CN111808201B - 重组人胰高血糖素样肽-1类似物融合蛋白的制备方法 - Google Patents
重组人胰高血糖素样肽-1类似物融合蛋白的制备方法 Download PDFInfo
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Abstract
本公开提供一种重组人胰高血糖素样肽‑1类似物融合蛋白的制备方法，以含有重组人胰高血糖素样肽‑1类似物融合蛋白的酵母工程菌发酵液上清为对象，在辛酸钠和氨甲环酸存在的条件下进行加热灭酶，减少了目的蛋白在分离纯化过程中的降解和生物活性损失，提高了注射用重组人胰高血糖素样肽‑1类似物融合蛋白的稳定性。
Description
技术领域
本发明属于生物制药领域，具体涉及一种基因工程菌重组表达外源目的蛋白的分离纯化方法，更具体涉及一种重组人胰高血糖素样肽-1类似物融合蛋白的制备方法。
背景技术
胰高血糖素样肽-1（GLP-1）及其类似物例如Exendin-4被广泛的用来研究治疗2型糖尿病，由于GLP-1多肽在体内被蛋白酶二肽基肽酶Ⅳ(DPP-IV)所迅速失活，在血浆中的半衰期很短，使其难以在临床上被广泛使用；而Exendin-4对DPP-Ⅳ的酶降解不敏感，其稳定性增加，但分子量较低（4187.61D），在体内半衰期短，所以每天必须注射两次，这阻碍了在临床上使用。目前许多努力用来解决这个技术难题，例如缓释微球、PEG修饰，脂肪酸链修饰，白蛋白融合。其中白蛋白融合技术在保持目的蛋白的生物学和治疗功能的同时，通过与人白蛋白融合大大提高了其在体内半衰期。尽管GLP-1类制剂及其衍生物用于治疗糖尿病是现实可行的，但是因为糖尿病患者一经诊断后必须长期持续给药，终生接受治疗，因此对于该类制剂的安全性、经济性和使用便利性的要求都是极其迫切的。
Albugon是LaurieL.Baggio等设计的一种GLP-1/HSA融合蛋白，就是在其两者间插入一个额外的GLP-1分子作为间隔物，但其生物学活性仅仅保留了约1%；生物学活性的下降导致临床剂量的大幅增加（LaurieL.Baggio, Qingling Huang, Theodore J. Brown, andDaniel J. Drucker, DIABETES Vol.53：2492-2500 （2004））。GLP-1/HSA融合蛋白制剂临床剂量的增加不仅导致抗体生成的风险加大，而且还进一步推高了原本就高昂的重组GLP-1/HSA融合蛋白单位剂量生产和使用成本。
为解决上述问题，CN201310331182.6通过融合蛋白分子结构优化和设计，提供了与GLP-1/HSA融合蛋白更加稳定的GLP-1类似物融合蛋白。然而，上游的重组蛋白分子设计受限于结构-功能的构效关系，难以在确保功能的情况下消除全部降解位点。杨扬等通过对宿主菌进行基因工程改造，通过构建蛋白酶缺失的宿主菌来提高目的蛋白的表达和生物活性（“巴斯德毕赤酵母PEP4基因的剔除及对基因工程菌外源蛋白质表达的影响”，《食品与药品》 2007年第4期）。然而，宿主菌表达的酶种类繁多、酶合成代谢网络及调控复杂，通过基因改造的效果往往被宿主菌的代偿机制抵消，大规模的基因改造又可能影响对外源蛋白的表达能力。基于上述现有技术存在的不足，GLP-1类制剂生产制备过程中急需更多提高重组GLP-1融合蛋白稳定性、降低成本的方法。
发明内容
针对胰高血糖素样肽-1（GLP-1）类似物融合蛋白生产过程中目的蛋白易降解、有效活性回收率低、融合蛋白产品不稳定的技术问题，本发明在上游优化重组人胰高血糖素样肽-1类似物融合蛋白分子设计的基础上，对重组菌发酵、目的蛋白诱导表达、产物分离纯化等下游生产工艺进行系统分析和研究。首先，通过大量的前期分析，发现胰高血糖素样肽-1（GLP-1）类似物融合蛋白的降解始于发酵末期、贯穿于整个生产工艺。进而，针对宿主菌蛋白酶和胰高血糖素样肽-1（GLP-1）类似物融合蛋白在结构、生物活性、理化形状等方面的差异，尝试对发酵液进行预处理。最终，通过对预处理添加剂、理化条件等的优化，确定了在辛酸钠和氨甲环酸存在的条件下进行加热灭酶，减少了目的蛋白的降解和生物活性损失。
具体而言：
