CN100493615C - Monomodified PEGylated insulin and its preparation method - Google Patents

Monomodified PEGylated insulin and its preparation method Download PDF

Info

Publication number
CN100493615C
CN100493615C CN 200610118923 CN200610118923A CN100493615C CN 100493615 C CN100493615 C CN 100493615C CN 200610118923 CN200610118923 CN 200610118923 CN 200610118923 A CN200610118923 A CN 200610118923A CN 100493615 C CN100493615 C CN 100493615C
Authority
CN
Grant status
Grant
Patent type
Prior art keywords
insulin
modified
concentrated
reaction
peg
Prior art date
Application number
CN 200610118923
Other languages
Chinese (zh)
Other versions
CN1970086A (en )
Inventor
印春华
敏 张
窦怀智
Original Assignee
复旦大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Grant date

Links

Abstract

本发明公开了单修饰的聚乙二醇化胰岛素及其制备方法。 The present invention discloses a PEGylated insulin and a preparation method of a single modified. 本发明的单修饰的聚乙二醇化胰岛素以碳氮键相连,分子量范围6550~10800。 Polyethylene glycol mono-modified insulin of the present invention is connected to carbon-nitrogen bond, a molecular weight range of 6550 to 10800. 其按如下步骤制备:将胰岛素溶于pH3.0~5.0醋酸盐缓冲液,加入mPEG-醛和NaBH<sub>3</sub>CN,搅拌,加入小分子氨基酸终止反应,透析浓缩后上SP Sepharose FF层析柱,用含NaCl的缓冲液进行梯度洗脱,收集的峰3洗脱液,浓缩,透析,冷冻干燥,得PheB1-PEG-胰岛素。 Which was prepared as follows: The insulin was dissolved in pH3.0 ~ 5.0 acetate buffer, added mPEG- aldehyde and NaBH <sub> 3 </ sub> CN, with stirring, the reaction was terminated by adding a small amino acid molecules, after dialysis concentrate SP Sepharose FF column performed with a buffer containing NaCl gradient elution, peak 3 eluate collected, concentrated, dialyzed and lyophilized to yield PheB1-PEG- insulin. 与现有技术相比,本发明的PheB1-PEG-胰岛素保留了胰岛素的原有活性且提高其物理生物稳定性,该制备方法操作简便,适于工业化大生产。 Compared with the prior art, PheB1-PEG- insulin according to the present invention retain the original activity of insulin and increase the physical stability of the biological, the preparation method is simple and suitable for industrial mass production.

Description

一种单傪饰的聚乙二酵化胰岛素及其制备方法 A single-fermentation polyethylene insulin and a preparation method of decoration Ca

技术顦域 Technical 顦 domain

本发明属聚合物修饰蛋白质多肽的技术领域,具体涉及单修饰的聚乙二醉化胰岛素及其制备方法。 The present invention belongs to the field of polymer modification techniques of proteins polypeptides, particularly relates to a single polyethylene glycol-modified insulin and a preparation method of intoxication. 背暴技术 Back violent Technology

胰岛素(insulin)是脊椎动物胰脏P细胞分泌的一种重要的控制血糖的多肽调节激素。 Insulin (insulin) are vertebrate cells secrete pancreatic polypeptide P an important control blood sugar-regulating hormone. 来源于不同种属的胰岛素的一级结构稍有差异。 Insulin from different species of the primary structure is slightly different. 1955年Sanger等阐明牛胰岛素的一级结构。 1955 Sanger et elucidate the primary structure of bovine insulin. 它由含有51个氨基酸的两条肽链构成,其中A链含有21个氨基酸残基,B链含有30 个氨基酸残基,A链和B链间通过二个二硫键共价连接,另外,A链本身还有一个链内二硫键。 It consists of two peptide chains comprising 51 amino acids, wherein the A-chain containing 21 amino acid residues of the B chain containing 30 amino acid residues connected by two disulfide covalent A and B chains, in addition, a chain itself there is a disulfide bond. 二级结构主要有a-螺旋和P-折叠两种构型。 A- helical secondary structure mainly P- and folded two configurations. 其分子量为5734。 A molecular weight of 5734. 自从1921年问世以来,其一直是胰岛素依赖型糖尿病患者的首选药物,但临床应用中还存在没有解决的问题, 如消化过程导致胰岛素的口服给药难以实现;肾脏的快速清除和体内蛋白水解作用导致糖尿病患者需频繁注射胰岛素,而长期反复注射会使体内产生抗体,甚至引起过敏和胰岛素抵抗;外源的胰岛素会引起免疫反应;胰岛素六聚体化会导致起效滞后;纤维化作用使胰岛素制剂缺乏稳定性并降低其活性。 Since its 1921 inception, it has been the drug of choice in patients with insulin-dependent diabetes, but clinical application has not solved the problem, such as the digestive process leading to oral administration of insulin difficult to achieve; rapid clearance and kidney in vivo proteolysis lead to diabetes patients need frequent injections of insulin, long-term repeated injections make the body produce antibodies, insulin resistance and even cause allergies; exogenous insulin can cause an immune response; insulin hexamer cause onset of hysteresis; fibrosis insulin lack of stability of the formulation and decrease its activity.

聚合物修饰的方法被尝试用来解决上述问题,如Myere, SR等用脂肪酸对胰岛素进行修饰(Diabetes, 1997, 46: 637); Kim, SW等用单甲氧聚乙二醇(mPEG)对胰岛素进行修饰(Bioconjugate Chemistry , 2000 , 11: 195-201; Advanced Drug Delivery Reviews, 2002, 54: 505-530): Calceti, R等用聚乙二醇(PEG)化胰岛素结合水凝胶开发出新型胰岛素口服给药系统(European Journal of Pharmaceutical Sciences, 2004, 22: 315-323)。 Polymer modified methods are used to attempt to solve the above problems, such as Myere, SR and other modified insulin (Diabetes, 1997, 46: 637) with a fatty acid; Kim, SW etc. by the monomethoxy polyethyleneglycol (mPEG) of insulin modified (Bioconjugate Chemistry, 2000, 11: 195-201; Advanced Drug Delivery Reviews, 2002, 54: 505-530): Calceti, R and the like with polyethylene glycol (PEG) hydrogel insulin binding develop new insulin for oral administration (European Journal of Pharmaceutical Sciences, 2004, 22: 315-323). 胰岛素的PEG化可从胰岛素结构上解决其临床应用存在的问题。 PEG insulin may solve the problem on their clinical use the insulin structure. PEG化可以提髙胰岛素稳定性,增加其抵抗瞎解的能力,消除其免疫原性和抗原性,并降低肾脏淸除速率延长体循环时间。 PEG insulin Gao of stability can be improved to increase its ability to resist solution blind, eliminating antigenicity and immunogenicity, and reduce the rate of kidney Qing prolonged circulation time of addition. 胰岛素单体具有三个游离氨基,分别为Al位Gly, Bl位Phe, B29位Lys。 Insulin possesses three free amino group monomer, respectively, Al bit Gly, Bl bit Phe, B29 bits Lys. Al位和B29 位被修饰后,胰岛素活性部分丧失或不能显著改善胰岛素的物理化学性质。 Al bits and the modified insulin position B29 or partial loss of activity not significantly improve the physicochemical properties of insulin. 因此Kim,SW 等研究了胰岛素的定位修饰技术(Advanced Drug Delivery Reviews, 1999, 35: 289-360)。 So Kim, SW and other studies localization of insulin modification techniques (Advanced Drug Delivery Reviews, 1999, 35: 289-360). 该方法先用Boc (苄甲氧基)保护未修饰的氨基,再以酸軒为活性基团的PEG对目标位点进行PEG化,反应在DMF或DMSO中进行并且保持高pH。 The first method for maintaining a high pH and in the DMF or DMSO with Boc (methoxybenzyl) amino-protected unmodified, then the acid Xuan target loci of the PEG is a PEG reactive group, the reaction. Boc对非修饰位点的保护将提髙生产成本,使步骤复杂化。 Boc protection of the non-modified sites provide Gao production costs, complicated steps. 酸酐和氨基的反应速度较慢,产率较低。 An amino acid anhydride and the reaction rate is slow, lower yields. 另外,有机溶剂和离pH可致胰岛素活力降低,且不利于产品的后处理。 Further, from the organic solvent, and pH can cause reduced insulin activity, and not conducive to the product after treatment. Harris, JM等制备了以醒基为活性基团的mPEG,为简化PEG化蛋白质的反应步骤及提髙PEG化蛋白质复合物的稳定性提供了可能(Advanced Drug Delivery Reviews, 2002, 54: 459-476)。 Group to wake up to an active group mPEG Harris, JM et prepared, may be provided (Advanced Drug Delivery Reviews reaction step to simplify the PEG protein extract and protein Gao PEG complex stability, 2002, 54: 459- 476). Kinstler, O.等利用蛋白质N端氨基具有较低pKa的特点,用mPEG-酵对其进行定位修饰并成功制备了N末端单修饰PEG化重组人粒细胞集落刺激因子(AG-CSF) (Advanced Drug Delivery Reviews, 2002, 54: 477-485)。 Kinstler, O. and other use of protein having a characteristic N-terminal amino group of lower pKa, its positioning and successfully modified with mPEG- yeast N-terminal of the single PEG modified recombinant human granulocyte colony stimulating factor (AG-CSF) Preparation of (Advanced Drug Delivery Reviews, 2002, 54: 477-485). 郁正艳等(中国专利, 20041008卯50.8,公开日:2005年8月10日)发明了一种单甲氧基聚乙二醉-胰岛素复合物,胰岛素和单甲氧基聚乙二醇通过前者的游离氨基与后者的具有活性的丙醛基团连接成的酰胺键连接在一起,分子量介于10.8〜25.8KD。 Yuzheng Yan et al (China patent 20041008 d 50.8, Publication date: August 10, 2005) invented a monomethoxy polyethylene glycol drunk - insulin compound, insulin and monomethoxy polyethyleneglycol by the former the free amino group of the latter with propionic aldehyde group having an active connection to the amide linkage with a molecular weight between 10.8~25.8KD. 发明内容 SUMMARY

