215088, peak height is 128628, compare and to get with Fig. 1 result: after the ionic strength of moving phase improves 5 times, the NGF peak area rises to original 4.3 times, and peak height rises to original 4.4 times;
Fig. 3 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is 0.07M biphosphate sodium water solution;
Fig. 4 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is the 2M ammonium acetate aqueous solution, the presentation of results of Fig. 3 and Fig. 4: when ionic strength during at 0.07～2.0M, nerve growth factor main peak and albumin main peak degree of separation are all greater than 1.5, there is not auxiliary material peak interference problem, but the equal quantitative Analysis of nerve growth factor main peak area in the preparation;
Fig. 5 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is the 0.10M aqueous sodium persulfate solution;
Fig. 6 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is 0.75M sodium citrate salt buffer, the presentation of results of Fig. 5 and Fig. 6: when moving phase intermediate ion intensity is preferably 0.10M～0.75M, nerve growth factor main peak and albumin main peak degree of separation are all greater than 1.5, there is not auxiliary material peak interference problem, simultaneously nerve growth factor main peak area response value increases to some extent in the preparation, equally can quantitative Analysis;
Fig. 7 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is 0.15M Tris-acetate buffer solution;
Fig. 8 has shown the HPLC chromatographic peak collection of illustrative plates when moving phase is 0.5M sodium acetate salt buffer; The presentation of results of Fig. 7 and Fig. 8: with moving phase intermediate ion intensity more preferably during 0.15M～0.5M, nerve growth factor main peak area value maximum in the preparation.
Embodiment
Embodiment 1: adopt the mobile phase of 0.07mol/L sodium dihydrogen phosphate aqueous solution.
One chromatographic condition:
(1) instrument: use day island proper Tianjin Shimadzu LC-10Avp high performance liquid chromatograph;
(2) gel chromatographic columns: TSK G3000SWXL 7.8 * 3.0mm, internal diameter 0.5 μ m (TOSOH Japan);
(3) column temperature: room temperature;
(4) detect wavelength: 214nm;
(5) flow velocity: 0.8ml/min;
(6) moving phase pH value: 7.0.
Two experimental procedures
(1) sample preparation: get one in nerve growth factor preparation, the accurate moving phase 0.5ml that adds, jog makes dissolving, as need testing solution.
(2) reference substance is handled: precision is measured the mouse nerve growth factor reference substance and (is got mouse submandibular gland, go out NGF through ion-exchange chromatography and molecular sieve separation and purification, by " Chinese biological goods rules 2000 editions " requirement method check purity＞98%) an amount of, make the solution that contains 20 μ g among every 1ml approximately with moving phase, in contrast product solution.
(3) get reference substance solution 20 μ l, inject liquid chromatograph, the record chromatogram.Obtain parameters such as retention time, peak height, peak area.
The result: nerve growth factor main peak area A=914086, reference substance area A=903234, with calculated by peak area, sample size is 101.2% with external standard method.Nerve growth factor main peak and albumin main peak degree of separation are greater than 1.5, and sample main peak retention time is consistent with the reference substance retention time.HPLC chromatographic peak collection of illustrative plates is seen accompanying drawing 3.
Embodiment 2: adopting the 2.0mol/L ammonium acetate aqueous solution is moving phase.
One chromatographic condition: with embodiment 1.
Two experimental procedures
(1) sample and reference substance are handled with embodiment 1.
(2) accurate need testing solution and each 20 μ l of reference substance solution of drawing inject liquid chromatograph respectively, the record chromatogram.Obtain parameters such as retention time, peak height, peak area.
The result: nerve growth factor main peak area A=923186, reference substance area A=921096, with calculated by peak area, sample size is 100.2% with external standard method.Nerve growth factor main peak and albumin main peak degree of separation are greater than 1.5, and sample main peak retention time is consistent with the reference substance retention time.HPLC chromatographic peak collection of illustrative plates is seen accompanying drawing 4.