一方面，本发明提供一种重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，包括工程菌构建、诱导表达、重组人胰高血糖素样肽-1类似物融合蛋白纯化；
其中，在诱导表达步骤和重组人胰高血糖素样肽-1类似物融合蛋白纯化步骤之间包括对诱导表达发酵液上清进行预处理的步骤，所述预处理是在添加辛酸钠和氨甲环酸的条件下对工程菌诱导表达的发酵液上清进行加热处理。
进一步，本发明所述重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其中，预处理步骤中辛酸钠和氨甲环酸的摩尔比为1-2：1-2，终浓度分别为10-20mM。
进一步，本发明所述重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其中，预处理步骤加热温度为65-70℃，优选68℃；处理时间为30min-2h。
进一步，本发明所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其中，将工程菌诱导表达的发酵液上清调节至pH 6.5-7.5之间进行预处理。
更进一步，本发明所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其中所述重组人胰高血糖素样肽-1类似物融合蛋白包括GLP-1类似物、载体蛋白、可选的位于二者之间的蛋白接头。
优选的，所述GLP-1类似物如SEQ ID NO:1所示，所述载体蛋白为HSA，所述蛋白接头选自SEQ ID NO:2-4之一。
更优选的，所述人胰高血糖素样肽-1类似物融合蛋白的氨基酸序列如SEQ ID NO:5所示。
更进一步，本发明所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其中，所述诱导表达包括甘油培养阶段和甲醇诱导阶段；
甘油流加培养至菌体湿重达216-260g/L时，停止流加甘油，开始流加甲醇诱导发酵，直至发酵液中重组人胰高血糖素样肽-1类似物融合蛋白的表达量达0.3-0.7mg/mL。
更进一步，本发明所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其特征在于：所述重组人胰高血糖素样肽-1类似物融合蛋白纯化包括Blue SepharoseFast Flow 3.1L亲和层析、苯基琼脂糖疏水层析（Phenyl Sepharose High Performance）、DEAE琼脂糖阴离子层析（DEAE Sepharose Fast Flow）、以及G25脱盐层析。
另一方面，本发明还提供由本发明所述方法制备的重组人胰高血糖素样肽-1类似物融合蛋白，其与通过不包含所述预处理步骤（而其它步骤相同）的制备方法获得的重组人胰高血糖素样肽-1类似物融合蛋白相比，具有更少的降解产物。
第三方面，本发明还提供辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其中，所述辛酸钠和氨甲环酸的摩尔比为1-2：1-2，所述重组蛋白位于酵母发酵液上清中。
进一步，本发明所述辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其中，所述重组蛋白为重组人胰高血糖素样肽-1类似物融合蛋白，所述辛酸钠和氨甲环酸降低重组人胰高血糖素样肽-1类似物融合蛋白在分离纯化过程中的降解。
进一步，本发明所述辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其中，对含重组人胰高血糖素样肽-1类似物融合蛋白的发酵液上清在添加辛酸钠和氨甲环酸的条件下进行加热处理。
进一步，本发明所述辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其中，辛酸钠和氨甲环酸的摩尔比为1：1，终浓度分别为10-20mM。
进一步，本发明所述辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其中，加热温度为65-70℃，优选68℃；加热时间为30min-2h。
