CN102875663B - Purification method of lixisenatide - Google Patents

Purification method of lixisenatide Download PDF

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CN102875663B
CN102875663B CN 201210363634 CN201210363634A CN102875663B CN 102875663 B CN102875663 B CN 102875663B CN 201210363634 CN201210363634 CN 201210363634 CN 201210363634 A CN201210363634 A CN 201210363634A CN 102875663 B CN102875663 B CN 102875663B
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phase
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lixisenatide
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CN102875663A (en
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覃亮政
刘建
马亚平
袁建成
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深圳翰宇药业股份有限公司
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Abstract

本发明涉及一种利西拉来的纯化方法,其包括以下步骤:步骤1:采用高效液相色谱法对利西拉来粗肽进行纯化,所述高效液相色谱法以苯基硅烷键合硅胶为固定相,以D-酒石酸盐的甲醇/磷酸盐缓冲溶液为流动相A相,以乙腈为流动相B相进行梯度洗脱;步骤2:采用高效液相色谱法对步骤1所得到的馏分进行转盐纯化,所述高效液相色谱法以烷基硅烷键合硅胶为固定相,以冰醋酸溶液为流动相A相,以乙腈为流动相B相进行梯度洗脱;步骤3:收集溶液冻干。 The present invention relates to a method for purifying lixisenatide, which comprises the following steps: Step 1: HPLC method lixisenatide crude peptide was purified by high performance liquid chromatography to the chemically bonded phenyl silica gel as the stationary phase, D- tartrate in methanol / phosphate buffer solution as mobile phase a phase to B phase acetonitrile as mobile phase gradient elution; step 2: high performance liquid chromatography obtained in step 1 transferring salt fraction purified by HPLC to said silane bonded silica gel as the stationary phase, mobile phase was acetic acid to phase A, phase B acetonitrile as mobile phase gradient elution; step 3: collect solution lyophilized.

Description

利西拉来的纯化方法 Purification methods lixisenatide

技术领域 FIELD

[0001] 本发明属于药物化学领域,具体涉及利西拉来的纯化方法。 [0001] The present invention belongs to the field of pharmaceutical chemistry, specifically relates to a method of purification lixisenatide.

背景技术 Background technique

[0002] 糖尿病是一种全球性的高发病,其主要分为I型和II型,后者占糖尿病患者总数的90%以上。 [0002] Diabetes is a global high incidence, which is divided into type I and type II, which accounted for 90% of diabetic patients.

[0003] 通过胰高血糖素样肽I (GLP-1)受体拮抗剂利西拉来(Lixisenatide)联合基础胰岛素来治疗II型糖尿病患者,可显著改善其血糖控制,并提高其餐后血糖(PPG)的控制。 [0003] By glucagon-like peptide I (GLP-1) receptor antagonist lixisenatide (lixisenatide) combined basal insulin to treat type II diabetes, can significantly improve glycemic control and to improve postprandial glucose (PPG) control.

[0004] 利西拉来是由43个氨基酸残基组成的长链多肽,而且肽序列中有如Ser、His、Pr0等在多肽固相化学合成过程中易异构化的氨基酸而导致利西拉来粗肽中含有很多异构体杂质。 [0004] lixisenatide long-chain polypeptide consisting of 43 amino acid residues, and the like in a peptide sequence polypeptide solid phase chemical synthesis of amino acids easily isomerized Ser, His, Pr0 like lead lixisenatide to crude peptide contains many isomer impurity. 因此利西拉来的分离纯化成为该药品制备工艺中的技术难点,尤其是大规模制备的利西拉来的纯化已成为制约其产业化的瓶颈之一。 Therefore, separation and purification of lixisenatide become technical difficulties in the manufacturing process of the drug, especially in large scale production of lixisenatide to purification has become one of the bottlenecks of its industrialization.

发明内容 SUMMARY

[0005] 本发明的目的是提供一种利西拉来的纯化方法,尤其是通过多肽固相化学合成方法而大规模制备的利西拉来的纯化方法。 [0005] The object of the present invention is to provide a purification method lixisenatide lixisenatide and particularly large scale preparation of the polypeptide by solid phase chemical synthesis methods for the purification process.

[0006] 本发明的利西拉来的纯化方法包括以下步骤: [0006] The present invention is lixisenatide purification method comprising the steps of:

[0007] 步骤1:采用高效液相色谱(HPLC)方法对利西拉来粗肽进行纯化,所述HPLC方法以苯基硅烷键合硅胶为固定相,以D-酒石酸盐的甲醇/磷酸盐缓冲溶液为流动相A相,以乙腈为流动相B相进行梯度洗`脱; [0007] Step 1: high performance liquid chromatography (HPLC) method lixisenatide crude peptide was purified by the HPLC method phenyl silane bonded silica gel as stationary phase D- tartrate in methanol / phosphate a buffer solution as mobile phase A phase, and acetonitrile as mobile phase B gradient elution `off phase;

[0008] 步骤2:采用HPLC方法对步骤I所得到的馏分进行转盐纯化,所述HPLC方法以烷基硅烷键合硅胶为固定相,以冰醋酸溶液为流动相A相,以乙腈为流动相B相进行梯度洗脱; [0008] Step 2: HPLC method step I fraction was subjected to transfer purified salt, the HPLC method alkyl silane bonded silica gel as the stationary phase, mobile phase was acetic acid to the A-phase, acetonitrile mobile phase B phase gradient elution;

[0009] 步骤3:收集溶液冻干。 [0009] Step 3: Collect solution was lyophilized.

[0010] 本发明通过采用以苯基硅烷键合硅胶为固定相,在流动相中加入D-酒石酸盐和质子给予体甲醇的HPLC方法纯化,可以一次性将利西拉来粗肽中的异构体杂质和其他难分离杂质很好地分离而去除。 [0010] HPLC purification method of the present invention, the body of methanol to be administered by using phenylsilane bonded silica gel as the stationary phase, the mobile phase was added D- tartrate and protons, it can be disposable lixisenatide crude peptide iso isomers difficult to separate impurities and other impurities are well separated and removed. 然后利用反相HPLC方法转成醋酸盐API,提高了产品的收率和纯度。 Using reverse phase HPLC method was then converted into acetate API, improved product yield and purity. 此外,该纯化方法操作简便,有利于实现规模化制备的利西拉来的纯化。 Moreover, this purification method is simple, facilitate production scale purification lixisenatide achieved.

附图说明 BRIEF DESCRIPTION

[0011] 图1实施例1中的利西拉来纯化后的色谱图。 Chromatogram after lixisenatide purified in Example 1. [0011] FIG.

