CN112649538A - 多肽混合物高效液相色谱分析方法 - Google Patents
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Abstract
本发明涉及一种多肽混合物高效液相色谱分析方法,具体而言,该方法包括如下步骤,步骤(1)配置待测醋酸格拉替雷溶液;步骤(2)以阳离子交换液相色谱法,对待测样本进行梯度洗脱,步骤(3)检测分析共聚物各组分对应峰面积,通过与对照品比对,确定待测样本各组分含量范围是否合格。
Description
本申请是申请号201580079194.1,申请日为2015年4月28日,发明名称为“多肽混合物高效液相色谱分析方法”的中国专利申请的分案申请。
技术领域
本发明属于生物医药领域,具体而言,涉及一种多肽混合物高效液相色谱分析方法。
背景技术
醋酸格拉替雷(Glatiramer acetate,缩写为GA)是一种人工合成多肽类混合物(相对分子量:4700-11000道尔顿),由L-丙氨酸、L-谷氨酸、L-酪氨酸和L-赖氨酸四个氨基酸所组成的随机聚合物,长为45-100个氨基酸。各氨基酸摩尔比大约为0.392-0.462,0.129-0.153,0.086-0.100,0.300-0.374。该药物由以色列药厂梯瓦制药(TEVA)所研发制造,商品名为于1996年获美国FDA核准用于治疗多发性硬化症,目前有注射用水针和注射用冻干粉针两种产品,均用于皮下注射。
醋酸格拉替雷是一个连续性很强的共聚物,其结构如下式所示:
(Glu,Ala,Lys,Tyr)x.xCH3COOH
(C5H9NO4.C3H7NO2.C6H14N2O2.C9H11NO3)x.
xC2H4O2
CAS-147245-92-9
对于仿制该药厂商,只能基于该药的一些固有性质来考察仿制醋酸格拉替雷与对照制剂醋酸格拉替雷的组分差异。
本发明基于本品在不同缓冲溶液中所带电荷、极性、非极情况,采用阴离子交换色谱、阳离子交换色谱、反相色谱,进行阶梯式梯度洗脱的方式对比分析试制样品与市售商品制剂(对照品)的差异。
基于醋酸格拉替雷是一个连续性很强的共聚物,很难用一种分离方法弄清各个组分。现有的分析方法基于组分间分子量差异用体积排阻法进行简单分离分析(将单一峰分为若干组分收集后对其组分再进行分析),可见文献方法较为繁琐。开发能有效分离醋酸格拉替雷各组分的分析方法是非常有必要的。
发明内容
本发明首先涉及一种采用高效液相色谱法对醋酸格拉替雷进行线性或阶梯式梯度洗脱,并分析该共聚物各组分含量是否合格的方法。
该方法包括如下步骤,
步骤(1)配置待测醋酸格拉替雷溶液;
步骤(2)以阴离子交换液相色谱法、阳离子交换液相色谱法或反相液色谱法,对待测样本进行梯度洗脱,
步骤(3)检测分析共聚物各组分对应峰面积,通过与对照品比对,确定待测样本各组分含量范围是否合格。
步骤(2)所述的洗脱梯度为
步骤(2)所述采用阴离子交换液相色谱法的色谱条件为:
采用羧基键合聚苯乙烯-二乙烯苯颗粒为填料的色谱柱,所述的色谱柱填料颗粒粒径为1.7-10μm;
以含10-50mM的三羟基氨基甲烷盐酸盐溶液为流动相A,所述流动相A用NaOH溶液调节pH值至10-12;
以含10-50mM三羟基氨基甲烷盐酸盐、0.5-1.5M氯化钠溶液为流动相B,所述流动相B用盐酸溶液调节pH值至8-10;
进样量5-50uL,样品浓度1-20mg/ml,
流速0.5-1.5mL/min,洗脱时间50-250min;
洗脱梯度为:
采用不同形式的洗脱梯度进行洗脱,
柱温25-50℃;
所述的洗脱梯度为:
总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱,前N-1步梯度中流动相A的比例由100%渐次降至50%,流动相B的比例由0渐次升至50%,第N步时流动相A比例为100%,流动相B为0;
优选的5≤N≤15,更优选的8≤N≤12,最优选的,N=10。
步骤(2)所述采用阳离子交换液相色谱法的色谱条件为:
采用叔铵基键合聚苯乙烯-二乙烯苯颗粒为填料的色谱柱,所述的色谱柱填料颗粒粒径为1.7-10μm;
以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA溶液为流动相A,用NaOH溶液调pH值至4-6;
以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA、1-2.5M的NaCl的溶液为流动相B,用NaOH溶液调pH值至5-7;
进样量5-50uL,样品浓度1-20mg/ml,