进一步，本发明所述辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其中，将含重组人胰高血糖素样肽-1类似物融合蛋白的发酵液上清调节至pH 6.5-7.5之间进行加热。
更进一步，本发明所述辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其中所述重组人胰高血糖素样肽-1类似物融合蛋白包括GLP-1类似物、载体蛋白、可选的位于二者之间的蛋白接头。
优选的，所述GLP-1类似物如SEQ ID NO:1所示，所述载体蛋白为HSA，所述蛋白接头选自SEQ ID NO:2-4之一。
更优选的，所述人胰高血糖素样肽-1类似物融合蛋白的氨基酸序列如SEQ ID NO:5所示。
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“胰高血糖素样肽-1（GLP-1）”是回肠内分泌细胞分泌的一种脑肠肽，目前主要作为2型糖尿病药物作用的靶点。由于GLP-1可抑制胃排空，减少肠蠕动，故有助于控制摄食，减轻体重。在19例肥胖患者中进行的前瞻性安慰剂对照、随机、双盲、交叉试验中，GLP-1皮下注射给药可增加患者餐后的饱腹感，并使每餐的饮食量平均减少15%。但由于GLP-1是多肽，不能口服给药。
“融合蛋白”是通过DNA重组技术得到的多个基因重组后的表达产物。构建融合蛋白的基本方法是将具有特定功能的天然或人工编码的多肽序列模块化，并使用基因编码的DNA序列模板合成，随后将第1个蛋白的终止密码子删除，再接上带有终止密码子的第2个蛋白基因，以实现两个基因的共同表达。通过控制每一个功能肽模块在整体蛋白材料中的确切位置和密度，人们便能够根据实际需要改变融合蛋白的组成。
“蛋白接头”也称连接肽( Linker )，是将不同功能的蛋白进行连接融合的多肽，连接肽的设计和选择对融合蛋白的表达,稳定性及功能活性至关重要。在构建融合蛋白中，一个关键的问题是两蛋白间的接头序列，即连接肽。它的长度对蛋白质的折叠和稳定性非常重要。如果接头序列太短，可能影响两蛋白高级结构的折叠，从而相互干扰；如果接头序列太长，又涉及免疫原性的问题，因为接头序列本身就是新的抗原。目前对于连接肽序列的设计还没有可靠的选择标准。依赖于蛋白质的一级结构来预测其二级结构已经产生了重大的进步，但是对于序列和结构之间的关系的了解还是很有限的。
“人血清白蛋白”（ Human Serum Albumin ,HSA）,是人血浆中的蛋白质，其非糖基化的单链多肽包含585个氨基酸，分子量为66kD。在血浆中其浓度为42g/L，约占血浆总蛋白的60%。在体液中人血清白蛋白可以运输脂肪酸、胆色素、氨基酸、类固醇激素、金属离子和许多治疗分子等，同时维持血液正常的渗透压。人血清白蛋白(HSA)是血液中各种疏水性分子的天然载体,以其无毒、无免疫原性、组织相容性好等优点成为药物输送领域的研究热点。
“蛋白酶”（Protease），是水解蛋白质肽链的一类酶的总称。按其降解多肽的方式分成内肽酶和端肽酶两类。前者可把大分子量的多肽链从中间切断，形成分子量较小的朊和胨；后者又可分为羧肽酶和氨肽酶，它们分别从多肽的游离羧基末端或游离氨基末端逐一将肽链水解生成氨基酸。
“蛋白表达系统”（Protein expression system）是指用模式生物如细菌、酵母、动物细胞或者植物细胞表达外源基因蛋白的一种分子生物学技术。蛋白表达系统包括宿主、外源基因、载体和辅助成分组成的体系，通过这个体系可以实现外源基因在宿主中表达的目的。本发明所述重组GLP-1类似物融合蛋白的基因序列可以通过本领域技术人员熟知的表达系统表达，包括但不限于用重组噬菌体、质粒等载体转化的细菌，用酵母表达载体转化的酵母，用真菌载体转化的丝状真菌、用病毒载体感染的昆虫细胞、动物细胞等等。在本发明的一个具体实施例中，表达系统选用巴斯德毕赤酵母（Pichiapastoris）分泌形式表达，巴斯德毕赤酵母具有高水平表达、成本低、以及具有真核表达系统的蛋白质加工、折叠、翻译后修饰的优点。在具体生产中，细胞可以在实验室中通过摇瓶培养，或者通过发酵罐发酵培养（包括连续、分批、补料分批和固体状态发酵）。
与现有技术相比，本发明的技术方案具有以下优点：