本发明所霈解决的技术问题之一是提供一种单修饰的聚乙二醉化胰岛素;本发明所需解决的技术问题之二是提供一种单修饰的聚乙二醇化胰岛素的制备方法,以克服现有技术的不足和缺陷。 Pei one of the technical problem solved by the present invention is to provide a mono-modified polyethylene drunk insulin; the present invention is required to solve the second technical problem is to provide a mono-modified PEGylated insulin preparation, to overcome the shortcomings and deficiencies of the prior art.

本发明的构思如下: Concept of the invention as follows:

在酸性条件下,由于a-氨基与其他亲核类基团相比具有较低的pKa值,使得醛与a-贫基的偶合具有高选择性。 Under acidic conditions, since the a- and other nucleophilic amino group having a lower class pKa value as compared with an aldehyde such coupling lean a- group with high selectivity. 亲电类PEG衍生物与氨基酸残基的偶合在很大程度上取决于氨基酸残基的亲核性。 Coupling of PEG derivatives to electrophilic class of amino acid residues depends largely on the nucleophilic amino acid residues. 亲核反应只有在蛋白质水溶液的pH值接近或略高于蛋白质的pKa值时才会发生,所以每个残基的反应活性还取决于相邻的氨基酸残基。 Nucleophilic reactions occur only at pH values ​​close to or slightly higher than the protein solution pKa value of proteins, the activity of each residue of the reaction also depends on the adjacent amino acid residues. 虽选择性仍达不到100%, 但修饰剂与赖氨酸反应的不均一性大大降低。 Although selectivity is still less than 100%, but a variation of lysine react with the modifying agent is greatly reduced. 通过Schiff^的形成,醛可偶联到伯胺上,从而得到稳定的仲胺。 By forming Schiff ^, the aldehyde can be coupled to a primary amine, resulting in a stable secondary amine. 与其它亲电活性基团不同,醛基只与伯胺反应。 Other electrophilic with different active groups, aldehyde groups reacted only with primary amines. 利用mPEG-醛修饰胰岛素可避免对B29位Lys的修饰,但胰岛素具有A, B两条肽链,它们的末端有两个伯胺基团,通过控制反应条件可获得选择性修饰B1位的PEG化胰岛素,而避免mPEG-醛对Al位的修饰。 Modified insulin can be avoided by using mPEG- aldehyde modified position B29 Lys, but insulin A, B, two peptide chains, their ends have two primary amine groups, can be obtained by controlling the reaction conditions are modified to selectively position B1 PEG insulin, and to avoid mPEG- aldehyde modification of Al bits.

本发明的技术方案如下: Aspect of the present invention is as follows:

本发明的单修饰的聚乙二醇化胰岛素,mPEG与胰岛素以碳氮键相连,其通式如下: Mono-modified polyethylene glycol according to the present invention, insulin, and insulin of mPEG coupled to carbon-nitrogen bond, having the general formula:

HH HH

CHrO-(CH2CH20)n-(CH2)m—C—N—PheBl-insulin CHrO- (CH2CH20) n- (CH2) m-C-N-PheBl-insulin

H H

其中m为l〜3,分子量范围为6550〜10800, n为16-115。 Wherein m is l~3, a molecular weight in the range 6550~10800, n is 16-115.

所说的mPEG分子量为750〜5000,可避免较大的mPEG分子修饰胰岛素因空间位阻效应降低其生物活性。 He said molecular weight of 750~5000 mPEG, mPEG molecules can be modified to avoid large steric hindrance due to insulin decrease its biological activity. 本发明的单修饰的聚乙二醇化胰岛素,其制备方法如下: Polyethylene glycol mono-modified insulin of the present invention, was prepared as follows:

(1) 将1份胰岛素溶于pH3.0~5.0醋酸盐缓冲液(含NaCl),加入1〜10份mPEG-醛和5〜30份NaBH3CN, 4〜37'C搅拌0.5〜24小时后,加入小分子氨基酸终止反应;这里的份数以摩尔计; (1) insulin was dissolved in 1 part of pH3.0 ~ 5.0 acetate buffer (with NaCl), was added aldehyde and 5~30 1~10 parts mPEG- parts NaBH3CN, 4~37'C stirred for 0.5~24 hours , a small molecule acid was added to terminate the reaction; parts herein on a molar basis;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1M NaCl的pH值为2.0-4.5的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with a buffer containing 0~1M NaCl pH value 2.0 to 4.5, the absorption peak of 220nm UV detector;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素(PheBl陽PEG-insulin)。 (4) The collected peak 3 eluate was concentrated, dialyzed and freeze-dried to obtain a single-modified PEGylated insulin (PheBl male PEG-insulin).

所说的反应中醋酸盐缓冲液的pH值为3.0~5.0,优选的pH值为3.6~4.5,最优选的pH 值为3.8~4.2。 The reaction of said acetate buffer pH value of 3.0 to 5.0, preferably a pH value of 3.6 to 4.5, and most preferably a pH of 3.8 to 4.2. pH偏低,氨基质子化程度高,无法被mPEG-醛修饰,同时,强酸条件下NaBH3CN不稳定;pH偏高,接近胰岛素等电点(5.8)时,胰岛素的溶解度较低,反应无法进行,另外,碱性条件修饰缺乏特异性。 low pH, high degree of protonation of an amino group, an aldehyde mPEG- can not be modified, while NaBH3CN unstable under strongly acidic conditions; high pH close to the isoelectric point of insulin (5.8), the solubility of insulin is low, the reaction can not be carried out, In addition, the lack of specificity of the modified alkaline conditions.

mPEG-醛和胰岛素的用量比例(摩尔)为1~10:1,优选的比例为1~5:1,最优选的比例为2:1。 mPEG- aldehyde and an amount ratio (molar) insulin 1 to 10: 1, preferably a ratio of 1 to 5: 1, most preferably the ratio is 2: 1. mPEG-醛和胰岛素的用量比例过低,胰岛素不能充分被修饰,过高,则其他位点被更多修饰。 The amount and ratio of aldehyde mPEG- low insulin, insulin can not be sufficiently modified, too, the other site is more modified.

