CN107522774B - 一种醋酸格拉替雷制备过程中哌啶残留量的实时控制方法 - Google Patents
一种醋酸格拉替雷制备过程中哌啶残留量的实时控制方法 Download PDFInfo
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Abstract
本发明提供了一种醋酸格拉替雷制备过程中哌啶残留量的实时控制方法,以超滤方法对格拉替雷粗品溶液进行纯化,同时监测pH值至9.9‑10.0时停止纯化,酸化以获得哌啶残留量低于0.1%(0.05%,更优选0.03%)的醋酸格拉替雷。本发明的方法能够使醋酸格拉替雷产品中的哌啶残留量降低,纯化时间缩短。
Description
技术领域
本发明涉及一种多肽药物的制备方法,具体涉及一种醋酸格拉替雷制备过程中哌啶残留量的实时控制方法
背景技术
醋酸格拉替雷是一种用于治疗多发性硬化症的的人工合成多肽混合物(M.M.Mouradain,Pharmacology&Therapeutics,98,245-255,2003)。醋酸格拉替雷(又叫共聚物-1)是一个由丙氨酸、谷氨酸、赖氨酸和酪氨酸组成的随机聚合物。它的氨基酸摩尔比大约是0.392~0.462:0.129~0.153:0.300~0.374:0.086~0.100,平均分子量大约4700~11000道尔顿。醋酸格拉替雷的结构式是:
(Glu,Ala,Lys,Tyr)x·xCH3COOH
醋酸格拉替雷或共聚物-1的合成方法已经在美国专利3849550,5800808,5981589,48898,6054430,6342476,6362161等专利中描述。合成方法是通过L-丙氨酸、L-酪氨酸、L-谷氨酸-γ-苄酯、L-ε-三氟乙酰基-赖氨酸的N-甲酸酐(NCA)在无水1,4-二氧六环中,使用二乙胺引发进行随机聚合,以产生受保护的多肽。γ-苄基基团的脱保护是通过室温下在溴化氢/乙酸中搅拌受保护的多肽来实现的。该条件也可以切割共聚物。下一步通过哌啶处理可以脱除ε-三氟乙酰基。最后通过超滤技术纯化共聚物并冻干,得到醋酸格拉替雷。因此,现有技术包括:聚合、两次去保护、纯化和冻干步骤。
哌啶在《联合国禁止非法贩运麻醉药品和精神药物公约》中出现在表2中,其被归属为麻醉药品和精神药品的前体,所以其生物活性不容小视。哌啶在醋酸格拉替雷的合成过程中的最后一步化学反应中,被用作脱除赖氨酸残基上的-三氟乙酰基的脱除剂,所以醋酸格拉替雷产品中哌啶的残留量就成为了很重要的一个质量指标。在醋酸格拉替雷原研厂家——以色列梯瓦公司发表的文献中没有有关控制醋酸格拉替雷中哌啶的方法报道。
因为合成醋酸格拉替雷的最后一步化学反应是用哌啶脱除三氟乙酰基格拉替雷中赖氨酸残基上的ε-三氟乙酰基,然后进行纯化和冻干操作。而哌啶的沸点达到了106℃,在冻干环节很难除去。所以纯化环节就成了唯一可能去除残留哌啶,使其维持一个可以接受范围内的机会。
本发明的目的就是通过纯化环节在线检测哌啶的残留量,使产品醋酸格拉替雷中哌啶的残留量低于0.10%
发明内容
为了克服上述问题,本发明提供了一种醋酸格拉替雷的制备方法,其包括以下步骤:
1)合成全保护的格拉替雷,
2)脱除除赖氨酸侧链保护基以外的其他保护基,得到三氟乙酰基格拉替雷,
3)以哌啶水溶液脱除三氟乙酰基,得到格拉替雷粗品溶液;
4)以超滤方法对格拉替雷粗品溶液进行纯化,同时监测pH值至9.9-10.0时停止纯化,并加醋酸至pH值5.5-5.8;
5)冻干步骤4)所得溶液,获得哌啶残留量低于0.1%(0.05%,更优选0.03%)的醋酸格拉替雷。
其中,步骤4)中还包括了以气相色谱法和/或离子色谱法辅助实时检测哌啶残留量的步骤。
其中,
所述气相色谱法的检测条件为:柱温起始温度为40℃维持5min,然后以每分钟20℃的速率升至200℃,维持9分钟min;以N2为载气,流速为2.0ml/min;N2,28ml/min;H2,30ml/min;空气,300ml/min;分流比20∶1;采用顶空进样,样品炉加热温度为80℃;样品加热平衡时间为30min,进样针温度为90℃;采用氢火焰离子化检测器(FID);进样口温度为200℃;检测器温度为250℃;