第一、本发明在优化重组人胰高血糖素样肽-1类似物融合蛋白分子设计的基础上，进一步通过对下游发酵和纯化工艺的改进来减少目的蛋白的降解、提高目的蛋白的稳定性。通过对目的蛋白降解发生的阶段进行研究，确定在诱导发酵结束后进行蛋白酶灭活处理，这个阶段发酵液中蛋白酶活性最高、含量最大，纯化前进行预处理不仅最大程度的减少蛋白酶对目的蛋白的降解，而且在后续纯化步骤中能够有效终产物中添加剂的含量。
第二、本发明在对宿主菌蛋白酶和胰高血糖素样肽-1（GLP-1）类似物融合蛋白在结构、生物活性、理化形状等方面进行系统分析的基础上，结合上游的诱导发酵工艺、下游的分离纯化工艺，通过理化条件的协同，在既能使分子量大、活性高度依赖复杂空间结构的蛋白酶失活，又能避免分子量小、空间结构相对简单的胰高血糖素样肽-1（GLP-1）类似物融合蛋白不可逆变性的条件下进行预处理。
第三、对于发酵液上清预处理的操作条件、参数进行了优化选择，本发明提供的预处理方法既能够减少重组胰高血糖素样肽-1（GLP-1）类似物融合蛋白的降解、又不会对重组胰高血糖素样肽-1（GLP-1）类似物融合蛋白的生物活性产生显著不良影响，对于提高注射用重组人胰高血糖素样肽-1类似物融合蛋白（酵母）的产品稳定性、降低单位剂量成本具有重要意义。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。而且在整个附图中，用相同的参考符号表示相同的部件。在附图中：
图1：辛酸钠在不同加热时间下对目的蛋白降解的抑制作用（68℃）。
泳道1 未处理发酵液对照; 泳道2 加热1h; 泳道3 加热2h; 泳道4加热3h; 泳道5 加热4h; 泳道6 阳性对照。
图2：氨甲环酸在不同加热时间下对目的蛋白降解的抑制作用（68℃）。
泳道1未处理发酵液对照; 泳道2 加热1h; 泳道3 加热2h; 泳道4加热3h; 泳道5加热4h。
图3：同时加入辛酸钠和氨甲环酸在不同加热条件下对目的蛋白降解的抑制作用。
泳道1 发酵液原始对照; 泳道2 75℃加热10min; 泳道3 70℃加热10min; 泳道470℃加热20min; 泳道5 70℃加热30min; 泳道6 65℃加热1h ; 泳道7 65℃加热2h; 泳道8 65℃加热3h; 泳道9 65℃加热4h。
图4：同时加入辛酸钠和氨甲环酸在不同pH值条件下对目的蛋白降解的抑制作用（加热温度68℃）。
泳道1 pH5.0; 泳道2 pH5.5; 泳道3 pH 6.0; 泳道4 pH 6.5; 泳道5 pH 7.0;泳道6 pH 7.5。
图5：辛酸钠和氨甲环酸的浓度对目的蛋白降解的抑制作用（加热温度68℃，pH7.0）。
泳道1; 对照，泳道2 3mM; 泳道3 5mM; 泳道4 7mM; 泳道5 10mM; 泳道6 15mM;泳道7 20mM。
图6：发酵液预处理对终产品生物活性的影响。
“○”代表发酵液经过预处理的试验样本；
“□”代表发酵液未经预处理的对照样本。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
实施例1：表达重组人胰高血糖素样肽-1类似物融合蛋白工程菌的构建
根据CN201310331182.6号发明专利申请的方法制备GLP-1类似物融合蛋白表达工程菌。简言之，人工合成GLP-1类似物-E3-HSA N端部分基因片段，PCR扩增获得HSA编码基因序列，通过重叠PCR获得GLP-1类似物-E3-HSA融合基因，测序鉴定其序列如SEQ ID NO:5所示。通过XhoI、NotI双酶切导入pPIC9质粒获得GLP-1类似物-E3-HSA/ pPIC9重组质粒。将GLP-1类似物-E3-HSA/ pPIC9重组质粒用SalI线性化后电转化至毕赤酵母GS115感受态细胞，筛选鉴定，获得阳性转化子，经过实验室摇瓶规模诱导表达分析。
实施例2：表达重组人胰高血糖素样肽-1类似物融合蛋白工程菌的规模化发酵培养和诱导表达方法
1. 配制培养基
分别称取二水硫酸钙46.5g，硫酸钾910.0g，硫酸镁383.0g，甘油4000.0g，量取85%磷酸1335.0ml加入不锈钢桶中，加水溶解后加入207.0gKOH和20.0ml GPE消泡剂溶解后用加纯化水至不锈钢桶中固定位置定容90L。