NaBH3CN和胰岛素的用量(摩尔)比例为5~30:1,优选的比例为12~20:1,最优选的比例为15:1。 NaBH3CN and an amount (mole) ratio of insulin is 5 to 30: 1, preferably a ratio of 12 to 20: 1, most preferred ratio is 15: 1. NaBH3CN和胰岛素的用量比例过低,Schiff碱无法充分还原,过高,则会使反应体系pH升高,降低修饰的特异性。 The amount of insulin is too low and the ratio of NaBH3CN, Schiff base can not be reduced sufficiently, is too high, the reaction system was elevated pH will decrease modified specificity.

所说的反应温度为4〜37°C,优选温度为室温。 Said reaction temperature is 4~37 ° C, preferably at room temperature. 反应温度过低会使反应速度慢,但过高则会导致蛋白质变性。 The reaction temperature is too low will slow response, but too high will lead to protein denaturation.

所说的反应时间为0.5〜24小时,优选的反应时间为5〜8小时。 He said reaction time is 0.5~24 hours, preferably the reaction time is 5 ~ 8 hours. 低于0.5小时,大量胰岛素不能被修饰,但超过24小时,则双修饰产物比例增大。 Less than 0.5 hours, a large number of insulin can not be modified, but more than 24 hours, the doubly modified product ratio increases.

所说的洗脱pH值为2.0〜4.5,优选的pH值为3.8〜4.2。 The pH of the elution of said 2.0~4.5, preferably a pH of 3.8~4.2. pH值过高,分离物无法与填料充分结合,各组分不能被完全分离。 high pH, ​​the separation was not fully integrated with the filler, the components can not be completely separated.

本发明的优点在于: Advantage of the present invention:

(1) 单修饰的聚乙二醇化胰岛素(PheBI-PEG-insulin),可保持胰岛素原有的生物活性; 增强其生物稳定性,抵抗蛋白酶的水解作用;提高其物理稳定性,降低纤维化作用;降低其免疫原性;延长降血糖作用时间,提高胰岛素的生物利用度; (1) Single-modified PEGylated insulin (PheBI-PEG-insulin), insulin can maintain the original biological activity; enhanced biological stability, resistance to protease hydrolysis; enhance its physical stability, decreased fibrosis ; reduce their immunogenicity; prolonged hypoglycemic effect time, increase the bioavailability of insulin;

(2) 单修饰的聚乙二醇化胰岛素(PheBl-PEG-insulin),使用mPEG—醛修饰胰岛素, 所生成的CC和CN键较稳定,不存在使用早期PEG化试剂,生成不稳定酯键连接等问题;(3)本发明的单修饰的聚乙二醇化胰岛素(PheBl-PEG-insulin)的制备方法,通过控制pH、反应时间及投料比可使得对胰岛素Bl位的定位修饰达到90%左右。 (2) a modified single PEGylated insulin (PheBl-PEG-insulin), aldehyde-modified insulin using mPEG-, the generated CC and CN bonds more stable, there is no early PEG reagents used to produce an ester bond labile and so on; (3) a single modified according to the present invention is pEGylated insulin (PheBl-PEG-insulin) preparation, by controlling the pH, reaction time and feed ratio may be so positioned on insulin Bl bit modification reaches 90% . 该方法操作简单, 成本较低,且适合于工业化大生产。 This method is simple, low cost, and is suitable for industrial production. 耩鸛说明 Stork explained plow

图1为实施例11中步骤(2)的反应混合物的高效液相色谱图,表明该混合物中以单修饰的聚乙二酵化胰岛素为主,约占80%。 FIG 1 is a step in Example 11 (2) of FIG. HPLC of the reaction mixture, the mixture showed a single-modified polyethylene-based fermentation insulin, about 80%.

图2为实施例11中步骤(2)的反应混合物的离子交换色谱图,峰1为穿透峰含过量mPEG,峰2含2mPEG—insulin,峰3含PheBl-PEG-insuiin,峰4含未反应的胰岛素。 Figure 2 is an ion reaction procedure of Example 11 (2) a mixture of Embodiment FIG exchange chromatography, a peak having a peak in excess of mPEG to penetrate, peak 2 contains 2mPEG-insulin, peak 3 containing PheBl-PEG-insuiin, not containing peaks 4 insulin reaction.

图3为图2中各洗脱峰纯化产物的高效液相色谱图,各图中仅有单一的色谱峰,表明已得到纯度较高的单修饰的聚乙二醇化胰岛素(PheBl-PEG2000-insulin)。 FIG 3 is a HPLC chromatogram of each elution peak 2 in FIG purification of the product, only a single peak in each of the figures, show that has been modified with high purity mono PEGylated insulin (PheBl-PEG2000-insulin ).

图4为单修饰的聚乙二醇化胰岛素(?}^8卜?£02000-^01的的基质辅助激光解吸电离-串联飞行时间质谱图,表明PheB卜PEG2000-insulin的分子量为7800。 ?? 4 is a mono-modified PEGylated insulin (Bu ^ 8} £ 02000- ^ 01 of matrix assisted laser desorption ionization - time of flight tandem mass spectrum, PheB Bu PEG2000-insulin showed a molecular weight of 7800.

图5为单修饰的聚乙二醇化胰岛素(?1^81-?£050004加^11)的基质辅助激光解吸电离-串联飞行时间质谱图,表明PheBl-PEG5000-insulin的分子量为10800。 FIG 5 is a single PEGylated insulin-modified matrix assisted laser desorption ionization (1 ^ 81- £ 050004 plus 11 ^??) - time of flight tandem mass spectrum indicated a molecular weight PheBl-PEG5000-insulin was 10,800.

图6为单修饰的聚乙二醇化胰岛素(PheBl-PEG2000-insulin)的片段分析图,其中(a) PheBl-PEG2000-insulin经胰蛋白酶酶切,而未经过二硫苏糖醇(DTT)处理,胰蛋白酶酵切位点在B末端的精氨酸处,若B29位Lys被修饰则酶切产物中应存在分子量约为2800 的片段,结果显示未见该修饰片段,仅有分子量约为900的B末端片段,表明B末端未被PEG修饰。 FIG 6 is a mono-modified PEGylated insulin (PheBl-PEG2000-insulin) in FIG fragment analysis, wherein (a) PheBl-PEG2000-insulin by trypsin digestion, without going through dithiothreitol (DTT) Processing , yeast trypsin cleavage site at the arginine at the end B, the Lys at position B29 is modified if the cleavage product fragment of approximately 2800 molecular weight should be present, the results show no modification segment, only a molecular weight of about 900 terminal fragment of B, B is not indicate the end of PEG modification. (b) PheBl-PEG2000-insulin经DTT处理,不进行醃切,由于胰岛素A、 B链的分子量相差较大,如果其A、 B链同时被修饰,分子量鉴定中应有两个明显的钟形区域, 结果显示处理后的样品存在A链(分子量约为2600), B链(分子量约为3500,可能是处理过程导致连接的PEG链脱落)和连接PEG的B链(分子量约为5500),表明PEG连接在B链上,而未连接在A链上。 (B) PheBl-PEG2000-insulin by treatment with DTT, pickled not cut, since the insulin A, B chain molecular weight difference between the larger, if it is A, the B chain is modified at the same time, there should be two distinct molecular identification bell region, the result display process in the sample a chain (molecular weight about 2600), B-chain (molecular weight of about 3500, may be connected to the process leading to detachment of the PEG chain) and the connection of the B chain of PEG (molecular weight of about 5500), It showed that PEG attached to B-chain, but not connected to the A chain. (c〉 PheBl-PEG2000-insulin酶切后用DTT处理,分子量测定显示有B末端(分子量约为900), A链(分子量约为2600)及除去B末端连接PEG 的B链(分子量约为4500)。综合三个图谱,可判定PEG修饰在胰岛素的B1位。 (C> PheBl-PEG2000-insulin after enzyme treatment with DTT, showed a molecular weight measurement terminal B (molecular weight about 900), A-chain (molecular weight about 2600), and terminal B is connected to remove the B chain of PEG (molecular weight of about 4500 ). integrated three maps, PEG modification can be determined in the position B1 insulin.

