CN116621964A - 一种制备人促血小板生成素的方法及人促血小板生成素 - Google Patents
一种制备人促血小板生成素的方法及人促血小板生成素 Download PDFInfo
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Abstract
一种制备人促血小板生成素的方法,其特征在于,包括如下步骤:步骤1,制备修饰用TPO;步骤2,通过SCM‑PEG40K对TPO进行修饰反应,其中SCM‑PEG40K为带有活性基团为琥珀酰亚胺乙醇酯(SCM)的PEG作为修饰剂,其分子量为40kDa,设置TPO溶液的蛋白质浓度为0.5‑2.0mg/ml,设置TPO与SCM‑PEG40K的摩尔比为1:3‑1:20,并且将反应时间设定为2‑18小时;步骤3,对步骤2所获得的TPO‑PEG40KSCM产物进行纯化。还涉及相应的人促血小板生成素。
Description
技术领域
本发明涉及生物药物领域,特别是一种制备人促血小板生成素的方法以及由此获得的人促血小板生成素。
背景技术
TPO(血小板生成素)是一种人体内源的生长因子,对巨核细胞增殖、分化、成熟起调节作用,是治疗血小板减少症的重要研究方向和途径。沈阳三生制药有限公司生产的重组TPO(商品名:特比澳)已上市多年,用于化疗相关的血小板减少症(CIT)和原发免疫性血小板减少症(ITP),被认为是第一代促血小板生成剂。但重组TPO在人体的半衰期较短,临床需每天注射,连续给药,患者顺应性差。因此,多家制药企业在开发以长效化为目的的第二代TPO产品。主要长效化的技术包括PEG修饰和融合表达两种。前者如Amgen公司将TPO蛋白N端含163个氨基酸的活性结构域进行PEG修饰,修饰后的蛋白显著延长了半衰期,在临床试验上实现了10天给药一次。但由于该活性结构域采用大肠杆菌表达,缺少糖基化修饰,在人体中产生了中和抗体,并且中和抗体与内源TPO发生交叉反应,造成严重的副作用,最终导致临床试验失败。融合表达多采用将TPO拟肽或具有升血小板活性的肽段与Fc融合的办法,通过Fc融合增大蛋白分子量,降低肽段的肾排泄,达到长效化的目的。如已获FDA批准用于ITP治疗的罗米司亭,即为一种人促血小板生成素模拟肽-Fc融合蛋白,临床使用频次为每周给药一次,患者治疗的便利度获得极大的提高。
本专利用PEG对全长TPO蛋白进行修饰,TPO为CHO细胞重组表达获得,具有完整的糖基化修饰,较大肠杆菌表达的TPO结构域的免疫原性更低,安全性更好,通过对修饰剂的对比和筛选,获得的修饰分子在大鼠中的半衰期延长了2倍,达到了长效化的目的,为人体试验奠定了基础。
发明内容
因此为了解决上述的技术问题,本发明提出了如下解决方案:
一种制备人促血小板生成素的方法,包括如下步骤:步骤1,制备修饰用TPO;步骤2,通过SCM-PEG40K对TPO进行修饰反应,其中SCM-PEG40K为带有活性基团为琥珀酰亚胺乙醇酯(SCM)的PEG作为修饰剂,其分子量为40kDa,设置TPO溶液的蛋白质浓度为0.5-2.0mg/ml,设置TPO与SCM-PEG40K的摩尔比为1:3-1:20,并且将反应时间设定为2-18小时;步骤3,对步骤2所获得的TPO-PEG40KSCM产物进行纯化。
根据本发明的一个优选实施例,在所述步骤3中包括步骤3.1阴离子交换层析;步骤3.2将阴离子交换层析的洗脱液进一步用分子筛层析。