2.灭菌
将配好的培养基用蠕动泵加入发酵罐中，进行实消灭菌，灭菌条件为115℃、30min。发酵罐灭菌结束后用支路蒸汽将补料管道和移种管道在线灭菌20min。
3.移种前准备
实消结束后，泵入氨水调节pH至4.5～4.7，泵入220ml已过滤除菌的PTM1微量盐溶液。取发酵罐内少量样品，离线检测pH值，若发酵罐上显示有偏差（≤pH1.00）,对显示值进行校正。若偏差＞pH1.00，应重新进行配料灭菌，并对pH电极进行检查确保无故障。设置300rpm转速，200slpm通气量，待DO稳定后校准100%。校准后将转速和通气量分别设置200rpm和100slpm的初始值，准备接种。
4.移种
移种前将移种管道从种子罐至发酵罐的方向在线灭菌20min以上。待种子罐种子液长至适合浓度后，通过调整通气量使得种子罐罐压大于发酵罐罐压至少2～5psig，打开种子罐出料阀和发酵罐移种阀使菌液通过压力差压至发酵罐中。
5. 甘油培养阶段
移种后控制温度30±1℃、pH5.0±0.1，过程中手动调整转速和通气量，保持DO大于30%。培养13～15h后原培养基中的甘油耗完，表现为溶氧突然上升，湿重应为100～130g/L。此时开始流加含12ml/L PTM1溶液的50%（w/w）甘油。初始速率约1L/h。甘油补加速率每小时提高250～500ml。最高至约2.5L/h后不再增加，每2h测定湿重。待湿重增至210～260g/L后，停甘油。
6.甲醇诱导阶段
停止流加甘油10～15min后开始以300ml/h速率流加甲醇，并控制温度在半小时内降至22℃。然后以5～7L/h泵速加入约7～9L YP补料。补加完YP后将pH设置为6.0。以300～500ml/h速率泵入甲醇5～7h后，停甲醇。此时菌体已经基本适应甲醇，停甲醇后应很快出溶氧峰。之后按照表1调节甲醇速率（根据溶氧的实际变化可稍做变动）。
表1甲醇流加速率
诱导过程中调整转速和通气量维持DO在30%左右，当压缩空气不足以维持溶氧开始以一定比例通纯氧来维持。每2h停止流加甲醇做DO spike，验证是否处于甲醇补料限速状态。如DO spike不明显需待多余甲醇耗完，出溶氧峰再次以相同泵速流加甲醇2h，再验证是否处于甲醇限速状态，直至明显做出DO spike。
诱导24h后再次加入YP补料约12L；诱导48h时加入YP补料约15L。诱导45～48h时根据体积情况需再次加入YP补料前带放30～40L发酵液。发酵液按离心工序操作规程离心后上清暂存4℃，之后随放罐上清一并交接至纯化。带放后下调甲醇泵速使得在相同搅拌速率和通气量下维持DO大于20%。
培养和发酵过程中每4h取样1次镜检和测定菌体湿重，诱导约60h后结束发酵，菌体湿重约为500g/L，并测定蛋白表达量，表达量在0.3-0.7mg/ml。
实施例3：重组人胰高血糖素样肽-1类似物融合蛋白的纯化方法
1、亲和BlUE层析
填料：填料Blue Sepharose Fast Flow 3.1L，柱高10cm，柱子BPG 200/500。
溶液：平衡液BUFFER A（10mmol/L NaAc-HAc pH5.0+5mmol/L EDTA-2Na +0.15mol/L NaCl）。
洗涤液：BUFFER B1（25% 乙二醇）。
洗脱液：BUFFER B2（20mmol/L Tris-HCl pH8.0+1mol/L NaCl+25%乙二醇+5mmol/L EDTA-2Na）。
先用平衡液BUFFER A平衡层析柱2-3CV(柱体积)，整个层析过程流速保保持50-80cm/h。平衡结束开始上样，上样载量控制在6-10mg/ml,上样完成后，先用2～2.5CV的BUFFER A平衡柱子，接着用2～2.5CV的BUFFER B1洗涤柱子，然后再用2～2.5CV的BUFFERB2洗脱，当UV280nm吸收达到1000mAu时用干消的瓶子收集洗脱峰，当UV280nm吸收小于1200mAu时停止收集。
2、苯基琼脂糖疏水层析
填料：填料Phenyl Sepharose High Performance，柱高10cm，柱子BPG 200/500。
平衡液BUFFER A（20mmol/L PB pH6.8，0.58mol/L 硫酸铵）