具体实施方式 Detailed ways

[0012] 本发明的利西拉来的纯化方法包括以下步骤: [0012] The present invention is lixisenatide purification method comprising the steps of:

[0013] 步骤1:采用HPLC方法对利西拉来粗肽进行纯化,所述HPLC方法以苯基硅烷键合硅胶为固定相,以D-酒石酸盐的甲醇/磷酸盐缓冲溶液为流动相A相,以乙腈为流动相B相进行梯度洗脱; [0013] Step 1: using HPLC method lixisenatide crude peptide was purified by the HPLC method phenyl silane bonded silica gel as stationary phase D- tartrate in methanol / phosphate buffer solution as mobile phase A phase to phase B acetonitrile as mobile phase gradient elution;

[0014] 步骤2:采用HPLC方法对步骤I所得到的馏分进行转盐纯化,所述HPLC方法以烷基硅烷键合硅胶为固定相,以冰醋酸溶液为流动相A相,以乙腈为流动相B相进行梯度洗脱; [0014] Step 2: HPLC method step I fraction was subjected to transfer purified salt, the HPLC method alkyl silane bonded silica gel as the stationary phase, mobile phase was acetic acid to the A-phase, acetonitrile mobile phase B phase gradient elution;

[0015] 步骤3:收集溶液冻干。 [0015] Step 3: Collect solution was lyophilized.

[0016] 所述步骤I中的HPLC方法的流动相A相中的D-酒石酸盐的摩尔浓度优选为10mM~50mM。 Molar concentration of the flow of the HPLC method [0016] Step I is the phase D- tartrate salt A phase is preferably 10mM ~ 50mM.

[0017] 所述步骤I中的HPLC方法的流动相A相中的D-酒石酸盐优选为一种或多种选自D-酒石酸钠、D-酒石酸钾、D-酒石酸铵的D-酒石酸盐。 The flow of the HPLC method [0017] of the steps I-phase phase D- tartrate salt A is preferably one or more selected from sodium tartrate D-, D- potassium tartrate, D- tartrate ammonium D- tartrate .

[0018] 所述步骤I中的HPLC方法的流动相A相中的磷酸盐的摩尔浓度优选为20mM"50mMo Molar concentration of the flow of the HPLC method [0018] of the steps A phase I phosphate phase is preferably 20mM "50mMo

[0019] 所述步骤I中的HPLC方法的流动相A相中的磷酸盐优选为一种或多种选自磷酸 The flow of the HPLC method [0019] of the steps I-phase phase A phosphate is preferably one or more selected from phosphoric acid

钠、磷酸氢二钠、磷酸二氢钠、磷酸钾、磷酸氢二钾、磷酸二氢钾、磷酸铵或磷酸二氢铵的磷酸盐。 Sodium, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, dihydrogen phosphate, ammonium phosphate or ammonium dihydrogen phosphate.

[0020] 所述步骤I中的HPLC方法的流动相A相溶液的pH优选为2.0-4.0。 [0020] The pH of the mobile phase A HPLC method step I with the solution is preferably 2.0-4.0.

[0021 ] 所述步骤I中的HPLC方法的流动相A相中的甲醇占整个流动相A相的体积比优选为10~20% (v/v)。 Methanol flow of the HPLC method [0021] Step I is the phase of the total volume of the phase A mobile phase A phase ratio is preferably 10 ~ 20% (v / v).

[0022] 所述步骤I中的HPLC方法的梯度按照流动相A相的百分比为 [0022] The gradient HPLC method of step I using the percentage of mobile phase A phase was

Figure CN102875663BD00041

,根据保留时间可适当调整。 , Can be adjusted based on retention time.

[0023] 所述步骤2中的HPLC方法的流动相A相中的冰醋酸水溶液的体积百分浓度优选为0.1-0.3% (v/v)。 [0023] The flow of the HPLC method in Step 2 with glacial acetic acid aqueous solution A phase volume percent concentration is preferably 0.1-0.3% (v / v).

[0024] 所述步骤2中的HPLC方法的固定相优选为八烷基硅烷键合硅胶、六烷基硅烷键合硅胶或四烷基硅烷键合硅胶。 [0024] The HPLC method in Step 2 is preferably stationary phase octadecyl silane bonded silica, silane bonded silica six or tetraalkyl silane bonded silica gel.

[0025] 所述步骤2中的HPLC方法的梯度为95%A+5%B Gradient HPLC method described in [0025] Step 2 95% A + 5% B

Figure CN102875663BD00042

,根据保留 According reserved

时间可适当调整。 Time can be appropriately adjusted.

[0026] 本发明的纯化方法操作简单,从而使得操作成本低。 [0026] The purification process of the present invention is simple, so that the low operating costs. 此外,本发明的纯化方法中的利西拉来收率高,从而适合于利西拉来的大规模产业化生产,并且经纯化得到的利西拉来纯度高、杂质含量低,因此具有可观的经济实用价值和广泛的应用前景。 Furthermore, the purification method of the present invention lixisenatide high yield, as appropriate for lixisenatide scale industrial production, and was lixisenatide give high purity, low impurity content, and therefore with considerable economic and practical value and broad application prospects.

[0027] 实施例 [0027] Example

[0028] 以下通过实施例对本发明进行详细说明。 [0028] The following detailed description of the present invention through examples.

[0029] 在以下实施例中,利西拉来含量的测定按照高效液相色谱法(中国药典2010年版二部附录VD)进行。 [0029] In the following examples, the assay performed in accordance with the content of lixisenatide high performance liquid chromatography (China Pharmacopoeia 2010 Appendix VD).

[0030] 色谱条件与系统适用性试验 [0030] Chromatographic conditions and system suitability test

[0031] 所用色谱柱以苯基硅烷键合硅胶作填充剂,色谱柱规格为4.6*250mm,所填充的苯基硅烷键合硅胶的粒径为5μπι;以0.lmol/L磷酸铵缓冲液(取磷酸二氢铵11.5g,加水至1000ml,用磷酸调节pH为2.5)为流动相A相,以色谱纯乙腈为流动相B相,按下表1进行梯度洗脱;流速为1.0ml/分钟,检测波长为220nm,柱温为50°C ;理论板数按利西拉来峰计算应不低于5000。 [0031] In the phenylsilane bonded silica as filler by column chromatography, column dimensions of 4.6 * 250mm, filled phenylsilane bonded silica having a particle size 5μπι; to 0.lmol / L of ammonium phosphate buffer (11.5 g of ammonium dihydrogen phosphate, add water to 1000ml, adjusted to pH 2.5 with phosphoric acid) as mobile phase A phase to phase HPLC grade acetonitrile mobile phase B, the following table 1 gradient elution; flow rate was 1.0ml / min, detection wavelength 220nm, column temperature was 50 ° C; number of theoretical plates calculated lixisenatide peak should not be less than 5,000. [0032] 利西拉来含量的测定方法 [0032] Determination of content to lixisenatide

[0033] 准确称取适量利西拉来粗品,加水制成含2.0mg/ml利西拉来粗品的溶液作为供试品溶液,另准确称取适量利西拉来对照品,加水制成含1.0mg/ml利西拉来对照品的溶液作为对照品溶液;取对照品溶液和供试品溶液各20 μ I分别注入液相色谱仪,记录色谱图,以峰面积按外标法计算,即得到利西拉来的含量。 [0033] lixisenatide accurately weighed amount of the crude product, made by adding water containing 2.0mg / ml solution of crude lixisenatide as the test solution, the other lixisenatide accurately weighed amount of reference substance, add the water containing 1.0mg / ml solution was used as reference lixisenatide reference solution; reference solution and test solution were injected into each of 20 μ I liquid chromatograph, record the chromatograms, peak area calculation by external standard method, to obtain lixisenatide content.

