WO2007068168A1 - Procede pour la determination de contenu de facteur de croissance neuronal - Google Patents

Procede pour la determination de contenu de facteur de croissance neuronal Download PDF

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WO2007068168A1
WO2007068168A1 PCT/CN2006/002021 CN2006002021W WO2007068168A1 WO 2007068168 A1 WO2007068168 A1 WO 2007068168A1 CN 2006002021 W CN2006002021 W CN 2006002021W WO 2007068168 A1 WO2007068168 A1 WO 2007068168A1
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mobile phase
solution
buffer
growth factor
nerve growth
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PCT/CN2006/002021
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French (fr)
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Zhiwen Zhou
Hongshan Zhang
Hongwei Wang
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Beijing Sannuo Jiayi Biological Technology Co., Ltd.
Staidson (Beijing) Pharma Co., Ltd.
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Publication of WO2007068168A1 publication Critical patent/WO2007068168A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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]

Definitions

  • the present invention relates to a novel high performance liquid phase gel chromatography method for determining the amount of nerve growth factor in a preparation. Background technique
  • NGF is one of the most important active components of the nervous system. It can maintain the survival of sympathetic and sensory nerve cells, affect the survival of central neurons, promote the differentiation of nerve cells, and protect the normal function of the nervous system. Important role, China has approved nerve growth factor preparations for drug sales and use.
  • the content of NGF in the existing nerve growth factor preparation is 4 ⁇ 3 ( ⁇ g/branch, using human serum albumin as a protective agent, since human albumin and NGF are both proteins, the content of human serum albumin in the preparation It is much higher than NGF and affects the quantitative determination of NGF.
  • various testing methods such as Lowry method, ultraviolet absorption method, Bradford method and ELIS A method have been tried, but human albumin cannot be excluded.
  • the interference is limited by the sensitivity of the detection method itself.
  • the ionic strength of the mobile phase will greatly affect the elution of NGF on the column.
  • the ionic strength of the mobile phase is 0.01 ⁇ 0.07M phosphate buffer.
  • the detection limit of 10 ⁇ ⁇ / ⁇ 1 ionic strength of the mobile phase will be more preferably 0.15 ⁇ 0.5 ⁇ phosphate buffer, the detection limit of 2 ⁇ ⁇ / ⁇ 1, increased sensitivity 5 times, even at very low levels of NGF formulation, the quantification can be accurately detected, improve the accuracy of analysis.
  • the salt solution and the buffer are used as the mobile phase, and the ionic strength in the mobile phase can be controlled to achieve accurate quantitative analysis.
  • the present invention adopts the following technical solutions:
  • a method for determining the amount of nerve growth factor in a preparation which is a gel chromatography method using a high performance liquid chromatography, and the chromatographic conditions are:
  • the mobile phase pH 6. 5 ⁇ 7. 2, the concentration is 0. 07 ⁇ 2.0mol / L salt solution or buffer.
  • the mobile phase is preferably: aqueous sodium dihydrogen phosphate solution, aqueous ammonium acetate solution, aqueous sodium sulfate (potassium) solution, sodium citrate (potassium) salt buffer solution, aqueous ammonium formate 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.
  • the content of NGF in the preparation is determined by gel chromatography in high performance liquid chromatography.
  • the basic principle is to separate the molecular weight of various substances flowing through the column. The larger the molecular weight, the shorter the retention time. . Since each substance has a specific retention time under certain conditions, the position of the target peak and related parameters can be known by the retention time on the chromatogram, and the content of the target can be calculated by the above parameters.
  • the most basic principle of choosing a gel column is that its detection range contains the molecular weight of the substance to be tested.
  • the molecular weight of the NGF monomer is 13.6 kD
  • the molecular weight of the dimer is 26 kD.
  • the gel column having a molecular weight detection range of 10,000 to 50,000 Daltons is selected as the analytical column.
  • the wavelength selection criteria are related to the light absorption properties of the substance to be tested. For a protein or polypeptide, the maximum absorption wavelength is fixed. By ultraviolet scanning, it was found that NGF has light absorption at a wavelength of 205 to 220 nm, which can be used as a detection wavelength.