Embodiment 3: adopting the 0.10mol/L aqueous sodium persulfate solution is moving phase.
One chromatographic condition: with embodiment 1.
Two experimental procedures
(1) sample and reference substance are handled with embodiment 1.
(2) accurate need testing solution and each 20 μ l of reference substance solution of drawing inject liquid chromatograph respectively, the record chromatogram.Obtain parameters such as retention time, peak height, peak area.
The result: nerve growth factor main peak area A=3147076, reference substance area A=3098921, with calculated by peak area, sample size is 101.6% with external standard method.Nerve growth factor main peak and albumin main peak degree of separation are greater than 1.5, and sample main peak retention time is consistent with the reference substance retention time.HPLC chromatographic peak collection of illustrative plates is seen accompanying drawing 5.Embodiment 4: adopting 0.75mol/L sodium citrate salt buffer is moving phase.
One chromatographic condition: with embodiment 1.
Two experimental procedures
(1) product and reference substance are handled with embodiment 1.
(2) accurate need testing solution and each 20 μ l of reference substance solution of drawing inject liquid chromatograph respectively, the record chromatogram.Obtain parameters such as retention time, peak height, peak area.
The result: nerve growth factor main peak area A=3044833, reference substance area A=3001632, with calculated by peak area, sample size is 101.4% with external standard method.Nerve growth factor main peak and albumin main peak degree of separation are greater than 1.5, and sample main peak retention time is consistent with the reference substance retention time.HPLC chromatographic peak collection of illustrative plates is seen accompanying drawing 6.
Embodiment 5: adopting the 0.15mol/LTris-acetate buffer solution is moving phase.
One chromatographic condition: with embodiment 1.
Two experimental procedures
(1) product and reference substance are handled with embodiment 1.
(2) accurate need testing solution and each 20 μ l of reference substance solution of drawing inject liquid chromatograph respectively, the record chromatogram.Obtain parameters such as retention time, peak height, peak area.
The result: nerve growth factor main peak area A=3444833, reference substance area A=3405687, with calculated by peak area, sample size is 101.1% with external standard method.Nerve growth factor main peak and albumin main peak degree of separation are greater than 1.5, and sample main peak retention time is consistent with the reference substance retention time.HPLC chromatographic peak collection of illustrative plates is seen accompanying drawing 7.
Embodiment 6: adopting 0.5mol/L sodium acetate salt buffer is moving phase.
One chromatographic condition: with embodiment 1.
Two experimental procedures
(1) product and reference substance are handled with embodiment 1.
(2) accurate need testing solution and each 20 μ l of reference substance solution of drawing inject liquid chromatograph respectively, the record chromatogram.Obtain parameters such as retention time, peak height, peak area.
The result: nerve growth factor main peak area A=3463376, reference substance area A=3410533, with calculated by peak area, sample size is 101.5% with external standard method.Nerve growth factor main peak and albumin main peak degree of separation are greater than 1.5, and sample main peak retention time is consistent with the reference substance retention time.HPLC chromatographic peak collection of illustrative plates is seen accompanying drawing 8.
Claims (9)
1. the method for the nerve growth factor content in the high performance liquid chromatogram gel chromatography preparation, chromatographic condition is: (1) gel chromatographic columns: the molecular weight detection scope is 10000～500000 dalton, the aperture is the gel column of 200～300 dusts; (2) column temperature: room temperature; (3) detect wavelength: 205～220nm; (4) flow velocity: 0.6～1.0ml/min; (5) moving phase: the pH value is 6.5～7.2 salt solusion or damping fluid; It is characterized in that: the molarity of moving phase is 0.10～2.0mol/L.
2. method according to claim 1 is characterized in that: described gel chromatographic columns is TSKG3000SW XL, SHODEX Protein KW-804, BSA-7 or Phenomenex K3.
3. method according to claim 1 is characterized in that: the pH value of described moving phase is 7.0.