[0034] 表1利西拉来含量测定的色谱法中的洗脱梯度 [0034] The gradient elution chromatography lixisenatide Table 1 Content Determination

[0035] [0035]

Figure CN102875663BD00051

[0036] 实施例1 [0036] Example 1

[0037] 将利西拉来粗肽4.0g (含利西拉来1.08g)用纯化水200ml溶解,过滤,收集滤液备用。 [0037] 4.0 g of the crude peptide lixisenatide (containing 1.08 g of lixisenatide) was dissolved with 200ml of purified water, collected by filtration and the filtrate set aside.

[0038] 纯化色谱条件: [0038] Purification Chromatographic conditions:

[0039] 高效液相色谱仪型号:Waters 2545 [0039] HPLC type: Waters 2545

[0040] 色谱柱:50X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0040] Column: 50X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0041]流速:80ml/分钟。 [0041] flow rate: 80ml / min.

[0042]检测波长:280nm。 [0042] Detection wavelength: 280nm.

[0043] 流动相A相:IOmM D-酒石酸铵和30mM磷酸二氢铵的20%甲醇/80%水溶液(v/v),用磷酸调pH为2.0。 [0043] Phase A Mobile phase: 20% IOmM D- ammonium tartrate and ammonium dihydrogen phosphate 30mM methanol / 80% aqueous (v / v), adjusted to pH 2.0 with phosphoric acid.

[0044] 流动相A相配制过程:称取18.4g D-酒石酸铵和34.5g磷酸二氢铵,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到IOL血清瓶,加入2L色谱纯甲醇后加纯化水至IOL刻度,用磷酸调pH为2.0。 [0044] Mobile phase A phase formulation process: Weigh 34.5g 18.4g D- ammonium tartrate and ammonium dihydrogen phosphate, an appropriate amount of purified water was dissolved after through 0.45 μ m membrane filter, the filtrate collected all IOL serum bottle, was added 2L chromatography purified water was added to the mark after IOL pure methanol, pH was adjusted to 2.0 with phosphoric acid.

[0045] 流动相B相:色谱纯乙腈。 [0045] Mobile Phase B Phase: HPLC grade acetonitrile.

[0046]梯度: [0046] Gradient:

Figure CN102875663BD00052

[0047]上样量:2.0g(100mL)。 [0047] The loading amount: 2.0g (100mL).

[0048] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0048] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment.

[0049] 分两次进样,重复以上操作。 [0049] The injections twice, repeat the above operation.

[0050] 转盐色谱条件:[0051] 高效液相色谱仪型号:Waters 2545 [0050] Chromatographic conditions turn salt: [0051] HPLC type: Waters 2545

[0052] 色谱柱:50 X 250mm,内装反相C8色谱填料,该填料的粒径为10 μ m。 [0052] Column: 50 X 250mm, built-C8 reversed-phase chromatography packing, the particle size of the filler is 10 μ m.

[0053]流速:80ml/分钟。 [0053] flow rate: 80ml / min.

[0054]检测波长:280nm。 [0054] Detection wavelength: 280nm.

[0055] 流动相A相:0.10%冰醋酸(v/v)溶液。 [0055] Phase A Mobile phase: 0.10% glacial acetic acid (v / v) solution.

[0056] 流动相B相:色谱纯乙腈。 [0056] Mobile Phase B Phase: HPLC grade acetonitrile.

[0057]梯度 [0057] Gradient

Figure CN102875663BD00061

[0058]上样体积:200ml。 [0058] The loading volume: 200ml.

[0059] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0059] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至20ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 20ml lyophilization.

[0060] 冻干后得白色粉末状固体精肽0.66g。 [0060] to give fine white powder after lyophilization peptides 0.66g. 纯度98.47%,各杂质含量均小于0.5%。 A purity of 98.47% of the impurity content of less than 0.5%. 纯化收率61.1% (以粗品中利西拉来含量计算),总收率16.5%。 Purification Yield 61.1% (calculated to the content of crude lixisenatide), total yield of 16.5%. 结果见图1,其中横坐标代表以分钟计的运行时间,纵坐标代表峰高。 The results shown in Figure 1, where the abscissa represents the run time in minutes, ordinate represents high peaks. 统计结果见表2。 Statistical results are shown in Table 2.

[0061] 表2利西拉来固体精肽的色谱检测结果 [0061] Table 2 lixisenatide chromatography on a detection result of solid fine peptide

[0062] [0062]

Figure CN102875663BD00062

[0063] 实施例2 [0063] Example 2

[0064] 将利西拉来粗肽6.0g (含利西拉来1.62g)用纯化水300ml溶解,过滤,收集滤液备用。 [0064] 6.0 g of the crude peptide lixisenatide (containing 1.62 g of lixisenatide) was dissolved with 300ml of purified water, collected by filtration and the filtrate set aside.

[0065] 纯化色谱条件: [0065] Purification Chromatographic conditions:

[0066] 高效液相色谱仪型号:Waters 2545 [0066] HPLC type: Waters 2545

[0067] 色谱柱:50X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0067] Column: 50X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0068]流速:80ml/ 分钟。 [0068] flow rate: 80ml / min.

[0069]检测波长:280nm。 [0069] Detection wavelength: 280nm.

[0070] 流动相A相:40mM D-酒石酸钠和20mM磷酸二氢铵的15%甲醇/85%水溶液(v/v),用磷酸调pH为3.0。 [0070] Phase A Mobile Phase: 15% 40mM D- 20mM sodium tartrate and ammonium dihydrogen phosphate methanol / aqueous 85% (v / v), adjusted to pH 3.0 with phosphoric acid.

[0071] 流动相A相配制过程:称取92.0g D-酒石酸钠和23.0g磷酸二氢铵,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到IOL血清瓶,加入1.5L色谱纯甲醇后加纯化水至IOL刻度,用磷酸调pH为3.0。 [0071] Mobile phase A phase during formulation: weighed 0.45 μ m membrane filter, the filtrate collected after all take 23.0g 92.0g D- sodium tartrate and ammonium dihydrogen phosphate, an appropriate amount of purified water was dissolved IOL serum bottle, was added 1.5L purified water was added to the mark IOL after chromatography pure methanol, pH was adjusted to 3.0 with phosphoric acid.