图7为实施例11和实施例20中的PheBl-PEG2000-insulin和PheBl-PEG5000-insulin 的降血糖作用,表明PheBl-PEG-insulin保留了胰岛素的原有的生物活性,且延长降血糖作用的时间。 11 and FIG. 7 is a hypoglycemic action PheBl-PEG2000-insulin and PheBl-PEG5000-insulin in Example 20. Example indicating PheBl-PEG-insulin retain the original biological activity of insulin and extended the hypoglycemic effect time.

图8为实施例11和实施例20中的PheB卜PEG2000-insulin和PheBl-PEG5000-insulin 的抵抗胰蛋白雜的雜解作用,同胰岛素相比,PheB卜PEG-insulin可降低胰蛋白酶的肆解作用。 8 and FIG. 11 is a resistance to trypsin and PheB Bu PEG2000-insulin PheBl-PEG5000-insulin in Example 20 of the embodiment of the hybrid protein heteroaryl photolysis embodiment, as compared with the insulin, PheB Bu PEG-insulin solutions can store decrease trypsin effect. 图9为实施例11和实施例20中的PheBl-PEG2000-insulin和PheBl-PEG5000-insulin 的抵抗a-糜蛋白酶的酶解作用,同胰岛素相比,PheBl-PEG-insulin可降低a-糜蛋白酵的酶解作用。 9 is an embodiment 11 of 20 PheBl-PEG2000-insulin and enzymatic hydrolysis PheBl-PEG5000-insulin resistance and a- chymotrypsin embodiment, as compared with the insulin, PheBl-PEG-insulin reduces a- chymotrypsin enzymatic hydrolysis of yeast.

图10为实施例11和实施例20中的PheBl-PEG2000-insuIin和PheBl-PEG5000-i加ulin 的远紫外圆二色谱图,PheBl-PEG-insulin的[e] 223比值增大,表明两个胰岛索单体缔合为二聚体时形成的反平行p-折叠结构减少,PEG化抑制单体的寡聚化。 FIG 10 is a PheBl-PEG2000-insuIin 20 and far UV circular PheBl-PEG5000-i plus ulin dichroism FIG Example 11 and Example, PheBl-PEG-insulin to [e] 223 ratio is increased, showed two insulin reduction monomers associate to form dimers when antiparallel p- folding structure, PEG-suppressing oligomerization of monomers.

图11为实施例U和实施例20中的PheBl-PEG2000-insulin和PheBl-PEG5000-insulin 的近紫外圆二色谱图,PheBl-PEG-insulin的273 rnn谱带主要是由胰岛素二聚体中两个单体之间的侧链芳香环以及六聚体中的侧链芳香环形成,[e】273负值的幅度随着PEG化程度的提高而降低,表明PEG化能够在一定程度上抑制胰岛素单体的寡聚化。 Example 11 is a near-UV circular and U PheBl-PEG2000-insulin Example 20 and PheBl-PEG5000-insulin dichroism FIG embodiment, PheBl-PEG-insulin band of 273 rnn mainly by two insulin dimers the side chain of an aromatic ring between monomeric and hexameric form an aromatic ring in a side chain, [273] e negative amplitude with increasing degree of PEG is reduced, indicating that PEG of insulin can be suppressed to a certain extent oligomerization of the monomers.

(上述PEGxxxx表示分子量为xxxx的PEG,下同) 具体实施方式 (PEGxxxx above represents the molecular weight is xxxx of PEG, the same below) DESCRIPTION OF EMBODIMENTS

通过下面的具体实施例可进一步了解本发明。 Specifically by the following examples of the present invention may be further understood. 但它们不是对本发明的限定。 But they are not limitative of the present invention. 实施例l: Example l:

(1) 将1份胰岛素溶于pH3.0醋酸盐缓冲液(含NaCl),加入1份mPEG750-醛和10 份NaBH3CN, 20'C撹拌10小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.0 (containing NaCI), and 1 part 10 parts of an aldehyde mPEG750- NaBH3CN, 20'C Jiao stirred for 10 h, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓缩液上SPSepharose FF层析柱,用含0〜1MNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on SPSepharose FF chromatography column, eluting with a gradient of buffer pH3.8 containing a 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例2: Example 2:

(1) 将1份胰岛素溶于pH4.0醋酸盐缓冲液(含NaCl),加入2份mPEG750-醛和5 份NaBHbCN, 20'C搅拌8小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.0 (containing NaCI), was added 2 parts of an aldehyde and 5 parts mPEG750- NaBHbCN, 20'C After stirring for 8 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.0 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例3: Example 3:

(1)将1份胰岛素溶于pH3.8醋酸盐缓冲液(含NaCl),加入10份mPEG750-醆和30份NaBH3CN, 20'C搅拌0.5小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.8 (containing NaCI), was added 10 parts and 30 parts of NaBH3CN mPEG750- 醆, 20'C After stirring for 0.5 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH2.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH2.0 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例4: Example 4:

(1) 将l份胰岛素溶于pH4.5醋酸盐缓冲液(含NaCl),加入5份mPEG750-醛和10 份NaBH3CN, 20'C搅拌24小时后,加入小分子氨基酸终止反应; (1) The parts of insulin was dissolved in l pH4.5 acetate buffer (NaCI), was added 5 parts and 10 parts of NaBH3CN mPEG750- aldehyde, 20'C After stirring for 24 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SPS印harose FF层析柱,用含0〜1MNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a printed SPS harose FF chromatography, eluting with a gradient of buffer pH3.8 containing a 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例5: Example 5:

(1) 将1份胰岛素溶于pH3.6醋酸盐缓冲液(含NaCl),加入2份mPEG750-醛和15 份NaBH3CN, 20'C搅拌8小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.6 (containing NaCI), was added 2 parts of an aldehyde and 15 parts of NaBH3CN mPEG750-, 20'C After stirring for 8 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.5的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.5 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓缩,透析,冷冻千燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed and freeze dry in, i.e., obtain a single-modified PEGylated insulin, PheBl-PEG-insulin.

实施例6: Example 6:

(1) 将1份胰岛素溶于pH4.2醋酸盐缓冲液(含NaCl),加入6份mPEG750-醛和20 份NaBH3CN, 20"搅拌0.5小时后,加入小分子氨基酸终止反应: (1) insulin was dissolved in 1 part acetate buffer pH4.2 (containing NaCI), was added 6 parts and 20 parts of an aldehyde mPEG750- NaBH3CN, 20 "After stirring for 0.5 h, the reaction was terminated by adding a small amino acid molecule:

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH2.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值: (3) The solution was concentrated on a SP Sepharose FF column, eluting with a gradient of buffer pH2.0 containing a 0~1MNaCl, absorption peak at 220nm UV detection:

(4) 将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例7:(1) 将1份胰岛素溶于pH5.0醋酸盐缓冲液(含NaCl),加入1份mPEG750-醛和10 Example 7: (1) insulin was dissolved in 1 part acetate buffer pH5.0 (containing NaCl), and 1 part of aldehyde 10 mPEG750-

份NaBH3CN, 20'C搅拌24小时后,加入小分子氨基酸终止反应; Parts of NaBH3CN, 20'C After stirring for 24 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮: (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated:

(3) 将浓缩液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.2的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.2 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例8: Example 8:

(1) 将1份胰岛素溶于pH4.8醋酸盐缓冲液(含NaCl),加入3份mPEG750-醛和15 份NaBH3CN, 20'C搅拌10小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.8 (containing NaCI), was added 3 parts and 15 parts of NaBH3CN mPEG750- aldehydes, 20'C After stirring for 10 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓缩液上SPSepharoseFF层析柱,用含0〜lMNaCl的pH4.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on SPSepharoseFF chromatography, eluting with a gradient of buffer containing the pH4.0 0~lMNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例9: Example 9:

(1) 将1份胰岛素溶于pH4.0醋酸盐缓冲液(含NaCl),加入10份mPEG750"醛和30份NaBH3CN, 20'C搅拌5小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.0 (containing NaCI), was added 10 parts of mPEG750 "30 parts of NaBH3CN and an aldehyde, 20'C After stirring for 5 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓缩; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜lMNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH3.8 0~lMNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheB卜PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheB Bu PEG-insulin.