流速0.5-1.5mL/min,洗脱时间50-250min;
洗脱梯度为:
采用不同形式的洗脱梯度进行洗脱,
柱温25-50℃;
所述的洗脱梯度为:
总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱,前N-1步梯度中流动相A的比例由100%渐次降至0%,流动相B的比例由0渐次升至100%,第N步时流动相A比例为90%,流动相B为10%;
优选的5≤N≤15,更优选的8≤N≤12,最优选的,N=10。
步骤(2)所述采用反相液色谱法的色谱条件为:
采用C18、C12、C8、C4键合硅胶颗粒为填料的色谱柱,所述的色谱柱填料颗粒粒径为1.7-10μm;
以乙腈为流动相A;
以含30-80mM的硫酸铵的溶液为流动相B,用磷酸溶液调pH值至2-3;
或以0.1%三氟乙酸溶液作为流动相B;
进样量5-50uL,样品浓度1-20mg/ml,
流速0.5-1.5mL/min,洗脱时间50-250min;
洗脱梯度为:
采用不同形式的洗脱梯度进行洗脱,
柱温25-50℃;
所述的洗脱梯度为:
总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱,前N-1步梯度中流动相A的比例由5%渐次升至40%,流动相B的比例由95%渐次降至60%,第N步时流动相A比例为5%,流动相B为95%;
优选的5≤N≤15,更优选的8≤N≤12,最优选的,N=10。
步骤(3)所述的检测分析采用紫外检测器检测波长260-280nm;
采用荧光检测器时,其激发波长为230nm,发射波长为300nm。
附图说明
图1-1,实施例1检测样本色谱分析图。
图1-2,实施例2检测样本色谱分析图。
图1-3,实施例3检测样本色谱分析图。
图1-4,实施例4检测样本色谱分析图。
图1-5,实施例5检测样本色谱分析图。
图2-1,实施例7检测样本色谱分析图。
图2-2,实施例8检测样本色谱分析图。
图2-3,实施例9检测样本色谱分析图。
图2-4,实施例10检测样本色谱分析图。
图3-1,实施例12检测样本色谱分析图。
图3-2,实施例13检测样本色谱分析图。
图3-3,实施例14检测样本色谱分析图。
图3-4,实施例15检测样本色谱分析图。
图3-5,实施例16检测样本色谱分析图。
具体实施方式
实施例1
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸150mm长,4.6mm直径,色谱柱填料为聚苯乙烯-二乙烯苯颗粒,3μm粒径。流动相A是20mM的三羟基氨基甲烷盐酸盐,用NaOH溶液调pH值至11.2;流动相B是20mM的三羟基氨基甲烷盐酸盐,含1M的NaCl,用盐酸溶液调pH值至9.8,流动相A和B的比例依据下表1进行设置。分别取市售醋酸格拉替雷(下称对照品)、试制样品醋酸格拉替雷(下称试制样品),加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为0.8mL/min,柱温采用30℃。其各组分比例见表2,某一样品色谱分析图见附图1-1。
表1是实施例1流动相A与B的比例
表2是3批对照品与3批试制样品阴离子交换各组成分析比对结果
实施例2
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸150mm长,4.6mm直径,色谱柱填料为聚苯乙烯-二乙烯苯颗粒,5μm粒径。流动相A是10mM的三羟基氨基甲烷盐酸盐,用NaOH溶液调pH值至10;流动相B是10mM的三羟基氨基甲烷盐酸盐,含1.5M的NaCl,用盐酸溶液调pH值至8,流动相A和B的比例依据表1进行设置。分别取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量15μL,流速设为0.5mL/min,柱温采用25℃。某一样品色谱分析图见附图1-2。
实施例3
使用沃特世2695高效液相色谱仪2489紫外多波长检测器,波长为275nm,色谱柱尺寸150mm长,4.6mm直径,色谱柱填料为聚苯乙烯-二乙烯苯颗粒,10μm粒径。流动相A是50mM的三羟基氨基甲烷盐酸盐,用NaOH溶液调pH值至12;流动相B是50mM的三羟基氨基甲烷盐酸盐,含0.5M的NaCl,用盐酸溶液调pH值至10,流动相A和B的比例依据表1进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约10mg的溶液,供测试。进样量50μL,流速设为1.5mL/min,柱温采用50℃。某一样品色谱分析图见附图1-3