洗脱液BUFFER B（20mmol/L PB pH6.8）
样品预处理：将样品用3.2mol/L的硫酸铵或注射用水调节电导在80-100mS/cm。如果有偏差则用3.2mol/L的硫酸铵或注射用水调节。
先用平衡液BUFFER A平衡层析柱2-3CV(柱体积)，整个层析过程流速保保持50-80cm/h。平衡结束开始上样，上样载量控制在20-35mg/ml。
上样完成后，先用2.0～2.5CV的BUFFER A平衡柱子，再用80%梯度的BUFFER B 洗脱2～2.5CV，当UV280nm吸收达到800mAu时用干消的瓶子收集洗脱峰，当UV280nm吸收小于1000mAu时停止收集。
3、DEAE琼脂糖阴离子层析
填料：DEAE Sepharose Fast Flow，柱高10cm, BPG 140/50；
平衡液BUFFER A（20mmol/L PB pH6.8）；
洗脱液BUFFER B1（20mmol/L NaAc-HAc pH4.5）。
样品预处理：将疏水层析所得样品用注射用水稀释至电导5mS/cm以下。
先用平衡液BUFFER A平衡层析柱8-12CV(柱体积)，整个层析过程流速保保持50-80cm/h。平衡结束开始上样，上样载量控制在25-35mg/ml。
上样完成后，先用2CV的BUFFER A平衡柱子，再用BUFFER B1 洗脱5～7CV，当UV280nm吸收达到300mA时用干消的瓶子收集洗脱峰，当UV280nm吸收小于280mA时停止收集。
4、G25脱盐层析
填料：Sephadex Coarse G25 柱子：INDEX 100/950mm柱高70cm；
平衡液BUFFER A（10mM PB pH6.8）。
先用平衡液BUFFER A平衡层析柱1.5-2.5CV(柱体积)，整个层析过程流速保保持60-80cm/h。平衡结束开始上样，上样体积控制在柱体积1/5以下，上样完成后当UV280nm吸收达到500mAU时收集蛋白峰，当UV280nm吸收小于800mAU时停止收集，所得样品浓度在20mg/ml以上。
实施例4：重组人胰高血糖素样肽-1类似物融合蛋白工程菌发酵液的预处理
发酵液加热实验的操作方法：取20ml发酵液至50ml离心管中，称取适量辛酸钠、氨甲环酸加入到发酵液中缓慢摇匀，用稀盐酸或2M Tris 调节发酵液到指定的pH后，放置于水浴锅中加热，用温度计及时测量发酵液中的温度，达到设定温度后开始计时，加热完成后室温冷却后送样。
1、辛酸钠在不同加热时间下对目的蛋白降解的抑制作用
将实施例2制备的发酵液经过离心收集发酵液上清，加入20mM辛酸钠，调节pH至7.0，68±1℃加热1h、2h、3h、4h，加热结束后室温放置过夜，进行SDS-PAGE电泳分析，结果如图1所示。
图1中加热不同时间的样品没有明显差异，表明在仅加入辛酸钠加热的条件下无法抑制重组人胰高血糖素样肽-1类似物融合蛋白的降解。
2、氨甲环酸在不同加热时间下对目的蛋白降解的抑制作用
将实施例2制备的发酵液经过离心收集发酵液上清，加入20mM氨甲环酸，调节pH至7.0，68±1℃加热1h、2h、3h、4h，加热结束后室温放置过夜，进行SDS-PAGE电泳分析，结果如图2所示。
图2中加热不同时间的样品没有明显差异，表明在仅加入氨甲环酸加热的条件下无法抑制重组人胰高血糖素样肽-1类似物融合蛋白的降解。
3、同时加入辛酸钠和氨甲环酸在不同加热条件下对目的蛋白降解的抑制作用
将实施例2制备的发酵液经过离心收集发酵液上清，加入辛酸钠和氨甲环酸（摩尔比1：1）共20mM，调节pH至7.0，分别在75℃、70℃、65℃加热，加热时间如下：75℃加热10min；70℃加热10min、20min、30min；65℃加热1h、2h、3h、4h，加热后进行SDS-PAGE电泳分析，结果如图3所示。
图3中泳道2-4与泳道1的未处理发酵液对照样品无明显差异，泳道6-9与泳道1的未处理发酵液对照样品相比具有更少的降解条带，泳道5的目的产物条带浓度最高、降解产物条带最少。上述结果表明，即使在同时加入两种试剂的情况下，只有特定的加热时间和加热温度预处理条件下才能够抑制重组人胰高血糖素样肽-1类似物融合蛋白的降解，短时间的高温和长时间的低温都无法有效抑制目的蛋白的降解。