实施例10: Example 10:

(1) 将1份胰岛素溶于pH3.0醋酸盐缓冲液(含NaCl),加入1份mPEG2000-醛和5 份NaBH3CN, 20"C搅拌10小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.0 (containing NaCI), 1 part of aldehyde and 5 parts mPEG2000- NaBH3CN, 20 "C after 10 hours of stirring, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH3.8 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin. (1) 将1份胰岛素溶于pH4.0醋酸盐缓冲液(含NaCl),加入2份mPEG2000-醛和15份NaBH3CN, 20r搅拌8小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.0 (containing NaCI), was added 2 parts of an aldehyde and 15 parts of NaBH3CN mPEG2000-, 20r After stirring for 8 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓缩液上SPSepharose FF层析柱,用含0〜1MNaCl的pH4.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on SPSepharose FF chromatography column, eluting with a gradient of buffer pH4.0 containing a 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻千燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed and freeze dry in, i.e., obtain a single-modified PEGylated insulin, PheBl-PEG-insulin.

实施例12: Example 12:

(1) 将1份胰岛素溶于pH3.8醋酸盐缓冲液(含NaCl),加入10份mPEG2000-醛和30份NaBH3CN, 20'C搅拌0.5小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.8 (containing NaCI), was added 10 parts of an aldehyde and 30 parts of NaBH3CN mPEG2000-, 20'C After stirring for 0.5 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮: (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated:

(3) 将浓縮液上SPS印haroseFF层析柱,用含0〜lMNaCl的pH2.0的缓沖液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a printed haroseFF SPS chromatography, eluting with a gradient of buffer pH2.0 containing a 0~lMNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻千燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed and freeze dry in, i.e., obtain a single-modified PEGylated insulin, PheBl-PEG-insulin.

实施例13: Example 13:

(1) 将1份胰岛素溶于pH4.5醋酸盐缓冲液(含NaCl),加入5份mPEG2000-醛和10份NaBH3CN, 20'C搅拌24小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.5 (containing NaCI), was added 5 parts and 10 parts of NaBH3CN mPEG2000- aldehyde, 20'C After stirring for 24 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮: (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated:

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH3.8 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例14: Example 14:

(1) 将1份胰岛素溶于pH3.6醋酸盐缓冲液(含NaCl),加入2份mPEG2000-醛和15 份NaBH3CN, 20'C搅拌8小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.6 (containing NaCI), was added 2 parts of an aldehyde and 15 parts of NaBH3CN mPEG2000-, 20'C After stirring for 8 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.5的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.5 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素,PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例15: Example 15:

(1) 将1份胰岛素溶于pH4.2醋酸盐缓冲液(含NaCl),加入6份mPEG2000-醛和20 份NaBH3CN, 20'C搅拌0.5小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.2 (containing NaCI), was added 6 parts of 20 parts of NaBH3CN and an aldehyde mPEG2000-, 20'C After stirring for 0.5 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓缩液上SPSepharose FF层析柱,用含0〜1MNaCl的pH2.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on SPSepharose FF chromatography column, eluting with a gradient of buffer pH2.0 containing a 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻千燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed and freeze dry in, i.e., obtain a single-modified PEGylated insulin, PheBl-PEG-insulin.

实施例16: Example 16:

(1) 将1份胰岛素溶于pH5.0醋酸盐缓冲液(含NaCl),加入1份mPEG2000-醛和10份NaBH3CN, 20'C搅拌24小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH5.0 (containing NaCI), was added 1 part of 10 parts of NaBH3CN and an aldehyde mPEG2000-, 20'C After stirring for 24 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SPSepharoseFF层析柱,用含0〜lMNaCl的pH4.2的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on SPSepharoseFF chromatography, eluting with a gradient of buffer containing the pH4.2 0~lMNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻千燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed and freeze dry in, i.e., obtain a single-modified PEGylated insulin, PheBl-PEG-insulin.

实施例17: Example 17:

(1) 将1份胰岛素溶于pH4.8醋酸盐缓冲液(含NaCl),加入3份mPEG2000-醛和15份NaBH3CN, 20'C搅拌10小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.8 (containing NaCI), was added 3 parts and 15 parts of NaBH3CN mPEG2000- aldehydes, 20'C After stirring for 10 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.0 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻千燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-i腿lin。 (4) The collected peak 3 eluate was concentrated, dialyzed and freeze dry in, i.e., obtain a single-modified PEGylated insulin, PheBl-PEG-i leg lin.

实施例18: Example 18:

(1) 将1份胰岛素溶于pH4.0醋酸盐缓沖液(含NaCl),加入10份mPEG2000"醛和30份NaBH3CN, 20'C搅拌5小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.0 (containing NaCI), was added 10 parts of mPEG2000 "30 parts of NaBH3CN and an aldehyde, 20'C After stirring for 5 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓缩; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SPSepharose FF层析柱,用含0〜lMNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值;U)将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (3) The solution was concentrated on SPSepharose FF chromatography column, eluting with a gradient of buffer containing the pH3.8 0~lMNaCl, 220nm UV detector absorption peak; the U-) peak 3 collected eluate was concentrated, dialyzed , freeze-dried, to obtain mono pEGylated insulin-modified, PheBl-PEG-insulin. 实施例19: Example 19:

(1) 将1份胰岛素溶于pH3.0醋酸盐缓冲液(含NaCl),加入1份mPEG5000-醛和5 份NaBH3CN, 20'C搅拌10小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.0 (containing NaCI), 1 part of aldehyde and 5 parts of NaBH3CN mPEG5000-, 20'C After stirring for 10 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1M NaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, eluted with a gradient buffer of pH3.8 containing a 0~1M NaCl, the absorption peak of 220nm UV detector;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例20: Example 20:

(1) 将1份胰岛素溶于pH4.0醋酸盐缓冲液(含NaCl),加入2份mPEG5000-醛和15份N必H3CN, 20'C搅拌8小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.0 (containing NaCI), was added 2 parts of an aldehyde and 15 parts of N mPEG5000- will H3CN, 20'C After stirring for 8 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓缩; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.0 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl隱PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl hidden PEG-insulin.

实施例21: Example 21:

(1) 将1份胰岛素溶于pH3.8醋酸盐缓冲液(含NaCl),加入10份mPEG5000-醛和30份NaBH3CN, 20匸搅拌0.5小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.8 (containing NaCI), was added 10 parts of an aldehyde and 30 parts of NaBH3CN mPEG5000-, 20 Xi after stirring for 0.5 hour, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH2.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH2.0 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例22: Example 22:

(1) 将1份胰岛素溶于pH4.5醋酸盐缓冲液(含NaCl),加入5份mPEG5000-醛和10份N必H3CN, 20'C搅拌24小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.5 (containing NaCI), was added 5 parts and 10 parts of N mPEG5000- aldehyde will H3CN, 20'C After stirring for 24 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓缩; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SPS印haroseFF层析柱,用含0〜lMNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a printed haroseFF SPS chromatography, eluting with a gradient of buffer pH3.8 containing a 0~lMNaCl, 220nm UV detector absorption peak;

(4)将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醉化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin drunk, PheBl-PEG-insulin. 实施例23: Example 23:

(1) 将1份胰岛素溶于pH3.6醋酸盐缓冲液(含NaCl),加入2份mPEG5000-醛和15 份NaBH3CN, 20"C搅拌8小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH3.6 (containing NaCI), was added 2 parts and 15 parts of an aldehyde mPEG5000- NaBH3CN, 20 "C after stirring for 8 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.5的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.5 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醉化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin drunk, PheBl-PEG-insulin.