[0072] 流动相B相:色谱纯乙腈。 [0072] Mobile Phase B Phase: HPLC grade acetonitrile.

Figure CN102875663BD00071

[0074]上样量:3.0g(150ml)。 [0074] The loading amount: 3.0g (150ml).

[0075] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0075] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment.

[0076] 分两次进样,重复以上操作。 [0076] in two injections, repeat the above operation.

[0077] 转盐色谱条件: [0077] Chromatographic conditions turn salt:

[0078] 高效液相色谱仪型号:Waters 2545 [0078] HPLC type: Waters 2545

[0079] 色谱柱:50 X 2 50mm,内装反相C8色谱填料,该填料的粒径为10 μ m。 [0079] Column: 50 X 2 50mm, built-C8 reversed-phase chromatography packing, the particle size of the filler is 10 μ m.

[0080]流速:80ml/ 分钟。 [0080] flow rate: 80ml / min.

[0081]检测波长:280nm。 [0081] Detection wavelength: 280nm.

[0082] 流动相A相:0.15%冰醋酸(v/v)溶液。 [0082] Phase A Mobile phase: 0.15% glacial acetic acid (v / v) solution.

[0083] 流动相B相:色谱纯乙腈。 [0083] Mobile Phase B Phase: HPLC grade acetonitrile.

[0084]梯度:9S%A+5%B」2=^95%A+5%B [0084] Gradient: 9S% A + 5% B "2 = ^ 95% A + 5% B

Figure CN102875663BD00072

[0085]上样体积:200ml。 [0085] The loading volume: 200ml.

[0086] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0086] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至20ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 20ml lyophilization.

[0087] 冻干后得白色粉末状固体精肽0.93g。 [0087] to give fine white powder after lyophilization peptides 0.93g. 纯度98.62%,各杂质含量均小于0.5%。 A purity of 98.62% of the impurity content of less than 0.5%. 纯化收率57.4% (以粗品中利西拉来含量计算),总收率15.5%。 Purification Yield 57.4% (calculated to the content of crude lixisenatide), total yield of 15.5%.

[0088] 实施例3 [0088] Example 3

[0089] 将利西拉来粗肽6.0g (含利西拉来1.62g)用纯化水300ml溶解,过滤,收集滤液备用。 [0089] 6.0 g of the crude peptide lixisenatide (containing 1.62 g of lixisenatide) was dissolved with 300ml of purified water, collected by filtration and the filtrate set aside.

[0090] 纯化色谱条件: [0090] Purification Chromatographic conditions:

[0091] 高效液相色谱仪型号:Waters 2545 [0091] HPLC type: Waters 2545

[0092] 色谱柱:50X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0092] Column: 50X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0093]流速:80ml/ 分钟。 [0093] flow rate: 80ml / min.

[0094]检测波长:280nm。 [0094] Detection wavelength: 280nm.

[0095] 流动相A相:30mM D-酒石酸钾和40mM磷酸二氢钾的10%甲醇/90%水溶液(v/v),用磷酸调pH为2.5。 [0095] Phase A Mobile phase: 30mM D- 40mM potassium dihydrogen phosphate, potassium tartrate and 10% methanol / 90% aqueous (v / v), adjusted to pH 2.5 with phosphoric acid.

[0096] 流动相A相配制过程:称取56.1g D-酒石酸钾和54.4g磷酸二氢钾,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到IOL血清瓶,加入1.0L色谱纯甲醇后加纯化水至IOL刻度,用磷酸调pH为2.5。 [0096] Mobile phase A phase during formulation: weighed 0.45 μ m membrane filter, the filtrate collected all taken after IOL serum vials and 54.4g 56.1g D- tartrate, potassium dihydrogen phosphate, an appropriate amount of purified water was dissolved by adding 1.0L purified water was added to the mark IOL after chromatography pure methanol, pH was adjusted to 2.5 with phosphoric acid.

[0097] 流动相B相:色谱纯乙腈。 [0097] Mobile Phase B Phase: HPLC grade acetonitrile.

[0098]梯度: [0098] Gradient:

Figure CN102875663BD00081

[0099]上样量:3.0g(150ml)。 [0099] loading amount: 3.0g (150ml).

[0100] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0100] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment.

[0101] 分两次进样,重复以上操作。 [0101] in two injections, repeat the above operation.

[0102] 转盐色谱条件: [0102] Chromatographic conditions turn salt:

[0103] 高效液相色谱仪型号:Waters 2545 [0103] HPLC Model: Waters 2545

[0104] 色谱柱:50X250mm,内装反相C4色谱填料,该填料的粒径为10 μ m。 [0104] Column: 50X250mm, built C4 reverse phase chromatography packing, particle size of the filler is 10 μ m.

[0105]流速:80ml/ 分钟。 [0105] flow rate: 80ml / min.

[0106]检测波长:280nm。 [0106] Detection wavelength: 280nm.

[0107] 流动相A相:0.3%冰醋酸(v/v)溶液。 [0107] Phase A Mobile phase: 0.3% acetic acid (v / v) solution.

[0108] 流动相B相:色谱纯乙腈。 [0108] Mobile Phase B Phase: HPLC grade acetonitrile.

[0109] [0109]

Figure CN102875663BD00082

[0110]上样体积:250ml。 [0110] Volume loaded: 250ml.

[0111] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0111] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至20ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 20ml lyophilization.

[0112] 冻干后得白色粉末状固体精肽0.98g。 [0112] to give fine white powder after lyophilization peptides 0.98g. 纯度98.52%,各杂质含量均小于0.5%。 A purity of 98.52% of the impurity content of less than 0.5%. 纯化收率60.5% (以粗品中利西拉来含量计算),总收率16.3%。 Purification Yield 60.5% (calculated to the content of crude lixisenatide), total yield of 16.3%.

[0113] 实施例4 [0113] Example 4

[0114] 将利西拉来粗肽4.0g (含利西拉来1.08g)用纯化水200ml溶解,过滤,收集滤液备用。 [0114] 4.0 g of the crude peptide lixisenatide (containing 1.08 g of lixisenatide) was dissolved with 200ml of purified water, collected by filtration and the filtrate set aside.

[0115] 纯化色谱条件: [0115] Purification Chromatographic conditions:

[0116] 高效液相色谱仪型号:Waters 2545 [0116] HPLC Model: Waters 2545

[0117] 色谱柱:50X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m。 [0117] Column: 50X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m.

[0118]流速:80ml/分钟。 [0118] flow rate: 80ml / min.

[0119]检测波长:280nm。 [0119] Detection wavelength: 280nm.

[0120] 流动相A相:50mM D-酒石酸钠和50mM磷酸氢二钠的10%甲醇/90%水溶液(v/v),用磷酸调pH为3.5。 [0120] Phase A Mobile phase: 10% sodium tartrate and disodium hydrogen phosphate 50mM 50mM D-methanol / 90% aqueous (v / v), adjusted to pH 3.5 with phosphoric acid.