  • the gel column is preferably TSK G3000 SW XL, 7. 8 X 300, 5 ⁇ , (T0S0H, Japan), SH0DEX Protein KW-804 (SH0DEX, Japan) or BSA-7 (MN, Germany), Phenomenex K3 ( Phenomenex, USA). These columns have molecular weights ranging from 10,000 to 500,000 Daltons and pore sizes of 200 to 300 angstroms.
  • a preferred embodiment of the present invention is shown in the experiment, wherein the mobile phase has a pH of 7.0, a flow rate of 0.6 to 1. Oml/min, and a detection wavelength of 214 nm.
  • the above gel chromatography can be measured by an external standard method.
  • the external standard method includes the following steps:
  • the injection volume is 20 ⁇ 1.
  • the NGF peak area is 752103, the peak height is 28955, and the minimum detection limit is 10 ⁇ ⁇ / ⁇ 1.
  • the NGF peak area is 3215088, the peak height is 128628, and the minimum detection limit is 2 g/ml.
  • the lower the detection limit the higher the sensitivity and the more accurate the quantitative analysis. By means of the lowest detection limit, it can be calculated that the method of the invention is 5 times more sensitive than the control method. See Figure 1 and Figure 2.
  • the salt solution or the buffer solution with the ionic strength of 0.07 ⁇ 2.0M is the mobile phase, which can greatly improve the detection limit of the nerve growth factor in the preparation, and exclude the auxiliary materials such as human serum albumin in the preparation. Interference. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows the HPLC chromatogram of the mobile phase with 0.05M phosphate buffer.
  • Figure 2 shows the HPLC chromatogram of the mobile phase with 0.25M phosphate buffer.
  • the NGF peak area is 3215088 and the peak height is 128628.
  • the ionic strength of the mobile phase is increased by 5 times.
  • the NGF peak area was increased by 4.3 times, and the peak height was increased by 4.4 times;
  • Figure 3 shows the HPLC chromatogram of the mobile phase with 0.07M aqueous sodium dihydrogen phosphate solution
  • Figure 4 shows the HPLC chromatogram of the mobile phase with 2M aqueous ammonium acetate solution, and the results of Figure 3 and Figure 4 illustrate: When the intensity is 0.07 ⁇ 2.0M, the separation of the main peak of the nerve growth factor and the main peak of albumin is greater than 1.5.
  • Figure 5 shows the HPLC chromatogram of the mobile phase with a 0.10 M aqueous solution of sodium sulfate
  • Figure 6 shows the HPLC chromatogram of the mobile phase with 0.75 M sodium citrate buffer
  • the results of Figures 5 and 6 illustrate:
  • the ionic strength in the mobile phase is preferably 0.10M ⁇ 0.75M
  • the separation of the main peak of the nerve growth factor and the main peak of albumin is greater than 1.5
  • the main peak area response value of the nerve growth factor in the preparation increases. The same can be quantitatively calculated
  • Figure 7 shows the HPLC chromatogram of the mobile phase with 0.15 M Tris-acetate buffer
  • Figure 8 shows the HPLC chromatogram of the mobile phase with 0.5 M sodium acetate buffer
  • Figure 7 and Figure 8 show: When the ionic strength in the mobile phase is more preferably from 0.15 M to 0.5 M, the main peak area of the nerve growth factor in the preparation is the largest. The best way to implement the invention
  • Example 1 A mobile phase of 0.07 mol/L aqueous sodium dihydrogen phosphate solution was used.
  • Control of the reference substance Precisely measure the rat nerve growth factor reference substance (take the mouse submandibular gland, and separate and purify the NGF by ion exchange chromatography and molecular sieve, and verify the purity>98% according to the requirements of the Chinese Biological Products Regulation 2000 Edition. Appropriate amount, using a mobile phase to make a solution containing about 2 ( ⁇ g) per lml as a reference solution.
  • the external standard method was used to calculate the peak area, and the sample content was 100.2%.
  • the main peak of nerve growth factor and the main peak of albumin are more than 1. 5, and the retention time of the main peak of the sample is consistent with the retention time of the reference substance.
  • the chromatographic peak spectrum of HPLC is shown in Figure 4.
  • Example 3 A mobile phase of 0.10 mol/L aqueous sodium sulfate was used.
  • the external standard method was used to calculate the peak area, and the sample content was 101.6%.
  • the main peak of nerve growth factor and the main peak of albumin are more than 1. 5, and the retention time of the main peak of the sample is consistent with the retention time of the reference substance.