实施例24: Example 24:

(1) 将1份胰岛素溶于pH4.2醋酸盐缓冲液(含NaCl),加入6份mPEG5000-醛和20 份NaBH3CN, 20'C搅拌0.5小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.2 (containing NaCI), was added 6 parts of 20 parts of NaBH3CN and an aldehyde mPEG5000-, 20'C After stirring for 0.5 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1M NaCl的pH2.0的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, eluting with a gradient of 0~1M NaCl buffer containing the pH2.0, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例25: Example 25:

(1) 将1份胰岛素溶于pH5.0醋酸盐缓冲液(含NaCl),加入1份mPEG5000-醛和10份NaBH3CN, 20'C搅拌24小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH5.0 (containing NaCI), was added 1 part of 10 parts of NaBH3CN and an aldehyde mPEG5000-, 20'C After stirring for 24 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓缩; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

(3) 将浓縮液上SP Sepharose FF层析柱,用含0〜1MNaCl的pH4.2的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; (3) The solution was concentrated on a SP Sepharose FF column, gradient elution with buffer containing the pH4.2 0~1MNaCl, 220nm UV detector absorption peak;

(4) 将收集的峰3洗脱液浓縮,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin.

实施例26: Example 26:

(1) 将1份胰岛素溶于pH4.8醋酸盐缓冲液(含NaCl),加入3份mPEG5000-醛和15份NaBH3CN, 20'C搅拌10小时后,加入小分子氨基酸终止反应; (1) insulin was dissolved in 1 part acetate buffer pH4.8 (containing NaCI), was added 3 parts and 15 parts of NaBH3CN mPEG5000- aldehydes, 20'C After stirring for 10 hours, the reaction was terminated by adding a small amino acid molecules;

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮;(3) 将浓缩液上SPS印haroseFF层析柱,用含0〜lMNaCl的pHW.0的缓冲液进行梯度洗脱,220nrn紫外检测吸收峰值; (2) (1) The reaction solution is dialyzed to remove small molecules salt, concentrated; (3) on the printed haroseFF concentrate SPS chromatography column with a buffer containing a gradient of pHW.0 0~lMNaCl wash off, 220nrn UV detector absorption peak;

(4) 将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-i腿lin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-i leg lin.

实施例27: Example 27:

(1) 将1份胰岛素溶于pH4.0醋酸盐缓冲液(含NaCl),加入10份mPEG5000-醛和30份NaBH3CN, 20'C搅拌5小时后,加入小分子氨基酸终止反应: (1) insulin was dissolved in 1 part acetate buffer pH4.0 (containing NaCI), was added 10 parts of an aldehyde and 30 parts of NaBH3CN mPEG5000-, 20'C After stirring for 5 hours, the reaction was terminated by adding a small amino acid molecule:

(2) 将(1)的反应液进行透析,除去小分子盐分,浓縮; (2) (1) The reaction solution is dialyzed to remove small molecules salt, and concentrated;

G)将浓縮液上SPSepharoseFF层析柱,用含0〜lMNaCl的pH3.8的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值; G) The solution was concentrated on SPSepharoseFF chromatography, eluting with a gradient of buffer pH3.8 containing a 0~lMNaCl, 220nm UV detector absorption peak;

(4)将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素, PheBl-PEG-insulin。 (4) The collected peak 3 eluate was concentrated, dialyzed, lyophilized to obtain polyethylene glycol mono-modified insulin, PheBl-PEG-insulin. 实施例28 Example 28

将实施例ll中步骤(2)的反应混合物进行高效液相色谱分析。 The reaction mixture in Example ll in step (2) is analyzed by HPLC. 色谱条件如下:色谱柱为Di咖onsilC18柱(4.6x150 mm, 5 urn, Dikma, USA),用0.1% (v/v) TFA水溶液(洗脱剂A)和含0.1% (Wv) TFA的乙腈(洗脱剂B)进行梯度洗脱。 Chromatographic conditions were as follows: Column for the coffee onsilC18 Di column (4.6x150 mm, 5 urn, Dikma, USA), with 0.1% (v / v) TFA in water (eluent A) containing 0.1% (Wv) TFA in acetonitrile (eluent B) gradient. 0-25 min, 3345% B,随后平衡10min再进下一个样。 0-25 min, 3345% B, followed by the balance 10min and then into the next sample. 洗脱梯度如下:0-10 min, 33-39% B; 10-25 min, 3945%。 Elution gradient as follows: 0-10 min, 33-39% B; 10-25 min, 3945%. 流速为1 mhnin",检测波长为220 nm,柱温为室温。表明该混合物中以单修饰的聚乙二醇化胰岛素为主,约占80%。 实施例29 The flow rate was 1 mhnin ", the detection wavelength was 220 nm, column temperature was room temperature. The mixture showed a single modified PEGylated insulin, accounting for about 80%. Example 29

将实施例11中步骤(2)的反应混合物进行阳离子交换色谱分析。 The reaction mixture was embodiment the procedure of Example 11 (2) is cation exchange chromatography. SP S印harose阳离子交换色谱柱(10x2.5 cm, GE Healthcare)装柱,经0.05 mol'l'1 pH 4.0的醋酸盐缓冲液洗涤平衡约5个柱体积后,取上述样品滤液注入Waters 650E高级蛋白质纯化系统(上样体积为10ml)。 Printing harose SP S cation exchange chromatography column (10x2.5 cm, GE Healthcare) packed in a column, washed with equilibration acetate after about 5 column volumes of buffer 0.05 mol'l'1 pH 4.0, the filtrate was filled into the above samples taken Waters 650E advanced protein purification system (the injection volume 10ml). 流速为5ml/min,用含0〜0.5MNaCl的50mMpH4.0醋酸盐缓冲液梯度洗脱,220 nm紫外检测。 Flow rate 5ml / min, eluting with a gradient 50mMpH4.0 acetate buffer containing 0~0.5MNaCl a, 220 nm UV detection. 峰1为穿透峰含过量mPEG,峰2含2mPEG —insulin,峰3含PheBl-PEG-insu】in,峰4含未反应的胰岛素。 Peak 1 is a peak containing excessive penetration mPEG, peak 2 contains 2mPEG -insulin, peak 3 containing PheBl-PEG-insu} in, 4 peak containing unreacted insulin. 按实施例28中色谱条件检测各收集蜂。 Chromatographic conditions described in Example 28 detects each collecting bees. 实施例30 Example 30

将PheBl-PEG2000-insulin和PheBl-PEG5000-insulin洛解于5%乙腈,0.22 pm滤膜过滤。 The PheBl-PEG2000-insulin and Los PheBl-PEG5000-insulin solution in 5% acetonitrile, 0.22 pm membrane filter. 取样品溶液和等体积的a-氣基-4-羟基肉桂酸的饱和溶液(溶剂为0.1%甲酸溶液) 混合均匀,点样,待样品水分挥干后进行测定。 A sample solution, and a saturated solution of equal volume of a- gas-4-hydroxy cinnamic acid (0.1% formic acid solvent) were mixed uniformly, spotting, the sample was measured until the water evaporated. 4700型基质辅助激光解吸电离-串联飞行时间质谱仪(MALDI-TOF, AB,美国)采取线性工作模式,以337 nm的筑原子激光激发样品,每个样品至少激发60次,监测正电荷离子的产生,测定PheBl-PEG2000-insuUn和 4700 Matrix assisted laser desorption ionization - tandem mass spectrometry time of flight (MALDI-TOF, AB, USA) take linear operation mode, in order to build the 337 nm laser excitation of the sample atoms, at least 60 times for each sample excitation, monitoring positive charged ions generating, measuring and PheBl-PEG2000-insuUn

PheBl-PEG5000-insulin的分子量。 PheBl-PEG5000-insulin molecular weight.