[0121] 流动相A相配制过程:称取115g D-酒石酸钠和179g磷酸氢二钠,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到IOL血清瓶,加入1.0L色谱纯甲醇后加纯化水至IOL刻度,用磷酸调pH为3.5。 [0121] Mobile phase A phase formulation process: Weigh 115g D- sodium tartrate and 179g of disodium hydrogen phosphate, had proper amount of purified water was dissolved 0.45 μ m membrane filter, the filtrate collected all IOL serum bottle, was added 1.0L chromatography purified water was added to the mark IOL methanol, pH was adjusted to 3.5 with phosphoric acid.

[0122] 流动相B相:色谱纯乙腈。 [0122] Mobile Phase B Phase: HPLC grade acetonitrile.

[0123] [0123]

Figure CN102875663BD00091

[0124]上样量:2.0g(100mL)。 [0124] loading amount: 2.0g (100mL).

[0125] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0125] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment.

[0126] 分两次进样,重复以上操作。 [0126] in two injections, repeat the above operation.

[0127] 转盐色谱条件: [0127] Chromatographic conditions turn salt:

[0128] 高效液相色谱仪型号:Waters 2545 [0128] HPLC Model: Waters 2545

[0129] 色谱柱:50X250mm,内装反相C6色谱填料,该填料的粒径为10 μ m。 [0129] Column: 50X250mm, built-C6 reverse phase chromatography packing, particle size of the filler is 10 μ m.

[0130]流速:80ml/ 分钟。 [0130] flow rate: 80ml / min.

[0131]检测波长:2 80nm。 [0131] Detection wavelength: 2 80nm.

[0132] 流动相A相:0.20%冰醋酸(v/v)溶液。 [0132] Phase A Mobile phase: 0.20% glacial acetic acid (v / v) solution.

[0133] 流动相B相:色谱纯乙腈。 [0133] Mobile Phase B Phase: HPLC grade acetonitrile.

[0134]梯度: [0134] Gradient:

Figure CN102875663BD00092

20mm >68%A+32%B 20mm> 68% A + 32% B

[0135]上样体积:200ml。 [0135] Volume loaded: 200ml.

[0136] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0136] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至20ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 20ml lyophilization.

[0137] 冻干后得白色粉末状固体精肽0.67g。 [0137] to give fine white powder after lyophilization peptides 0.67g. 纯度98.60%,各杂质含量均小于0.5%。 A purity of 98.60% of the impurity content of less than 0.5%. 纯化收率62.0% (以粗品中利西拉来含量计算),总收率16.8%。 Purification Yield 62.0% (calculated to the content of crude lixisenatide), total yield of 16.8%.

[0138] 实施例5 [0138] Example 5

[0139] 将利西拉来粗肽4.0g (含利西拉来1.08g)用纯化水200ml溶解,过滤,收集滤液备用。 [0139] 4.0 g of the crude peptide lixisenatide (containing 1.08 g of lixisenatide) was dissolved with 200ml of purified water, collected by filtration and the filtrate set aside.

[0140] 纯化色谱条件: [0140] Purification Chromatographic conditions:

[0141] 高效液相色谱仪型号:Waters 2545 [0141] HPLC Model: Waters 2545

[0142] 色谱柱:50X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0142] Column: 50X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0143]流速:80ml/ 分钟。 [0143] flow rate: 80ml / min.

[0144]检测波长:280nm。 [0144] Detection wavelength: 280nm.

[0145] 流动相A相:20mM D-酒石酸钠和30mM磷酸氢二钠的10%甲醇/90%水溶液(v/v),用磷酸调pH为4.0。 [0145] Phase A Mobile Phase: 10% 20mM D- 30mM sodium tartrate and disodium hydrogen phosphate methanol / 90% aqueous (v / v), adjusted to pH 4.0 with phosphoric acid.

[0146] 流动相A相配制过程:称取46.0g D-酒石酸钠和42.6g磷酸氢二钠,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到IOL血清瓶,加入1.0L色谱纯甲醇后加纯化水至IOL刻度,用磷酸调pH为4.0。 [0146] Mobile phase A phase formulation process: Weigh 46.0g D- 42.6g sodium tartrate and disodium phosphate, had proper amount of purified water was dissolved 0.45 μ m membrane filter, the filtrate collected all IOL serum bottle, was added 1.0L purified water was added to the mark IOL after chromatography pure methanol, pH was adjusted to 4.0 with phosphoric acid. [0147] 流动相B相:色谱纯乙腈。 [0147] Mobile Phase B Phase: HPLC grade acetonitrile.

[0148]梯度: [0148] Gradient:

Figure CN102875663BD00101

[0149]上样量:2.0g(100mL)。 [0149] loading amount: 2.0g (100mL).

[0150] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0150] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment. [0151 ] 分两次进样,重复以上操作。 [0151] in two injections, repeat the above operation.

[0152] 转盐色谱条件: [0152] Chromatographic conditions turn salt:

[0153] 高效液相色谱仪型号:Waters 2545 [0153] HPLC Model: Waters 2545

[0154] 色谱柱:50X250mm,内装反相C4色谱填料,该填料的粒径为10 μ m。 [0154] Column: 50X250mm, built C4 reverse phase chromatography packing, particle size of the filler is 10 μ m.

[0155]流速:80ml/ 分钟。 [0155] flow rate: 80ml / min.

[0156]检测波长:280nm。 [0156] Detection wavelength: 280nm.

[0157] 流动相A相:0.20%冰醋酸(v/v)溶液。 [0157] Phase A Mobile phase: 0.20% glacial acetic acid (v / v) solution.

[0158] 流动相B相:色谱纯乙腈。 [0158] Mobile Phase B Phase: HPLC grade acetonitrile.

[0159]梯度: [0159] Gradient:

Figure CN102875663BD00102

[0160]上样体积:200ml。 [0160] Volume loaded: 200ml.

[0161] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0161] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至20ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 20ml lyophilization.

[0162] 冻干后得白色粉末状固体精肽0.65g。 [0162] to give fine white powder after lyophilization peptides 0.65g. 纯度98.52%,各杂质含量均小于0.5%。 A purity of 98.52% of the impurity content of less than 0.5%. 纯化收率60.2% (以粗品中利西拉来含量计算),总收率16.3%。 Purification Yield 60.2% (calculated to the content of crude lixisenatide), total yield of 16.3%.

[0163] 实施例6 [0163] Example 6

[0164] 将利西拉来粗肽4.0g (含利西拉来1.08g)用纯化水200ml溶解,过滤,收集滤液备用。 [0164] 4.0 g of the crude peptide lixisenatide (containing 1.08 g of lixisenatide) was dissolved with 200ml of purified water, collected by filtration and the filtrate set aside.