优选地,所述步骤1包括:合成TPO的编码DNA序列,克隆进入pCDNA3.1表达载体中;提取质粒用Lipofectamine 2000转染CHO细胞株,通过在培养基中加入嘌呤霉素筛选获得阳性细胞池;将阳性细胞池按0.5个细胞/孔的密度有限稀释至96孔板中,待单克隆细胞生长至50%汇合度时用ELISA试剂盒筛选高表达细胞克隆;挑取表达量最高的克隆在三角瓶中进行扩增培养;当细胞密度生长至2×10E6个/ml时,取400ml接种到含2L基础培养基的7L生物反应器中继续培养,生物反应器设定温度37℃,pH7.0,搅拌80rpm,溶氧50%;监测并补加葡萄糖至终浓度5g/l,培养后期添加补料等营养元素;生物反应器上培养10天后,停止培养,离心收集上清;将上清依次用阳离子层析、疏水层析、分子筛层析进行纯化,获得纯度>98%,比活性≥2.5×10E5U/mg的纯品蛋白。
本发明还涉及一种用上述的制备人促血小板生成素的方法制备的人促血小板生成素。
通过对多种PEG修饰剂的对比筛选,确定修饰效果最好的PEG分子量为40kDa,活性基团为SCM,用该PEG修饰后活性损失较少,同时可以获得单位点PEG修饰的TPO分子,修饰后分子的结构均一,有利于产物的分离纯化和鉴定,也有利于对产物的质量进行分析和控制,实现批间一致性,方便产业化。本专利研发的TPO-PEG修饰分子的体内半衰期比TPO显著延长,达到长效的效果,预计临床可5-7天注射给药一次。
通过PEG修饰延长TPO的半衰期。SCM活性基团可以与蛋白N端和赖氨酸的氨基进行反应,TPO分子中含有7个赖氨酸,均为潜在的修饰位点,容易发生多位点修饰。我们的实验也显示,当用分子量20K的SCM-PEG20K进行修饰时,产物不均一,含有多位点修饰产物。意外的是,当PEG分子量40K时,虽然还是带有SCM活性基团,但修饰产物却为单PEG修饰。
附图说明
图1为SDS-PAGE检测SCM-PEG20K和SCM-PEG40K的修饰反应(考马斯亮蓝染色)。
泳道1为用SCM-PEG20K修饰的结果;泳道2为用SCM-PEG40K修饰的结果。
图2为SDS-PAGE检测SCM-PEG40K修饰反应优化的结果(碘染色)。
泳道1-3分别为浓度0.5、1、2mg/ml的TPO与SCM-PEG40K反应;泳道4-6分别为TPO与SCM-PEG40K摩尔比1:3、1:10、1:20的修饰结果;泳道7-10分别为TPO与SCM-PEG40K反应1、2、4、18h的修饰结果。
图3为SDS-PAGE检测TPO-PEG40KSCM的纯化结果(碘染色)。
泳道1-3分别为Q HP层析的上样、流穿液和洗脱液;泳道4-11分别分子筛层析洗脱峰的分管收集。
图4为TPO-PEG40KSCM对环磷酰胺诱导小鼠血小板减少的恢复。
具体实施方式
1修饰用TPO的制备
合成TPO的编码DNA序列,克隆进入pCDNA3.1表达载体中。提取质粒用Lipofectamine 2000(Invitrogen公司)转染CHO细胞株,通过在培养基中加入嘌呤霉素筛选获得阳性细胞池。将阳性细胞池按0.5个细胞/孔的密度有限稀释至96孔板中,待单克隆细胞生长至50%汇合度时用ELISA试剂盒(R&D公司,货号DTP00B)筛选高表达细胞克隆。挑取表达量最高的克隆在三角瓶中进行扩增培养。当细胞密度生长至2×10E6个/ml时,取400ml接种到含2L基础培养基的7L生物反应器中继续培养,生物反应器设定温度37℃,pH7.0,搅拌80rpm,溶氧50%。监测并补加葡萄糖至终浓度5g/l,培养后期添加补料等营养元素。生物反应器上培养10天后,停止培养,离心收集上清。将上清依次用阳离子层析、疏水层析、分子筛层析进行纯化,获得纯度>98%,比活性≥2.5×10E5U/mg的纯品蛋白。
2SCM-PEG20K和SCM-PEG40K的修饰反应