  • the chromatographic peak spectrum of HPLC is shown in Figure 5.
  • the chromatographic peak spectrum of HPLC is shown in Figure 6.
  • the external standard method was used to calculate the peak area, and the sample content was 101.1%.
  • the main peak of nerve growth factor and the main peak of albumin are more than 1. 5, and the retention time of the main peak of the sample is consistent with the retention time of the reference substance.
  • the chromatographic peak spectrum of HPLC is shown in Figure 7.
  • Example 6 0.5 mol/L sodium acetate buffer was used as the mobile phase.
  • the external standard method was used to calculate the peak area, and the sample content was 101.5%.
  • the main peak of nerve growth factor and the main peak of albumin are more than 1. 5, and the retention time of the main peak of the sample is consistent with the retention time of the reference substance.
  • the peak spectrum of the HPLC chromatogram is shown in Figure 8.
  • the invention adopts a salt solution or a buffer solution having an ionic strength of 0.07 to 2.0M as a mobile phase, which can greatly improve the detection limit of nerve growth factor in the preparation, and at the same time exclude interference of excipients such as human serum albumin in the preparation.

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Description

测定神经生长因子含量的方法 技术领域
本发明涉及一种新的高效液相凝胶色谱法测定制剂中的神经生长因子含量的方法。 背景技术
神经生长因子（NGF)是神经系统最主要的活性成分之一， 它能维持交感神经和 感觉神经细胞的生存， 影响中枢神经元的存活， 促进神经细胞的分化， 对保护神经系 统的正常功能具有重要的作用，我国已经批准神经生长因子制剂作为药物销售和使用。
现有的神经生长因子制剂中 NGF的含量在 4〜3(^g/支，采用人血白蛋白作为保 护剂， 由于人血白蛋白和 NGF 都是蛋白质， 人血白蛋白在制剂中的含量大大高于 NGF,影响了 NGF的定量测定。为解决这个难题，人们尝试了多种检验方法，如 Lowry 法、 紫外吸收法、 Bradford法和 ELIS A法等， 但都因无法排除人血白蛋白的干扰或 受检测方法本身的灵敏度所限而失败。 我们曾经申报过用凝胶色谱法分析 NGF的含 量， 流动相为 0.01~0.07M磷酸盐缓冲液， 解决了 NGF含量无法定量分析的问题。 后来我们经过进一步大量实验发现， 流动相的离子强度将在很大程度上影响 NGF 在色谱柱上的洗脱程度。 已申报的专利中， 流动相的离子强度为 0.01〜0.07M磷酸 盐缓冲液，最低检测限为 10μ§/πι1,我们将流动相的离子强度更优选为 0.15〜0.5Μ磷 酸缓冲液时， 最低检测限为 2μ§/ιη1， 灵敏度提高了 5倍， 即使在制剂中 NGF含量很 低的情况下， 也能准确定量检出， 提高了分析的准确度。 发明公开