[0165] 纯化色谱条件: [0165] Purification Chromatographic conditions:

[0166] 高效液相色谱仪型号:Waters 2545 [0166] HPLC Model: Waters 2545

[0167] 色谱柱:50X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0167] Column: 50X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0168]流速:80ml/ 分钟。 [0168] flow rate: 80ml / min.

[0169]检测波长:280nm。 [0169] Detection wavelength: 280nm.

[0170] 流动相A相:IOmM D-酒石酸钠、IOmM D-酒石酸铵和IOmM磷酸二氢钠与IOmM磷酸二氢铵的10%甲醇/90%水溶液(v/v),用磷酸调pH为2.5。 [0170] Mobile Phase A Phase: IOmM D- sodium tartrate, IOmM D- ammonium tartrate and sodium dihydrogen phosphate and ammonium dihydrogen IOmM IOmM acid 10% methanol / 90% aqueous (v / v), pH was adjusted with phosphoric acid 2.5.

[0171] 流动相A相配制过程:称取23.0g D-酒石酸钠、18.4g D-酒石酸铵和12.0g磷酸二氢钠与11.5g磷酸二氢铵,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到IOL血清瓶,加入1.0L色谱纯甲醇后加纯化水至IOL刻度,用磷酸调pH为2.5。 [0171] Mobile phase A phase formulation process: Weigh 23.0g D- sodium tartrate, 18.4 g of D-tartaric acid and 12.0g of ammonium dihydrogen phosphate and sodium dihydrogen phosphate, ammonium 11.5g, after dissolving an appropriate amount of purified water was filtered through 0.45 μ m purified water was added to the mark IOL after membrane filtration, the filtrate was collected to all IOL serum vials, chromatography 1.0L methanol was added, pH was adjusted to 2.5 with phosphoric acid.

[0172] 流动相B相:色谱纯乙腈。 [0172] Mobile Phase B Phase: HPLC grade acetonitrile. [0173]梯度 [0173] Gradient

Figure CN102875663BD00111

[0174]上样量:2.0g(100mL)。 [0174] loading amount: 2.0g (100mL).

[0175] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0175] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment.

[0176] 分两次进样,重复以上操作。 [0176] in two injections, repeat the above operation.

[0177] 转盐色谱条件: [0177] Chromatographic conditions turn salt:

[0178] 高效液相色谱仪型号:Waters 2545 [0178] HPLC Model: Waters 2545

[0179] 色谱柱:50X250mm,内装反相C4色谱填料,该填料的粒径为10 μ m。 [0179] Column: 50X250mm, built C4 reverse phase chromatography packing, particle size of the filler is 10 μ m.

[0180]流速:80ml/ 分钟。 [0180] flow rate: 80ml / min.

[0181]检测波长:280nm。 [0181] Detection wavelength: 280nm.

[0182] 流动相A相:0.20%冰醋酸(v/v)溶液。 [0182] Phase A Mobile phase: 0.20% glacial acetic acid (v / v) solution.

[0183] 流动相B相:色谱纯乙腈。 [0183] Mobile Phase B Phase: HPLC grade acetonitrile.

[0184]梯度: [0184] Gradient:

Figure CN102875663BD00112

[0185]上样体积:200ml。 [0185] Volume loaded: 200ml.

[0186] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0186] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至20ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 20ml lyophilization.

[0187] 冻干后得白色粉末状固体精肽0.65g。 [0187] to give fine white powder after lyophilization peptides 0.65g. 纯度98.52%,各杂质含量均小于0.5%。 A purity of 98.52% of the impurity content of less than 0.5%. 纯化收率60.2% (以粗品中利西拉来含量计算),总收率16.3%。 Purification Yield 60.2% (calculated to the content of crude lixisenatide), total yield of 16.3%.

[0188] 实施例7 [0188] Example 7

[0189] 将利西拉来粗肽20g (含利西拉来5.70g)用纯化水500ml溶解,过滤,收集滤液备用。 [0189] The crude peptide lixisenatide 20g (lixisenatide containing 5.70 g) was dissolved with 500ml of purified water, collected by filtration and the filtrate set aside.

[0190] 纯化色谱条件: [0190] Purification Chromatographic conditions:

[0191] 高效液相色谱仪型号:Novasep LC150 [0191] HPLC Model: Novasep LC150

[0192] 色谱柱:100X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0192] Column: 100X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0193]流速:250ml/分钟。 [0193] flow rate: 250ml / min.

[0194]检测波长:280nm。 [0194] Detection wavelength: 280nm.

[0195] 流动相A相:20mM D-酒石酸钠和20mM磷酸二氢钠的10%甲醇/90%水溶液(v/v),用磷酸调pH为2.0。 [0195] Phase A Mobile Phase: 10% 20mM D- 20mM sodium tartrate and sodium dihydrogen phosphate methanol / 90% aqueous (v / v), adjusted to pH 2.0 with phosphoric acid.

[0196] 流动相A相配制过程:称取92.0g D-酒石酸钠和48.0g磷酸二氢钠,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到20L血清瓶,加入2.0L色谱纯甲醇后加纯化水至20L刻度,用磷酸调pH为2.0。 [0196] Mobile phase A phase formulation process: Weigh 48.0g 92.0g D- sodium tartrate and sodium dihydrogen phosphate, an appropriate amount of purified water was dissolved after through 0.45 μ m filter membrane and the filtrate collected all 20L serum bottle, was added 2.0L purified water was added to the 20L scale after chromatography pure methanol, pH was adjusted to 2.0 with phosphoric acid.

[0197] 流动相B相:色谱纯乙腈。 [0197] Mobile Phase B Phase: HPLC grade acetonitrile.

[0198]梯度:82%A+18%B—62%a+38%B[0199]上样量:10.0g (250ml)。 [0198] Gradient: 82% A + 18% B-62% a + 38% B [0199] sample volume: 10.0g (250ml).

[0200] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0200] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment. [0201 ] 分两次进样,重复以上操作。 [0201] in two injections, repeat the above operation.

[0202] 转盐色谱条件: [0202] Chromatographic conditions turn salt:

[0203] 高效液相色谱仪型号:Novasep LC150 [0203] HPLC Model: Novasep LC150

[0204] 色谱柱:100 X 250mm,内装反相C8色谱填料,该填料的粒径为10 μ m。 [0204] Column: 100 X 250mm, built-C8 reversed-phase chromatography packing, the particle size of the filler is 10 μ m.

[0205]流速:250ml/分钟。 [0205] flow rate: 250ml / min.

[0206]检测波长:280nm。 [0206] Detection wavelength: 280nm.

[0207] 流动相A相:0.1%冰醋酸(v/v)溶液。 [0207] Phase A Mobile phase: 0.1% acetic acid (v / v) solution.

[0208] 流动相B相:色谱纯乙腈。 [0208] Mobile Phase B Phase: HPLC grade acetonitrile.