4、同时加入辛酸钠和氨甲环酸在不同pH值条件下对目的蛋白降解的抑制作用
为了进一步优化预处理的条件，还对pH值进行了选择。将实施例2制备的发酵液经过离心收集发酵液上清，加入辛酸钠和氨甲环酸（摩尔比1：1）共20mM，分别调节pH值至5.0、5.5、6.0、6.5、7.0、7.5，68℃加热1h后进行SDS-PAGE电泳分析，结果如图4所示。
图4中与泳道1-3相比，泳道4-6具有更少的降解条带，上述结果表明发酵液上清在pH 6.5-7.5之间加热预处理更有利于抑制重组人胰高血糖素样肽-1类似物融合蛋白的降解。
5、辛酸钠和氨甲环酸的浓度对目的蛋白降解的抑制作用
药品生产工艺中添加剂的使用是一把双刃剑，非治疗活性添加剂既能发挥必要的作用，也容易给药品的纯度和安全性带来隐患。为了避免过度使用添加剂，对辛酸钠和氨甲环酸的浓度进行了优化。将实施例2制备的发酵液经过离心收集发酵液上清，加入辛酸钠和氨甲环酸浓度各3mM、5mM、 7mM、10 mM、15 mM、20mM，调节pH至7.0，在68℃加热1h后进行SDS-PAGE电泳分析，结果如图5所示。
图5中与泳道1-3相比，泳道4-6具有更少的降解条带，上述结果表明发酵液上清在辛酸钠和氨甲环酸浓度各10mM以上的条件下加热预处理更有利于抑制重组人胰高血糖素样肽-1类似物融合蛋白的降解。
根据上述优化的结果，最终选择最优的发酵液预处理条件为：发酵液离心收集上清以后，加入10mM氨甲环酸和10mM辛酸钠试剂后缓慢摇匀，用稀盐酸或2M Tris调节发酵液至pH 7.0后，68±2℃加热1h，后用冰醋酸将发酵pH调制5.0±0.1后进行后续层析。
实施例5：加热预处理条件对重组人胰高血糖素样肽-1类似物融合蛋白生物活性的影响
1、试验方法
GLP-1与细胞膜上的GLP-1 receptor（GLP-1R）结合激发细胞膜信号通路使胞内cAMP（环化腺苷酸）的浓度上升，作用于下游元件cAMP response element (CRE)，从而使其下游的luciferase（荧光素酶）基因表达，通过检测luciferase催化其底物产生的荧光而到达检测GLP-1生物活性的目的。本实验中采用稳定转染GLP-1R和cAMP调控表达荧光素酶报告基因的CHO DG44 2-A4-2-A8细胞进行生物学活性测定。具体操作如下：
（1）溶液配制
CHO DG44 2-A4-2-A8完全培养基：DMEM/F12培养基45ml，5ml FBS，500μl P/Ssolution(100×)，62.5μl G418（160mg/ml）。
（2）细胞铺板
取融合度达到90%的CHO DG44 2-A4-2-A8细胞，Trypsin-EDTA消化后，1000 rpm离心4 min，去上清，使用完全培养基重悬。调整细胞密度至5×105个/ml，100 μl/孔，置于细胞培养箱中培养过夜。
（3）加样
用完全培养基分别将样品及工作参考品分步预稀释至500nM。再4倍梯度稀释，共10个点。将已梯度稀释的工作参考品和供试品分别加入至96孔白色细胞板，100 μl/孔，每个浓度点设置复孔，置于细胞培养箱孵育约5 h。所述的样品（代号为GW002-DS）是对发酵液进行了如实施例4所述优化后预处理后纯化获得的产品；所述对照样品（代号为RS-GW00220160901）是同一批次发酵液，不经预处理直接纯化获得的产品。
（4）显色及读板
取出适量分装的Bright-GloTM Luciferase 底物避光融化，50 μl/孔加入细胞板中，避光孵育5 min。在酶标仪为上读取RLU，结果如图6所示；试验样品与对照样品的活性如表2所示。
表2试验样品与对照样品的活性测试
图6、表2的结果显示，经过发酵液预处理工艺获得的试验样品与未经发酵液预处理工艺获得的对照样品，在相同浓度下的生物活性曲线保持一致。上述结果表明，本发明优化的发酵液预处理步骤没有影响重组人胰高血糖素样肽-1类似物融合蛋白的分子结构和单位分子的生物活性。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
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<110> 迈威（上海）生物科技股份有限公司； 江苏迈威康新药研发有限公司