本发明的目的是提供一种新的测定制剂中的神经生长因子含量的方法。
在高效液相凝胶色谱分析中选用盐溶液和缓冲液作为流动相，同时控制流动相中 的离子强度可以达到准确定量分析的目的。 '
为实现上述目的， 本发明采用以下技术方案：
一种测定制剂中神经生长因子含量的方法，该方法为凝胶色谱法，使用高效液相 色谱仪检测， 色谱条件为：
( 1 ) 凝胶色谱柱：分子量检测范围为 10000〜500000道尔顿，孔径为 200〜300 埃的凝胶柱；
( 2) 柱温： 室温；
( 3 ) 检测波长: 205〜220 nm;
( 4) 流速： 0. 6〜1. Oml/min;
( 5) 流动相： pH值为 6. 5〜7. 2， 浓度为 0. 07〜2.0mol/L盐溶液或缓冲液。 其中流动相优选是： 磷酸二氢钠水溶液、醋酸铵水溶液、硫酸钠（钾）水溶液、 柠檬酸钠 （钾） 盐缓冲液、 甲酸铵水溶液、 氯化钠 （钾） 水溶液、 Tris-醋酸缓冲 液、 醋酸钠 （钾） 盐缓冲液或磷酸钠 （钾） 盐缓冲液； 更优选磷酸钠 （钾） 盐缓 冲液。
使用高效液相色谱法中的凝胶色谱法对制剂中 NGF的含量进行测定， 其基本 原理是通过流经色谱柱的各种物质的分子量的差异将其分离， 分子量越大， 保留 时间越短。 由于每种物质在一定条件下具有特定的保留时间， 因此可以通过色谱 图上的保留时间获知目的峰的位置及相关参数， 并通过上述参数计算出目的物的 含量。选用凝胶柱的最基本原则是其检测范围包含待检物质的分子量。 NGF单体的 分子量为 13. 6kD， 其二聚体的分子量为 26kD， 故本发明选用了分子量检测范围在 10000〜50000道尔顿的凝胶柱作为分析用色谱柱。 波长选取的标准与待检物质的 光吸收性质有关， 对一种蛋白质或多肽来讲， 其最大吸收波长是固定的。 通过紫 外扫描， 发现 NGF在 205〜220nm波长均有光吸收， 可作为检测波长。
所述凝胶色谱柱优选 TSK G3000 SW XL, 7. 8 X 300， 5 μ， (T0S0H, 日本）、 SH0DEX Protein KW-804 (SH0DEX, 日本）或 BSA- 7 (MN, 德国）， Phenomenex K3 (Phenomenex, 美国）。 这些柱子的分子量范围均在 10000〜500000道尔顿， 孔径为 200〜300埃。
通过实验， 摸索出本发明的较佳实施方案， 其中流动相的 pH值为 7. 0， 流速 为 0. 6〜1. Oml/min, 检测波长为 214nm。
以上凝胶色谱法可以使用外标法对制剂进行测定。
所述外标法包括如下步骤：
( 1 ) 作标准曲线： 取含量已知的 NGF纯品为标准品， 梯度稀释， 以磷酸缓冲 液为流动相， 过凝胶色谱柱， 在检测波长下检测， 记录色谱图， 获取保留时间、 峰高、 峰面积等参数， 得到标准曲线和线性范围；
( 2) 外标法测定 NGF含量： 在线性范围内选择合适的浓度， 配制对照溶液和 样品溶液， 在选定的色谱条件下进行测定， 记录色谱图， 按外标法以峰面积计算， 即得。
实验表明：
( 1 ) 同一份样品溶液， 进样量 20μ1， 当流动相为 0.05Μ磷酸盐缓冲液时， NGF 峰面积为 752103 , 峰高为 28955， 最低检测限为 10μ§/ηι1。 当流动相为 0.25Μ磷酸盐 缓冲液时， NGF峰面积为 3215088， 峰高为 128628， 最低检测限为 2 g/ml。 检测限 越低， 则灵敏度越高， 定量分析越准确。通过最低检测限可以算出本发明方法比对照 方法灵敏度提高 5倍。 见附图 1及附图 2。
(2) 离子强度为 0.07〜2.0M时， 神经生长因子主峰与白蛋白主峰分离度大于 1. 5， 不存在辅料峰干扰问题， 制剂中神经生长因子主峰面积均可定量计算。 见实施 例 1、 实施例 2。
(3) 我们将流动相中离子强度优选为 0.10M〜0.75M时， 制剂中神经生长因子 主峰面积响应值增加， 同样可以定量计算。 见实施例 3和实施例 4。
(4)我们将流动相中离子强度更优选为 0.15M〜0.5M时， 制剂中神经生长因子 主峰面积响应值最大， 灵敏度最高。 见实施例 5和实施例 6。