[0209] [0209]

Figure CN102875663BD00121

[0210]上样体积:500ml。 [0210] Volume loaded: 500ml.

[0211] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0211] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至50ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 50ml lyophilization.

[0212] 冻干后得白色粉末状固体精肽3.31g。 [0212] to give fine white powder after lyophilization peptides 3.31g. 纯度98.52%,各杂质含量均小于0.5%。 A purity of 98.52% of the impurity content of less than 0.5%. 纯化收率58.1% (以粗品中利西拉来含量计算),总收率16.6%。 Purification Yield 58.1% (calculated to the content of crude lixisenatide), total yield of 16.6%.

[0213] 实施例8 [0213] Example 8

[0214] 将利西拉来粗肽200g (含利西拉来57.0g)用纯化水5000ml溶解,过滤,收集滤液备用。 [0214] The 200 g of crude peptide lixisenatide (lixisenatide containing 57.0 g) was dissolved with 5000ml purified water, collected by filtration and the filtrate set aside.

[0215] 纯化色谱条件: [0215] Purification Chromatographic conditions:

[0216] 高效液相色谱仪型号:Novasep LC300 [0216] HPLC Model: Novasep LC300

[0217] 色谱柱:300X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0217] Column: 300X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0218]流速:2000ml/分钟。 [0218] flow rate: 2000ml / min.

[0219]检测波长:280nm。 [0219] Detection wavelength: 280nm.

[0220] 流动相A相:30mM D-酒石酸钠和30mM磷酸氢二钠的10%甲醇/90%水溶液(v/v ),用磷酸调pH为2.5。 [0220] Phase A Mobile Phase: 10% 30mM D- 30mM sodium tartrate and disodium hydrogen phosphate methanol / 90% aqueous (v / v), adjusted to pH 2.5 with phosphoric acid.

[0221] 流动相A相配制过程:称取1035g D-酒石酸钠和639g磷酸氢二钠,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到150L储液罐,加入15L色谱纯甲醇后加纯化水至150L刻度,用磷酸调pH为2.5。 [0221] Mobile phase A phase formulation process: Weigh 1035g D- sodium tartrate and 639g of disodium hydrogen phosphate, had proper amount of purified water was dissolved 0.45 μ m membrane filter, to collect all the filtrate reservoir 150L, ​​15L added chromatography purified water was added to the 150L scale methanol, pH was adjusted to 2.5 with phosphoric acid.

[0222] 流动相B相:色谱纯乙腈。 [0222] Mobile Phase B Phase: HPLC grade acetonitrile.

Figure CN102875663BD00122

[0224]上样量:100.0g (2500ml)。 [0224] sample volume: 100.0g (2500ml). [0225] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0225] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment.

[0226] 分两次进样,重复以上操作。 [0226] in two injections, repeat the above operation.

[0227] 转盐色谱条件: [0227] Chromatographic conditions turn salt:

[0228] 高效液相色谱仪型号:Novasep LC300 [0228] HPLC Model: Novasep LC300

[0229] 色谱柱:300 X 250mm,内装反相C8色谱填料,该填料的粒径为10 μ m。 [0229] Column: 300 X 250mm, built-C8 reversed-phase chromatography packing, the particle size of the filler is 10 μ m.

[0230]流速:2000ml/分钟。 [0230] flow rate: 2000ml / min.

[0231]检测波长:280nm。 [0231] Detection wavelength: 280nm.

[0232] 流动相A相:0.2%冰醋酸(v/v)溶液。 [0232] Phase A Mobile phase: 0.2% acetic acid (v / v) solution.

[0233] 流动相B相:色谱纯乙腈。 [0233] Mobile Phase B Phase: HPLC grade acetonitrile.

[0234]梯度: [0234] Gradient:

Figure CN102875663BD00131

[0235]上样体积:500ml。 [0235] Volume loaded: 500ml.

[0236] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0236] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至450ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 450ml lyophilization.

[0237] 冻干后得白色粉末状固体精肽35.3g。 [0237] to give fine white powder after lyophilization peptide 35.3g. 纯度98.59%,各杂质含量均小于0.5%。 A purity of 98.59% of the impurity content of less than 0.5%. 纯化收率61.9% (以粗品中利西拉来含量计算),总收率17.4%。 Purification Yield 61.9% (calculated to the content of crude lixisenatide), total yield of 17.4%.

[0238] 实施例9 [0238] Example 9

[0239] 将利西拉来粗肽4000g (含利西拉来1139g)用纯化水100L溶解,过滤,收集滤液备用。 [0239] The 4000 g of crude peptide lixisenatide (including lixisenatide 1139 g) was dissolved with purified water 100L collected by filtration and the filtrate set aside.

[0240] 纯化色谱条件: [0240] Purification Chromatographic conditions:

[0241] 高效液相色谱仪型号:Novasep LC450 [0241] HPLC Model: Novasep LC450

[0242] 色谱柱:450X250mm,内装苯基硅烷键合硅胶为固定相填料,该填料的粒径为10 μ m0 [0242] Column: 450X250mm, built phenylsilane bonded silica gel as stationary phase packing, the particle size of the filler is 10 μ m0

[0243]流速:5000ml/分钟。 [0243] flow rate: 5000ml / min.

[0244]检测波长:280nm。 [0244] Detection wavelength: 280nm.

[0245] 流动相A相:30mM D-酒石酸钠和30mM磷酸氢二钠的10%甲醇/90%水溶液(v/v ),用磷酸调pH为2.5。 [0245] Phase A Mobile Phase: 10% 30mM D- 30mM sodium tartrate and disodium hydrogen phosphate methanol / 90% aqueous (v / v), adjusted to pH 2.5 with phosphoric acid.

[0246] 流动相A相配制过程:称取2070g D-酒石酸钠和1280g磷酸氢二钠,适量纯化水溶解后过0.45 μ m滤膜过滤,滤液全部收集到300L储液罐,加入30L色谱纯甲醇后加纯化水至300L刻度,用磷酸调pH为2.5。 [0246] Mobile phase A phase formulation process: Weigh 2070g D- tartrate and sodium phosphate dibasic 1280g, through the proper amount of purified water was dissolved 0.45 μ m membrane filter, to collect all the filtrate reservoir 300L, 30L added chromatography purified water was added to the 300L scale methanol, pH was adjusted to 2.5 with phosphoric acid. 用完重复配制。 Repeat the preparation exhausted.

[0247] 流动相B相:色谱纯乙腈。 [0247] Mobile Phase B Phase: HPLC grade acetonitrile.

Figure CN102875663BD00132

[0249]上样量:250.0g(6250ml)。 [0249] sample volume: 250.0g (6250ml).