实施例31 Example 31

分别将PheBl-PEG2000-insulin经胰蛋白酶酶切,而未经过二硫苏糖醇(DTT)处理: 经DTT处理,不进行酶切或酶切后用DTT处理,按实施例30方法测定修饰位点。 Respectively PheBl-PEG2000-insulin by trypsin digestion, without going through dithiothreitol (DTT) treatment: After treatment with DTT, DTT without enzyme or enzyme treatment, measured according to the modified bits Example 30 point. 综合三个图谱,可判定PEG修饰在胰岛素的B1位。 Integrated three maps, PEG modification can be determined in the position B1 insulin. 实施例32 Example 32

取18只健康雄性小鼠,随机分为3组。 Eighteen healthy male mice were divided into 3 groups. 实验前禁食12h,可自由饮水。 Before the experiment fasting 12h, free access to water. 称重,皮下分别注射(0.5U.kg")胰岛素和三种修饰度的PEG化胰岛素溶液,于设定时间时自眼部经脉从取血200nl。待血液凝固后,于12,000 rpm离心4 min,精密量取20nl血清,按葡萄糖氧化酶法(GOD-PAP)测定血糖值。 实施例33 Weighing, were injected subcutaneously (0.5U.kg ") PEG insulin and insulin solution three kinds of modifications of the blood from the eye meridian at 200nl setting time until the blood coagulated by centrifugation at 12,000 rpm 4 min the precise amount of serum 20nl, by glucose oxidase (GOD-PAP) measured blood glucose level. Example 33

将胰岛素、PheBl-PEG2000-insulin和PheBl-PEG5000-i加ulin溶液(按胰岛素计0.2 mg/ml)和胰蛋白酶溶液U.2mg/ml)预先在37 。 Insulin, PheBl-PEG2000-insulin and PheBl-PEG5000-i ulin solution was added (in terms of insulin 0.2 mg / ml) and trypsin solution U.2mg / ml) 37 in advance. C水浴中平衡15 min。 C water bath equilibrated 15 min. 取胰岛素(或结合物)溶液取100pl,力卩150pl冷的酶终止液(0."/。TFA溶液),混匀,为零时样品。然后在5ml胰岛素(或结合物)溶液中加入50jil酶液,涡旋2s,分别于设定时间取样lOOnl, 加150 jil冷的酶终止液,立即混匀。样品12,000 rpm离心5 min,取20 jil注入HPLC仪进行检测。 实施例34 Take insulin (or conjugate) was taken 100 pL, enzyme force Jie 150pl cold stop solution (0. "/. TFA solution), mixed and the sample is zero. 50jil then added in 5ml of insulin (or conjugate) was enzyme solution, vortex 2s, respectively, to set the sampling time lOOnl, plus 150 jil enzyme cold stop solution, mix immediately. samples centrifuged 12,000 rpm 5 min, 20 jil taken injected into the HPLC instrument for testing. Example 34

将胰岛素、PheBl-PEG2000-insulin和PheBl-PEG5000-insulin溶液(按胰岛素计0.2 mg/ml)和a-糜蛋白酵溶液(1.6mg/ml)预先在37 。 Insulin, PheBl-PEG2000-insulin and PheBl-PEG5000-insulin solution (in terms of insulin 0.2 mg / ml) and yeast a- chymotrypsin solution (1.6mg / ml) at 37 in advance. C水浴中平衡15 min。 C water bath equilibrated 15 min. 取胰岛素(或结合物)取100nl,力H150nl冷的酶终止液(0.1。/。TFA溶液),混匀,为零时样品。 Take insulin (or a combination) take 100 nl, enzyme force H150nl cold stop solution (0.1./.TFA solution), mixed and the sample is zero. 然后在5ml胰岛素(或结合物)溶液中加入50jil酶液,涡旋2s,分别于设定时间取样100 加150 W冷的嗨终止液,立即混匀。 Then 5ml of insulin (or a combination) 50jil enzyme solution was added, vortexed 2s, respectively, to set the sampling time 100 plus 150 W Hi cold stop solution, mix immediately. 样品12,000 rpm离心5 min,取20 jxl注入HPLC仪进行检测。 Samples were centrifuged at 12,000 rpm 5 min, injected into the HPLC instrument 20 jxl taken for testing. 实施例35 Example 35

将胰岛素、PheBl-PEG2000-insulin和PheBl-PEG5000-insulin溶解于PBS,制备供试溶液。 Insulin, PheBl-PEG2000-insulin and PheBl-PEG5000-insulin was dissolved in PBS, prepared for the test solution. 0.22 nm滤膜过滤。 0.22 nm membrane filter. 取滤液,以PBS为参比,用U-3000分光光度计测定275 nm下的吸光度值并计算样品浓度。 The filtrate, and PBS as the reference, with the U-3000 spectrophotometer absorbance at 275 nm and the sample concentrations were calculated. 分别在250-320nm (近紫外)和190-250nm (远紫外)两个波段进行扫描,每个样品重复扫描5次。 It was scanned at 250-320nm (near ultraviolet) and 190-250 nm (far ultraviolet) two bands, the scan was repeated five times for each sample. 将得到的样品谱图减去PBS参比的谮图,利用随机软件减去噪音后得到最终的圆二色谱图。 The sample spectrum obtained by subtracting the reference malign FIG PBS, obtained by subtracting the final noise circular dichroism FIG using a random software. 将测得的椭圆率数据按下式【41转换为平均残基椭圆率(em): The measured ellipticity of the following formula [41 converts the data to mean residue ellipticity (em):

[e<«]=< exM)/(cxi) (式3.1)其中e [E < «] = <exM) / (cxi) (Formula 3.1) where e

是观测到的椭圆率值(mdeg) , M是胰岛素的平均残基分子量(g'mor1) , C是胰岛素浓 Is the observed ellipticity value (mdeg), M is the mean residue molecular weight of insulin (g'mor1), C is the concentration of insulin

度(g'mr') , l是光程长度(cm)。 Degree (g'mr '), l is the optical path length (cm).

上述实施例l-27所制备的单修饰的聚乙二醇化胰岛素,经检测表明与实施例11和20 制备的产品具有相类似的性能,这里不一一列举。 Mono-modified PEGylated insulin Example l-27 above prepared, tested and showed that the product prepared in Example 1 120 embodiment having similar properties, not enumerated here.

Claims (8)