4. method according to claim 1 is characterized in that: described flow velocity is 0.8ml/min.
5. method according to claim 1 is characterized in that: described detection wavelength is 214nm.
6. method according to claim 1 is characterized in that: the molarity scope is in the moving phase: 0.10～0.75mol/L salt solusion or damping fluid.
7. method according to claim 2 is characterized in that: the molarity scope is in the moving phase: 0.15～0.5mol/L salt solusion or damping fluid.
8. method according to claim 1 is characterized in that: moving phase is selected from a kind of in sodium ascorbyl phosphate or sylvite damping fluid, Tris-acetate buffer solution, acetic acid sodium salt or sylvite damping fluid, sodium citrate salt or sylvite damping fluid, sodium sulfate salt or aqueous solutions of potassium, sodium chloride salt or aqueous solutions of potassium, ammonium acetate aqueous solution, the ammonium formate aqueous solution.
9. method according to claim 8 is characterized in that: moving phase is sodium phosphate buffer.
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| CNB2005101303483A CN100419422C (en) | 2005-12-12 | 2005-12-12 | Method for determining nerve growth factor content |
| PCT/CN2006/002021 WO2007068168A1 (en) | 2005-12-12 | 2006-08-10 | A method for determining the nerve growth factor content |
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| CNB2005101303483A CN100419422C (en) | 2005-12-12 | 2005-12-12 | Method for determining nerve growth factor content |
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| CN100419422C true CN100419422C (en) | 2008-09-17 |
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| CN104931598B (en) * | 2014-03-21 | 2017-05-17 | 舒泰神（北京）生物制药股份有限公司 | Method for determining content of nerve growth factor (NGF) in nerve growth factor preparation |
| CN105223283B (en) * | 2014-07-01 | 2017-02-15 | 舒泰神（北京）生物制药股份有限公司 | Method for determining content of nerve growth factor |
| CN114137120A (en) * | 2021-11-29 | 2022-03-04 | 赛诺神畅医疗科技有限公司 | Method for detecting related substances in rapamycin drug stent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1566941A (en) * | 2003-07-08 | 2005-01-19 | 北京三诺佳邑生物技术有限责任公司 | Method for measuring nerve growth factor content in preparation |
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| IT1232157B (en) * | 1989-07-20 | 1992-01-25 | Fidia Farmaceutici | PURIFICATION OF THE NERVE GROWTH FACTOR THROUGH SUBUNITY EXCHANGE CHROMATOGRAPHY |
| CN1074448A (en) * | 1992-09-29 | 1993-07-21 | 中国医科大学 | The method of separation and purification of nerve growth factor from snake venom |
| CN1042833C (en) * | 1994-06-20 | 1999-04-07 | 中国人民解放军南京军区后勤部军事医学研究所 | Process for preparing growth factor of human nerve by embryonic down or decidual |
| TR199901734T2 (en) * | 1996-11-15 | 2000-01-21 | Genentech, Inc. | N�rotrofinlerin ar�t�lmas�. |
| CN1128811C (en) * | 1999-11-09 | 2003-11-26 | 边六交 | Method for directly enriching nerve growth factor from crude snake venom |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1566941A (en) * | 2003-07-08 | 2005-01-19 | 北京三诺佳邑生物技术有限责任公司 | Method for measuring nerve growth factor content in preparation |
Non-Patent Citations (2)
| Title |
|---|
| 反相高效液相色谱法测定猪脑提取物中神经生长因子的含量. 沙云菲等.复旦学报（医学版），第32卷第1期. 2005 |
| 反相高效液相色谱法测定猪脑提取物中神经生长因子的含量. 沙云菲等.复旦学报（医学版），第32卷第1期. 2005 * |
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Owner name: SHUTAISHEN( BEIJING ) BIOLOGY PHARMACY STOCK CO., Free format text: FORMER OWNER: BEIJING SANNUOJIAYI BIOTECHNOLOGY CO., LTD. Effective date: 20091211 |
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