本发明的优点是： 釆用离子强度为 0. 07〜2.0M的盐溶液或缓冲液为流动相， 能够大大提高制剂中神经生长因子的检测限， 同时排除了制剂中人血白蛋白等辅料 的干扰。 . 附图说明
图 1显示了流动相为 0.05M磷酸盐缓冲液时的 HPLC色谱峰图谱， NGF峰保留 时间： 13min, NGF峰面积为 752103, 峰高为 28955;
图 2显示了流动相为 0.25M磷酸盐缓冲液时的 HPLC色谱峰图谱， NGF峰面积 为 3215088， 峰高为 128628， 与图 1结果相比可得： 流动相的离子强度提高 5倍后， NGF峰面积提高为原来的 4.3倍， 峰高提高为原来的 4.4倍；
图 3显示了流动相为 0.07M磷酸二氢钠水溶液时的 HPLC色谱峰图谱； 图 4显示了流动相为 2M醋酸铵水溶液时的 HPLC色谱峰图谱， 图 3和图 4的 结果说明： 当离子强度在 0.07〜2.0M时， 神经生长因子主峰与白蛋白主峰分离度均 大于 1. 5， 不存在辅料峰干扰问题， 制剂中神经生长因子主峰面积均可定量计算； 图 5显示了流动相为 0.10M硫酸钠水溶液时的 HPLC色谱峰图谱； 图 6显示了流动相为 0.75M柠檬酸钠盐缓冲液时的 HPLC色谱峰图谱， 图 5和 图 6的结果说明： 将流动相中离子强度优选为 0.10M〜0.75M时， 神经生长因子主峰 与白蛋白主峰分离度均大于 1.5， 不存在辅料峰干扰问题， 同时制剂中神经生长因子 主峰面积响应值有所增加， 同样可以定量计算；
图 7显示了流动相为 0.15MTris—醋酸缓冲液时的 HPLC色谱峰图谱； 图 8显示了流动相为 0.5M醋酸钠盐缓冲液时的 HPLC色谱峰图谱； 图 7和图 8 的结果说明： 将流动相中离子强度更优选为 0.15M〜0.5M时， 制剂中神经生长因子 主峰面积值最大。 实施发明的最佳方式
实施例 1: 釆用 0.07mol/L磷酸二氢钠水溶液流动相。
一 色谱条件：
(1) 仪器： 使用日本岛津 Shiraadzu LC— ΙΟΑνρ高效液相色谱仪；
(2) 凝胶色谱柱： TSK G3000SWXL 7.8X3. Omra，内径 0.5 m(T0S0H 日本）；
(3) 柱温： 室温；
(4) 检测波长： 214 nrn;
(5) 流速： 0.8ml/min;
(6) 流动相 pH值： 7.0。 二 实验步骤
(1) 样品处理： 取神经生长因子制剂一支， 精密加入流动相 0.5ml， 轻摇使溶解， 作为供试品溶液。
(2) 对照品处理： 精密量取鼠神经生长因子对照品（取小鼠颌下腺， 经离子交换 层析和分子筛分离纯化出 NGF， 按 《中国生物制品规程 2000版》要求方法 检验纯度>98%) 适量， 用流动相制成每 lml中约含 2(^g的溶液， 作为对 照品溶液。
(3) 取对照品溶液 20W， 注入液相色谱仪， 记录色谱图。 获取保留时间、 峰高、 峰面积等参数。 结果： 神经生长因子主峰面积 A=914086， 对照品面积 A=903234， 用外标法以 峰面积计算，样品含量为 101.2%。神经生长因子主峰与白蛋白主峰分离度大于 1. 5, 样品主峰保留时间与对照品保留时间一致。 HPLC色谱峰图谱见附图 3。 实施例 2: 采用 2. Oraol/L醋酸铵水溶液为流动相。
一 色谱条件： 同实施例 1。
二 实验步骤
( 1 ) 样品和对照品处理同实施例 1。
(2) 精密吸取供试品溶液和对照品溶液各 20W，分别注入液相色谱仪，记录色谱 图。 获取保留时间、 峰高、 峰面积等参数。
结果： 神经生长因子主峰面积 Α=923186， 对照品面积 Α=921096， 用外标法以 峰面积计算，样品含量为 100.2%。神经生长因子主峰与白蛋白主峰分离度大于 1. 5， 样品主峰保留时间与对照品保留时间一致。 HPLC色谱峰图谱见附图 4。 实施例 3 : 采用 0. 10mol/L硫酸钠水溶液为流动相。
一 色谱条件： 同实施例 1。
二 实验步骤
( 1 ) 样品和对照品处理同实施例 1。
(2) 精密吸取供试品溶液和对照品溶液各 20W，分别注入液相色谱仪，记录色谱 图。 获取保留时间、 峰髙、 峰面积等参数。