  1. 1、一种仅在B1位Phe位点进行聚乙二醇修饰且单甲氧聚乙二醇与胰岛素以碳氮键相连的单修饰聚乙二醇化胰岛素的制备方法,其特征在于具体步骤如下:(1)将1份胰岛素溶于pH值为3.0~5.0含NaCl的醋酸盐缓冲液,加入1~10份mPEG-醛和5~30份NaBH3CN,4~37℃搅拌0.5~24小时,加入小分子氨基酸终止反应,其中份数以摩尔计;(2)将步骤(1)得到的反应液进行透析,除去小分子盐分,浓缩;(3)将浓缩液上SP Sepharose FF层析柱,用含0~1M NaCl的pH值为2.0~4.5的缓冲液进行梯度洗脱,220nm紫外检测吸收峰值;(4)将收集的峰3洗脱液浓缩,透析,冷冻干燥,即得到单修饰的聚乙二醇化胰岛素。 A Phe at position B1 in only site preparation and polyethylene glycol-modified polyethylene glycol and insulin is connected to a single carbon-nitrogen bond PEGylated insulin modified monomethoxy, characterized by the following steps : (1) insulin was dissolved in 1 part pH 3.0 to 5.0 acetate buffer containing NaCl, adding 1 to 10 parts by mPEG- aldehyde and 5 to 30 parts of NaBH3CN, stirred for 4 ~ 37 ℃ 0.5 ~ 24 hours. the reaction was terminated by adding a small amino acid molecules in which parts are by moles; the reaction solution (1) obtained in (2) the dialysis step, the salt is removed a small molecule, and concentrated; (3) the solution was concentrated on a SP Sepharose FF column, and a pH of 0 ~ 1M NaCl-containing buffer was 2.0 to 4.5 gradient elution, 220nm UV detector absorption peak; (4) the collected peak 3 eluate was concentrated, dialyzed and freeze-dried to obtain a modified single pEGylated insulin.
  2. 2、 根据权利要求1所述的单修饰的聚乙二醇化胰岛素的制备方法,其特征在于醋酸盐缓冲液的pH值为3.6~4.5。 2, a single method of preparing the modified PEGylated insulin according to claim 1, characterized in that the pH of the acetate buffer was 3.6 to 4.5.
  3. 3、 根据权利要求1所述的单修饰的聚乙二醇化胰岛素的制备方法,其特征在于醋酸盐缓冲液的pH值为3.8~4.2。 3, a single method of preparing the modified PEGylated insulin according to claim 1, characterized in that the pH of the acetate buffer was 3.8 to 4.2.
  4. 4、 根据权利要求1所述的单修饰的聚乙二醇化胰岛素的制备方法,其特征在于单甲氧聚乙二醇-醛和胰岛素的用量摩尔比例为1~5:1。 4. The process according to the modified single PEGylated insulin according to claim 1, wherein the monomethoxy-polyethylene glycol - amount of insulin and aldehyde molar ratio of 1 to 5: 1.
  5. 5、 根据权利要求1所述的单修饰的聚乙二醇化胰岛素的制备方法,其特征在于NaBH3CN和胰岛素的用量摩尔比例为12~20:1。 5. The process according to the modified single PEGylated insulin according to claim 1, characterized in that the amount of insulin molar ratio of NaBH3CN and 12 to 20: 1.
  6. 6、 根据权利要求1所述的单修饰的聚乙二醇化胰岛素的制备方法,其特征在于反应温度为室温。 6. The method of preparing a single polyethylene glycol-modified insulin as claimed in claim, wherein the reaction temperature is room temperature.
  7. 7、 根据权利要求1所述的单修饰的聚乙二醇化胰岛素的制备方法,其特征在于反应时间为5〜8小时。 7. The method of preparing a single polyethylene glycol-modified insulin as claimed in claim, characterized in that the reaction time was 5 ~ 8 hours.
  8. 8、 根据权利要求1所述的单修饰的聚乙二醇化胰岛素的制备方法,其特征在于上SP Sepharose FF层析柱洗脱时pH值为3.8~4.2。 8. The method for preparing a single polyethylene glycol-modified insulin according to claim 1, characterized in that the elution on SP Sepharose FF column a pH of 3.8 to 4.2.
CN 200610118923 2006-11-30 2006-11-30 Monomodified PEGylated insulin and its preparation method CN100493615C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200610118923 CN100493615C (en) 2006-11-30 2006-11-30 Monomodified PEGylated insulin and its preparation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 200610118923 CN100493615C (en) 2006-11-30 2006-11-30 Monomodified PEGylated insulin and its preparation method

Publications (2)

Publication Number Publication Date
CN1970086A true CN1970086A (en) 2007-05-30
CN100493615C true CN100493615C (en) 2009-06-03

Family

ID=38111232

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 200610118923 CN100493615C (en) 2006-11-30 2006-11-30 Monomodified PEGylated insulin and its preparation method

Country Status (1)

Country Link
CN (1) CN100493615C (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100589844C (en) 2008-05-20 2010-02-17 大连民族学院 Mono-modified polyethyleneglycol-insulin complexes and preparation method thereof
CN102952067A (en) * 2011-08-30 2013-03-06 苏州欣诺科生物科技有限公司 Pyridoxal derivative for pegylation modification of N terminal of protein and preparation method and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US5252714A (en) 1990-11-28 1993-10-12 The University Of Alabama In Huntsville Preparation and use of polyethylene glycol propionaldehyde
US6323311B1 (en) 1999-09-22 2001-11-27 University Of Utah Research Foundation Synthesis of insulin derivatives
CN1507357A (en) 2000-10-31 2004-06-23 Pr药品有限公司 Method and compositions for enhanced delivery of bioactive molecules
CN1538809A (en) 2001-06-04 2004-10-20 诺贝克斯公司 Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, used thereof and methods of making same
CN1651463A (en) 2004-12-02 2005-08-10 华东师范大学 Mono methoxy polyethylene glycol-insulin complex substance and its preparation method
CN1777440A (en) 2003-04-11 2006-05-24 Pr药品有限公司 Method for preparation of site-specific protein conjugates

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US5252714A (en) 1990-11-28 1993-10-12 The University Of Alabama In Huntsville Preparation and use of polyethylene glycol propionaldehyde
US6323311B1 (en) 1999-09-22 2001-11-27 University Of Utah Research Foundation Synthesis of insulin derivatives
CN1507357A (en) 2000-10-31 2004-06-23 Pr药品有限公司 Method and compositions for enhanced delivery of bioactive molecules
CN1538809A (en) 2001-06-04 2004-10-20 诺贝克斯公司 Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, used thereof and methods of making same
CN1777440A (en) 2003-04-11 2006-05-24 Pr药品有限公司 Method for preparation of site-specific protein conjugates
CN1651463A (en) 2004-12-02 2005-08-10 华东师范大学 Mono methoxy polyethylene glycol-insulin complex substance and its preparation method

Also Published As

Publication number Publication date Type
CN1970086A (en) 2007-05-30 application

Similar Documents

Publication Publication Date Title
US6436386B1 (en) Hydroxyapatite-targeting poly (ethylene glycol) and related polymers
US5438040A (en) Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
Marschütz et al. Oral peptide drug delivery: polymer–inhibitor conjugates protecting insulin from enzymatic degradation in vitro
US20050107297A1 (en) Novel poly(ethylene glycol) modified compounds and uses thereof
US6528485B1 (en) Site-specific preparation of polyethylene glycol-grf conjugates
US6174530B1 (en) Homogeneous polyoxime compositions and their preparation by parallel assembly
US4649191A (en) Conformationally constrained alpha-melanotropin analogs with specific central nervous system activity
US6602981B2 (en) Antinociceptive agent derivative
Krause et al. Location of an Amphipathic. alpha.-Helix in Peptides Using Reversed-Phase HPLC Retention Behavior of D-Amino Acid Analogs
US20060252916A1 (en) Modified glucagon-like peptide-1 analogs
US20110195895A1 (en) Fgf21 mutants and uses thereof
WO2007100535A2 (en) Oxyntomodulin derivatives
Boublik et al. Synthesis and hypertensive activity of neuropeptide Y fragments and analogs with modified N-or C-termini or D-substitutions
WO2005000339A2 (en) Melanocortin receptor 4(mc4) agonists and their uses
WO2008148839A2 (en) Long-acting polymeric prodrugs of exendin
US20020045731A1 (en) Peptide derivatives
WO1996041813A2 (en) Functionalized polymers for site-specific attachment
US5204328A (en) Peptides having atrial natriuretic factor activity
WO2012158965A2 (en) Improved peptide pharmaceuticals for insulin resistance
US5977310A (en) Peg-modified HGF
CN102875665A (en) Method for synthesizing liraglutide
Ryan et al. Conjugation of salmon calcitonin to a combed-shaped end functionalized poly (poly (ethylene glycol) methyl ether methacrylate) yields a bioactive stable conjugate
Esposito et al. PEGylation of growth hormone-releasing hormone (GRF) analogues
Veronese et al. New PEGs for peptide and protein modification, suitable for identification of the PEGylation site
WO2008012528A1 (en) N-terminal polysialylation

Legal Events

Date Code Title Description
C06 Publication
C10 Entry into substantive examination
C14 Grant of patent or utility model
C17 Cessation of patent right