所述离子色谱法的检测条件为:淋洗液为20mM的氢氧化钾溶液,抑制器电流为50mA,流速为1.0ml/min,柱温30℃,检测器为电导检测器。
其中,步骤1)为在极性非质子溶剂中,在引发剂存在下,聚合L-丙氨酸、L-酪氨酸、L-谷氨酸--苄酯、L--三氟乙酰基-赖氨酸的N-甲酸酐,以获得全保护的格拉替雷;以及
其中,步骤2)为将酸溶液加入到步骤1)中形成的全保护的格拉替雷中,以形成三氟乙酰基格拉替雷;
优选地,所述酸溶液为卤化氢/乙酸混合溶液;更优选为(20%-40%)溴化氢/乙酸混合溶液。
本发明的另一个方面提供了一种醋酸格拉替雷制备过程中哌啶残留量的实时控制方法,以超滤方法对格拉替雷粗品溶液进行纯化,同时监测pH值至9.9-10.0时停止纯化,酸化以获得哌啶残留量低于0.1%(0.05%,更优选0.03%)的醋酸格拉替雷。
其中,超滤方法所用的超滤膜为5KDa的超滤膜包。
其中,在pH值至9.9-10.0时,还包括以气相色谱法和/或离子色谱法辅助实时检测哌啶残留量的步骤。
具体实施方式
实施例1.合成受保护的格拉替雷
三口瓶中加入经金属钠处理过的1,4-二氧六环200mL,室温下加入L-丙氨酸NCA2.590g,L-酪氨酸NCA1.036g,L-谷氨酸-γ-苄酯NCA1.974g,L-ε-三氟乙酰基-赖氨酸NCA4.693g。搅拌30min,至体系澄清,加入二乙胺33mg。20~25℃下机械搅拌24小时。将反应液缓慢倒入400mL水中,产生大量白色固体,抽滤后,真空干燥得7.424g,收率92.8%。
实施例2.第一次脱保护/解聚
三口瓶中加入受保护的格拉替雷7.0g,31%溴化氢/乙酸混合液140mL,在22~24℃下搅拌22小时。将红棕色反应液倒入400mL水中,产生大量白色固体,抽滤,真空干燥得三氟乙酰基格拉替雷4.66g。
实施例3.脱三氟乙酰基
三口瓶中加入实施例2中所得三氟乙酰基格拉替雷4.00g,1M哌啶水溶液220ml,室温下搅拌24小时。
实施例4.超滤纯化/冻干
取实施例3中得到的溶液100ml,用赛托利斯的5KDa的超滤膜包进行超滤纯化,得到的溶液加醋酸酸至pH为5.5~5.8,搅拌1小时。冻干得白色粉末1.44g,检测产品哌啶残留量0.88%。
实施例5.超滤纯化/冻干
取实施例3中得到的溶液100ml,用赛托利斯的5KDa的超滤膜包进行超滤纯化,同时检测哌啶残留量。当pH=9.9-10.0时,定容100ml,取样,气相色谱法测定哌啶残留量为0.0512mmol/L,离子色谱法测定哌啶残留量为0.0345mmol/L。得到的溶液加冰乙酸至pH为5.5~5.8,搅拌1小时。冻干得白色粉末1.46g,检测产品哌啶残留量0.03%。实施例6.气相色谱法在线测哌啶残留量
精密量取3ml待测溶液置10ml顶空瓶中,轧盖后测定。色谱柱为Agilent CP-Sil8CB for Amines 30×0.32×(1.0);柱温起始温度为40℃维持5min,然后以每min20℃的速率升至200℃,维持9min;以氮气为载气,流速为2.0ml/min;N2,28ml/min;H2,30ml/min;空气,300ml/min;分流比20∶1;采用顶空进样,样品炉加热温度为80℃;样品加热平衡时间为30min,进样针温度为90℃;采用氢火焰离子化检测器(FID);进样口温度为200℃;检测器温度为250℃。精密量取供试品溶液上层气体各1ml注入气相色谱仪,记录色谱图。按外标法以峰面积计算
实施例7.离子色谱法在线测哌啶残留量
色谱柱为Dionex IonPacTM AS19RFICTMAnalytical 4×250mm,淋洗液为20mM的氢氧化钾溶液,抑制器电流为50mA,流速为1.0ml/min,柱温30℃,检测器为电导检测器。精密量取供试品溶液及对照品溶液各25ul注入离子色谱仪,记录色谱图。按外标法以峰面积计算。
实施例8.气相色谱法测醋酸格拉替雷中哌啶残留量