结果： 神经生长因子主峰面积 Α=3147076， 对照品面积 Α=3098921， 用外标法 以峰面积计算， 样品含量为 101.6%。 神经生长因子主峰与白蛋白主峰分离度大于 1. 5， 样品主峰保留时间与对照品保留时间一致。 HPLC色谱峰图谱见附图 5。 实施例 4: 釆用 0. 75mol/L柠檬酸钠盐缓冲液为流动相。
一 色谱条件： 同实施例 1。
二 实验步骤
( 1 ) 品和对照品处理同实施例 1。
(2) 精密吸取供试品溶液和对照品溶液各 20W，分别注入液相色谱仪，记录色谱 图。 获取保留时间、 峰高、 峰面积等参数。
结果： 神经生长因子主峰面积 A=3044833，对照品面积 A=3001632， 用外标法 以峰面积计算， 样品含量为 101.4%。 神经生长因子主峰与白蛋白主峰分离度大于 1. 5， 样品主峰保留时间与对照品保留时间一致。 HPLC色谱峰图谱见附图 6。
■实施例 5 : 采用 0. 15mol/LTris-醋酸缓冲液为流动相。
一 色谱条件： 同实施例 1。
二 实验步骤
( 1 ) 品和对照品处理同实施例 1。
(2) 精密吸取供试品溶液和对照品溶液各 20μΐ，分别注入液相色谱仪，记录色谱 图。 获取保留时间、 峰高、 峰面积等参数。
结果： 神经生长因子主峰面积 Α=3444833,对照品面积 Α=3405687， 用外标法 以峰面积计算， 样品含量为 101.1%。 神经生长因子主峰与白蛋白主峰分离度大于 1. 5， 样品主峰保留时间与对照品保留时间一致。 HPLC色谱峰图谱见附图 7。 实施例 6: 采用 0. 5mol/L醋酸钠盐缓冲液为流动相。
一 色谱条件： 同实施例 1。
二 实验步骤
( 1 ) 品和对照品处理同实施例 1。
(2) 精密吸取供试品溶液和对照品溶液各 20μ1，分别注入液相色谱仪，记录色谱 图。 获取保留时间、 峰高、 峰面积等参数。
结果： 神经生长因子主峰面积 Α=3463376，对照品面积 Α=3410533 , 用外标法 以峰面积计算， 样品含量为 101.5%。 神经生长因子主峰与白蛋白主峰分离度大于 1. 5， 样品主峰保留时间与对照品保留时间一致。 HPLC色谱峰图谱见附图 8。 工业应用性
本发明采用离子强度为 0. 07〜2.0M的盐溶液或缓冲液为流动相， 能够大大提 高制剂中神经生长因子的检测限， 同时排除了制剂中人血白蛋白等辅料的干扰。

Claims

权 利 要 求
、 一种高效液相凝胶色谱法测定制剂中的神经生长因子含量的方法， 色谱 条件为： （1 ) 凝胶色谱柱： 分子量检测范围为 10000〜500000道尔顿， 孔径为 200〜300埃的凝胶柱； （2 )柱温： 室温； （3 )检测波长： 205〜 220 nm; ( 4) 流速： 0. 6〜1· Oml/min; ( 5 ) 流动相： pH值为 6. 5〜7. 2 的盐溶液或缓冲液； 其特征在于： 流动相的离子强度为 0. 07〜2. 0M。 、 根据权利要求 1所述的方法，其特征在于：所述凝胶色谱柱是 TSK G3000 SW XL、 SHODEX Protein KW- 804、 BSA-7或 Phenomenex K3。
、 根据权利要求 1所述的方法， 其特征在于： 所述流动相的 pH值为 7. 0。 、 根据权利要求 1所述的方法， 其特征在于： 所述流速为 0.8ml/min。 、 根据权利要求 1所述的方法， 其特征在于： 所述检测波长为 214nm。 、 根据权利要求 1所述的方法， 其特征在于： 流动相中离子强度范围是： 0.10〜0.75M盐溶液和缓冲液。
、 根据权利要求 2所述的方法， 其特征在于： 流动相中离子强度范围是 :
0.15〜0.5M盐溶液和缓冲液。
、 根据权利要求 1 所述的方法， 其特征在于： 流动相选自磯酸钠盐或钾 盐缓冲液、 Tris-醋酸缓冲液、 醋酸钠盐或钾盐缓冲液、 柠檬酸钠盐或 钾盐缓冲液、 硫酸钠盐或钾水溶液、 氯化钠盐或钾水溶液、 醋酸铵水 溶液、 甲酸铵水溶液中的一种。
、 根据权利要求 8所述的方法， 其特征在于： 流动相是磷酸钠缓冲液。
PCT/CN2006/002021 2005-12-12 2006-08-10 Procede pour la determination de contenu de facteur de croissance neuronal WO2007068168A1 (fr)

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