[0250] 纯化过程:使色谱柱平衡5分钟后上样,运行梯度纯化,监测并分段收集目的峰馏分。 [0250] Purification process: that the loading column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the target segment peak fractions. 对所收集的馏分进行纯度检测(纯度检测的色谱条件与上述利西拉来含量测定的条件相同,以面积归一化法进行测定),将纯度大于等于98%的馏分除去大部分乙腈后用于转盐;将纯度大于等于70%小于98%的馏分除去大部分乙腈后回收并重复该纯化过程,再次收集纯度大于等于98%的馏分除去大部分乙腈后也用于转盐;纯度小于70%的馏分按废液处理。 The collected fraction (chromatographic purity test conditions the same conditions as the above-described assay lixisenatide content, by area normalization method was measured) detecting purity, a purity of greater than or equal to 98% after most of the acetonitrile was removed by fraction to turn salt; purity of 70% or more of the fraction less than 98% most of the acetonitrile is removed after the recovery and purification process is repeated, again collected purity not less than 98% after most of the acetonitrile fraction is also used for transferring salt removal; purity of less than 70 % fraction by waste liquid treatment.

[0251] 分16次进样,重复以上操作。 [0251] 16 minutes injections, repeat the above operation.

[0252] 转盐色谱条件: [0252] Chromatographic conditions turn salt:

[0253] 高效液相色谱仪型号:Novasep LC450 [0253] HPLC Model: Novasep LC450

[0254] 色谱柱:450 X 250mm,内装反相C8色谱填料,该填料的粒径为10 μ m。 [0254] Column: 450 X 250mm, built-C8 reversed-phase chromatography packing, the particle size of the filler is 10 μ m.

[0255]流速:5000ml/ 分钟。 [0255] flow rate: 5000ml / min.

[0256]检测波长:280nm。 [0256] Detection wavelength: 280nm.

[0257] 流动相A相:0.2%冰醋酸(v/v)溶液。 [0257] Phase A Mobile phase: 0.2% acetic acid (v / v) solution.

[0258] 流动相B相:色谱纯乙腈。 [0258] Mobile Phase B Phase: HPLC grade acetonitrile.

[0259]梯度 [0259] Gradient

Figure CN102875663BD00141

[0260]上样体积:2500ml。 [0260] Volume loaded: 2500ml.

[0261] 纯化过程:将色谱柱平衡5分钟后上样,运行梯度纯化,监测并收集目的峰馏分。 [0261] Purification process: loading the column equilibration for 5 minutes, run a gradient purified, monitoring and collecting the peak fraction purposes. 将目的峰馏分减压旋蒸浓缩至9000ml后冻干。 The object of the peak fraction was concentrated to rotary evaporation under reduced pressure to 9000ml lyophilization.

[0262] 冻干后得白色粉末状固体精肽704g。 [0262] after lyophilization as a white powdery solid fine peptide 704g. 纯度98.39%,各杂质含量均小于0.5%。 A purity of 98.39% of the impurity content of less than 0.5%. 纯化收率61.8% (以粗品中利西拉来含量计算),总收率17.6%。 Purification Yield 61.8% (calculated to the content of crude lixisenatide), total yield of 17.6%.

Claims (7)

1.一种利西拉来的纯化方法,其包括以下步骤: 步骤1:采用高效液相色谱法对利西拉来粗肽进行纯化,所述高效液相色谱法以苯基硅烷键合硅胶为固定相,以D-酒石酸盐的甲醇/磷酸盐缓冲溶液为流动相A相,以乙腈为流动相B相进行梯度洗脱,流动相A相中的磷酸盐的摩尔浓度为20mM~50mM,且洗脱梯度35πιιπ为(75~85) %A-> ( 55-65 ) %A; 步骤2:采用高效液相色谱法对步骤I所得到的馏分进行转盐纯化,所述高效液相色谱法以烷基硅烷键合硅胶为固定相,以冰醋酸溶液为流动相A相,以乙腈为流动相B相进行梯度洗脱,流动相A相中的冰醋酸溶液的体积百分浓度为0.1~0.3% ;步骤3:收集溶液冻干。 A lixisenatide purification method comprising the following steps: Step 1: HPLC method lixisenatide crude peptide was purified by high performance liquid chromatography to said phenylsilane bonded silica as stationary phase, D- tartrate in methanol / phosphate buffer solution as mobile phase a phase to B phase acetonitrile as mobile phase gradient elution, mobile phase phosphate molar concentration of phase a is 20mM ~ 50mM, and 35πιιπ elution gradient of (75 ~ 85)% A-> (55-65)% a; step 2: high performance liquid chromatography on fractions obtained in step I turn salt purified by the high performance liquid chromatography volume percentage concentration method with silane bonded silica gel as the stationary phase, mobile phase was acetic acid to phase a, phase B acetonitrile as mobile phase gradient elution, the mobile phase a solution of glacial acetic acid phase is 0.1 to 0.3%; step 3: collection solution was lyophilized.
2.权利要求1的方法,其中所述步骤I中高效液相色谱法的流动相A相中的D-酒石酸盐的摩尔浓度为IOmM~50mM。 Molar concentration method of claim 1, wherein said flowing step I phase HPLC D- tartrate salt A phase is IOmM ~ 50mM.
3.权利要求1的方法,其中所述步骤I中高效液相色谱法的流动相A相中的D-酒石酸盐为一种或多种选自D-酒石酸钠、D-酒石酸钾、D-酒石酸铵的D-酒石酸盐。 The method of claim 1, wherein said flowing step I phase HPLC D- tartrate salt A phase is one or more selected from sodium D- tartrate, potassium tartrate D-, D- D- tartrate ammonium tartrate.
4.权利要求1的方法,其中所述步骤I中高效液相色谱法的流动相A相中的磷酸盐为一种或多种选自磷酸钠、磷酸氢二钠、磷酸二氢钠、磷酸钾、磷酸氢二钾、磷酸二氢钾、磷酸铵或磷酸二氢铵的磷酸盐。 The method of claim 1, wherein said flowing step I by HPLC with phosphate phase A is one or more selected from sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium phosphate or ammonium dihydrogen phosphate.
5.权利要求1的方法,其中所述步骤I中高效液相色谱法的流动相A相溶液的pH为2.0 ~4.0。 The method of claim 1, wherein the pH of the mobile phase A step I by HPLC with a solution of 2.0 to 4.0.
6.权利要求1的方法,·其中所述步骤I中高效液相色谱法的流动相A相中的甲醇占整个流动相A相的体积比为10~20%。 6. The method of claim 1, wherein said step of flowing-I HPLC with methanol accounts for the entire phase A mobile phase A phase volume ratio of 10 to 20%.
7.权利要求1的方法,其中所述步骤2中高效液相色谱法的固定相为八烷基硅烷键合硅胶、六烷基硅烷键合硅胶或四烷基硅烷键合硅胶。 The method of claim 1, wherein said stationary phase HPLC of step 2 is octadecyl silane bonded silica, silane bonded silica six or tetraalkyl silane bonded silica gel.
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