取本品约150mg,精密称定,置10ml顶空进样瓶中,精密加10%的二甲亚砜3ml,轧盖后振摇,使样品溶解,作为供试品溶液。色谱柱为Agilent CP-Sil8CB for Amines 30×0.32×(1.0);柱温起始温度为40℃维持5min,然后以每分钟20℃的速率升至200℃,维持9min;以氮气为载气,流速为2.0ml/min;N2,28ml/min;H2,30ml/min;空气,300ml/min;分流比20∶1;采用顶空进样,样品炉加热温度为80℃;样品加热平衡时间为30min,进样针温度为90℃;采用氢火焰离子化检测器(FID);进样口温度为200℃;检测器温度为250℃。精密量取供试品溶液上层气体各1ml注入气相色谱仪,记录色谱图。按外标法以峰面积计算。
通过实施例5-8的结果能够说明pH值的实时监测能够更有效的确定超滤方法纯化的结束时间,pH值实时监测所采用的设备简单,结果能够实时监测,避免了纯化时间短,获得最终粉末产物后需要重新纯化的问题;同时,也避免了纯化时间过长导致效率降低的问题。
气相色谱法和离子色谱法能够对哌啶含量定量化,更精确的获得哌啶的含量,但是其使用的设备复杂,操作繁琐,相比于pH监测,其结果的获得需要一定的时间,因此,可以作为辅助的方法,在不影响试验进程的前提下,可选地进行辅助检测。
Claims (9)
1.一种醋酸格拉替雷的制备方法,其包括以下步骤:
1)合成全保护的格拉替雷,
2)脱除除赖氨酸侧链保护基以外的其他保护基,得到三氟乙酰基格拉替雷,
3)以哌啶水溶液脱除三氟乙酰基,得到格拉替雷粗品溶液;
4)以超滤方法对格拉替雷粗品溶液进行纯化,同时监测pH值至9.9-10.0时停止纯化,并加醋酸至pH值5.5-5.8;
5)冻干步骤4)所得溶液,获得哌啶残留量低于0.1%的醋酸格拉替雷,
其中,步骤4)中还包括了以气相色谱法和/或离子色谱法辅助实时检测哌啶残留量的步骤;
超滤方法所用的超滤膜为5KDa的超滤膜包。
2.根据权利要求1所述的制备方法,其中,步骤5)冻干步骤4)所得溶液,获得哌啶残留量低于0.05%的醋酸格拉替雷。
3.根据权利要求1-2任一项所述的制备方法,其中,
所述气相色谱法的检测条件为:柱温起始温度为40℃维持5min,然后以每分钟20℃的速率升至200℃,维持9 min;以N2为载气,流速为2.0ml/min;N2,28 ml/min;H2,30 ml/min;空气,300 ml/min;分流比20∶1;采用顶空进样,样品炉加热温度为80℃;样品加热平衡时间为30min,进样针温度为90℃;采用氢火焰离子化检测器(FID);进样口温度为200℃;检测器温度为250℃;
所述离子色谱法的检测条件为:淋洗液为20mM的氢氧化钾溶液,抑制器电流为50mA,流速为1.0ml/min,柱温30℃,检测器为电导检测器。
4.根据权利要求1-2任一项所述的制备方法,其中,步骤1)为在极性非质子溶剂中,在引发剂存在下,聚合L-丙氨酸、L-酪氨酸、L-谷氨酸-γ-苄酯、L-ε-三氟乙酰基-赖氨酸的N-甲酸酐,以获得全保护的格拉替雷。
5.根据权利要求1-2任一项所述的制备方法,其中,步骤2)为将酸溶液加入到步骤1)中形成的全保护的格拉替雷中,以形成三氟乙酰基格拉替雷。
6.根据权利要求5所述的制备方法,其中,所述酸溶液为卤化氢/乙酸混合溶液。
7.根据权利要求6所述的制备方法,其中,所述酸溶液为20%-40%的溴化氢/乙酸混合溶液。
8.一种醋酸格拉替雷制备过程中哌啶残留量的实时控制方法,以超滤方法对格拉替雷粗品溶液进行纯化,同时监测pH值至9.9-10.0时停止纯化,酸化以获得哌啶残留量低于0.1%的醋酸格拉替雷;
其中,在pH值至9.9-10.0时,还包括以气相色谱法和/或离子色谱法辅助实时检测哌啶残留量的步骤;
超滤方法所用的超滤膜为5KDa的超滤膜包。
9.根据权利要求8所述的实时控制方法,其中,酸化以获得哌啶残留量低于0.05%的醋酸格拉替雷。
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