选择带有活性基团为琥珀酰亚胺乙酸酯(SCM)的PEG作为修饰剂,PEG分子量分别为20kDa和40kDa。修饰反应条件为:取4ml浓度为1.0mg/ml的TPO溶液,按照TPO:修饰剂的摩尔比为1:5的比例分别加入SCM-PEG20K和SCM-PEG40K,室温下振荡反应18h。SDS-PAGE电泳检测修饰产物。两种不同分子量的修饰剂均能与TPO蛋白进行反应。SCM-PEG20K的修饰效率较高,TPO全部被PEG修饰,但产物包含有单PEG修饰和多PEG修饰。SCM-PEG40K修饰产物以单PEG修饰为主,虽然部分TPO未与修饰剂反应,但修饰的反应产物更均一,更有利于后续的纯化。因此,对SCM-PEG40K的修饰条件进一步优化。
3SCM-PEG40K修饰反应条件的优化
1)蛋白浓度的对比
分别取浓度为0.5、1.0、2.0mg/ml的TPO溶液,按照TPO:SCM-PEG40K摩尔比为1:5的比例加入SCM-PEG40K,室温下振荡反应18h。反应产物各取5ul进行SDS-PAGE电泳检测,修饰产物蛋白条带的灰度呈比例增加,表明0.5-2.0mg/ml的蛋白浓度条件下的反应效率较为一致。
2)修饰剂比例的对比
取浓度为1.0mg/ml的TPO溶液,按照TPO:SCM-PEG40K摩尔比为1:3、1:10、1:20的比例分别与SCM-PEG40K反应,室温下振荡反应18h。反应产物经SDS-PAGE电泳检测,结果可以看出,修饰产物TPO-PEG40KSCM的量随SCM-PEG40K加入量的增加而增多。即使TPO和SCM-PEG40K两者的摩尔比达到1:20,修饰产物仍以单PEG修饰为主。该比例条件下的TPO-PEG40KSCM得率最高。
3)反应时间的对比
取浓度为1.0mg/ml的TPO溶液,按照TPO:SCM-PEG40K摩尔比为1:20的比例加入SCM-PEG40K,室温下振荡反应,于1、2、4、18h分别取样进行电泳检测。反应1h时的修饰产物的量明显低于2、4、18h。因此,修饰反应的时间应大于2h。
4TPO-PEG40KSCM的纯化
TPO与SCM-PEG40K反应后,反应体系中除了含有产物TPO-PEG40KSCM外还包括有未反应的TPO和SCM-PEG40K。未反应的TPO和SCM-PEG40K影响TPO-PEG40KSCM的纯度,需进一步纯化去除。
1)TPO-PEG40KSCM的阴离子交换层析
取10mg TPO-PEG40KSCM修饰反应产物用10ml Q HP填料纯化。纯化平衡缓冲液为10mM PB,pH8.0。上样后用10倍柱体积的平衡缓冲液冲洗。洗脱采用10mM PB,0.5M NaCl,pH8.0的洗脱缓冲液等梯度洗脱,收集洗脱峰。对纯化各组分进行SDS-PAGE电泳检测,结果显示,上样和流穿液中未检测到TPO-PEG40KSCM,目的蛋白可以与填料很好地结合,冲洗液中含有未反应的PEG,洗脱液中未检测到未反应PEG,表明冲洗10个柱体积可以充分去除未反应的PEG。
2)TPO-PEG40KSCM的分子筛层析
将阴离子交换层析的洗脱液进一步用分子筛层析,选用20mm×700mm层析柱装Chromdex 75PG填料(博格隆公司产品),填料高度400mm。上样体积为填料体积的1/10,流速1ml/min,缓冲液为10mM PB,pH8.0。分管收集洗脱峰,超滤浓缩。
5活性测定
人巨细胞白血病细胞株MO7e细胞用含10%胎牛血清的DMEM培养基于37℃、5%CO2的条件下培养,每两天传代一次。将正常生长的MO7e细胞用培养基稀释至5×10E4个/ml的浓度,按照100ul/孔的量加至96孔培养板中,加入系列倍比稀释的TPO、TPO-PEG40KSCM,继续培养72h后加入40μl的显色剂(MTS/PMS 20:1),于37℃静置4h。用酶标仪测定每孔的吸收值,测定波长490nm,参比波长630nm,对数据以蛋白量为横坐标、吸收值为纵坐标进行四参数拟合绘图,分别计算TPO、TPO-PEG40KSCM的EC50值。结果显示TPO经修饰后的体外活性有所下降,TPO-PEG40KSCM的活性是TPO的48%左右。