<120> 重组人胰高血糖素样肽-1类似物融合蛋白的制备方法
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Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr
530 535 540
Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile
545 550 555 560
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu
565 570 575
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys
580 585 590
Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala
595 600 605
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala
610 615 620
Ala Ser Gln Ala Ala Leu Gly Leu
625 630
Claims (7)
1.一种重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，包括工程菌构建、诱导表达、重组人胰高血糖素样肽-1类似物融合蛋白纯化；
其特征在于，在诱导表达步骤和重组人胰高血糖素样肽-1类似物融合蛋白纯化步骤之间包括将工程菌诱导表达的发酵液调节至pH 6.5-7.5之间进行预处理的步骤，所述预处理是在添加辛酸钠和氨甲环酸的条件下对工程菌诱导表达的发酵液68℃加热处理1h；
其中，辛酸钠和氨甲环酸摩尔比为1:1，终浓度分别为10-20mM。
2.如权利要求1所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其特征在于：所述重组人胰高血糖素样肽-1类似物融合蛋白包括GLP-1类似物、载体蛋白、可选的位于二者之间的蛋白接头。
3.如权利要求2所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其特征在于：所述GLP-1类似物如SEQ ID NO:1所示，所述载体蛋白为HSA，所述蛋白接头选自SEQID NO:2-4之一。
4.如权利要求2所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其特征在于：所述人胰高血糖素样肽-1类似物融合蛋白如SEQ ID NO:5所示。
5.如权利要求1所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其特征在于：所述诱导表达包括甘油培养阶段和甲醇诱导阶段；其中甘油流加培养至菌体湿重216-260g/L时，停止流加甘油，开始流加甲醇诱导发酵，直至发酵液中重组人胰高血糖素样肽-1类似物融合蛋白的表达量达0.3-0.7mg/mL。
6.如权利要求1所述的重组人胰高血糖素样肽-1类似物融合蛋白的制备方法，其特征在于：所述重组人胰高血糖素样肽-1类似物融合蛋白纯化包括Blue Sepharose Fast Flow3.1L亲和层析、苯基琼脂糖疏水层析（Phenyl Sepharose High Performance）、DEAE琼脂糖阴离子层析（DEAE Sepharose Fast Flow）、以及G25脱盐层析。
7.辛酸钠和氨甲环酸在降低重组蛋白降解中的用途，其特征在于：将含有重组蛋白的酵母工程菌诱导表达发酵液上清调节至pH 6.5-7.5之间进行预处理，所述预处理是在添加辛酸钠和氨甲环酸的条件下对工程菌诱导表达的发酵液68℃加热处理1h；
其中，所述辛酸钠和氨甲环酸的摩尔比为1:1，终浓度分别为10-20mM；所述重组蛋白是重组人胰高血糖素样肽-1类似物融合蛋白。
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