6大鼠体内药代动力学
将体重200g的SD雄性大鼠分为两组,每组8只。两组分别按2ug/kg的给药量皮下注射TPO和TPO-PEG40KSCM。于给药前,给药后1、4、6、8、12、24、36、48、72、96h眼眶取血,分离血清。用人TPO Elisa测定试剂盒(R&D公司,货号DTP00B),分别以定量的TPO和TPO-PEG40KSCM为参考品绘制标准曲线,测定血药中TPO和TPO-PEG40KSCM浓度,计算药代动力学参数,TPO的平均消除半衰期为23h,TPO-PEG40KSCM的平均消除半衰期为41h,是未修饰TPO的1.8倍。
7TPO-PEG40KSCM对环磷酰胺诱导小鼠血小板减少的恢复
选6-8周龄的雄性BALB/c小鼠,于第1天按200mg/kg的量尾静脉注射环磷酰胺,于第2-6天,每天按30mg/kg的量尾静脉注射环磷酰胺。第7天通过尾静脉取血,测定血小板数量,降小板计数较造模前下降20%~30%的动物视为造模成功,按每组6只将模型小鼠分为3组。组1,每天皮下注射生理盐水;组2,每天皮下注射TPO;组3,每5天皮下注射TPO-PEG40KSCM。造模前、造模和给药过程中取血,测定血小板降低比例。结果显示,TPO-PEG40KSCM治疗组动物血小板下降比例最低,血小板回升速度也最快,TPO-PEG40KSCM具有良好的体内活性。
Claims (4)
1.一种制备人促血小板生成素的方法,其特征在于,包括如下步骤:步骤1,制备修饰用TPO;步骤2,通过SCM-PEG40K对TPO进行修饰反应,其中SCM-PEG40K为带有活性基团为琥珀酰亚胺乙醇酯(SCM)的PEG作为修饰剂,其分子量为40kDa,设置TPO溶液的蛋白质浓度为0.5-2.0mg/ml,设置TPO与SCM-PEG40K的摩尔比为1:3-1:20,并且将反应时间设定为2-18小时;步骤3,对步骤2所获得的TPO-PEG40KSCM产物进行纯化。
2.根据权利要求1所述的制备人促血小板生成素的方法,其特征在于,在所述步骤3中包括步骤3.1阴离子交换层析;步骤3.2将阴离子交换层析的洗脱液进一步用分子筛层析。
3.根据权利要求2所述的制备人促血小板生成素的方法,其特征在于,所述步骤1包括:合成TPO的编码DNA序列,克隆进入pCDNA3.1表达载体中;提取质粒用Lipofectamine 2000转染CHO细胞株,通过在培养基中加入嘌呤霉素筛选获得阳性细胞池;将阳性细胞池按0.5个细胞/孔的密度有限稀释至96孔板中,待单克隆细胞生长至50%汇合度时用ELISA试剂盒筛选高表达细胞克隆;挑取表达量最高的克隆在三角瓶中进行扩增培养;当细胞密度生长至2×10E6个/ml时,取400ml接种到含2L基础培养基的7L生物反应器中继续培养,生物反应器设定温度37℃,pH7.0,搅拌80rpm,溶氧50%;监测并补加葡萄糖至终浓度5g/l,培养后期添加补料等营养元素;生物反应器上培养10天后,停止培养,离心收集上清;将上清依次用阳离子层析、疏水层析、分子筛层析进行纯化,获得纯度>98%,比活性≥2.5×10E5U/mg的纯品蛋白。
4.一种用根据权利要求1-4中任一项所述的制备人促血小板生成素的方法制备的人促血小板生成素。
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