实施例4
使用沃特世2695高效液相色谱仪2489紫外多波长检测器,波长为275nm,色谱柱尺寸150mm长,4.6mm直径,色谱柱填料为聚苯乙烯-二乙烯苯颗粒,3μm粒径。流动相A是30mM的三羟基氨基甲烷盐酸盐,用NaOH溶液调pH值至10;流动相B是30mM的三羟基氨基甲烷盐酸盐,含1M的NaCl,用盐酸溶液调pH值至8,流动相A和B的比例依据下表3进行设置。分别取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为0.8mL/min,柱温采用40℃。某一样品色谱分析图见附图1-4。
表3实施例4流动相A与B的比例
时间(min) | 流动相A(%) | 流动相B(%) |
0 | 100 | 0 |
15 | 95 | 5 |
30 | 92.5 | 7.5 |
45 | 90 | 10 |
60 | 87.5 | 12.5 |
75 | 85 | 15 |
90 | 80 | 20 |
105 | 70 | 30 |
120 | 50 | 50 |
135 | 100 | 0 |
150 | 100 | 0 |
实施例5
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸150mm长,4.6mm直径,色谱柱填料为聚苯乙烯-二乙烯苯颗粒,3μm粒径。流动相A是20mM的三羟基氨基甲烷盐酸盐,用NaOH溶液调pH值至11.2;流动相B是20mM的三羟基氨基甲烷盐酸盐,含1M的NaCl,用盐酸溶液调pH值至9.8,流动相A和B的比例依据下表4进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为1mL/min,柱温采用30℃。某一样品色谱分析图见附图1-5,可见,在该洗脱条件下,不能对样品的组分进行有效分离。
表4.实施例5流动相A与B的比例
时间(min) | 流动相A(%) | 流动相B(%) |
0 | 100 | 0 |
10 | 100 | 0 |
30 | 0 | 100 |
40 | 0 | 100 |
41 | 100 | 0 |
55 | 100 | 0 |
实施例6
使用沃特世2695高效液相色谱仪2489紫外多波长检测器,波长为275nm,色谱柱尺寸150mm长,4.6mm直径,色谱柱填料为聚苯乙烯-二乙烯苯颗粒,3μm粒径。流动相A是20mM的三羟基氨基甲烷盐酸盐,用NaOH溶液调pH值至11.2;流动相B是20mM的三羟基氨基甲烷盐酸盐,含1M的NaCl,用盐酸溶液调pH值至9.8,流动相A和B的比例依据下表4进行设置。分别取对照品、试制样品,加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为0.8mL/min,柱温采用30℃。其各组分比例见表6。
表5是实施例6流动相A与B的比例
表6是3批对照品与3批试制样品阴离子交换各组成分析比对结果
阳离子交换液相色谱(实施例7-11)
实施例7
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒,5μm粒径。流动相A是20mM的2-(N-吗啉代)乙磺酸,含1mM EDTA,用NaOH溶液调pH值至5.2;流动相B是20mM的2-(N-吗啉代)乙磺酸,含2mM EDTA,含2M的NaCl,用NaOH溶液调pH值至5.8,流动相A和B的比例依据下表7进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为1mL/min,柱温采用30℃。某一样品色谱分析图见附图2-1。
表7是实施例7流动相A与B的比例
时间(min) | 流动相A(%) | 流动相B(%) |
0 | 90 | 10 |
15 | 90 | 10 |
15.1 | 80 | 20 |
30 | 80 | 20 |
30.1 | 70 | 30 |
45 | 70 | 30 |
45.1 | 60 | 40 |
60 | 60 | 40 |
60.1 | 50 | 50 |
75 | 50 | 50 |
75.1 | 40 | 60 |
90 | 40 | 60 |
90.1 | 30 | 70 |
105 | 30 | 70 |
105.1 | 20 | 80 |
120 | 20 | 80 |
120.1 | 0 | 100 |
135 | 0 | 100 |
135.1 | 90 | 10 |
150 | 90 | 10 |
表8是3批对照品与3批试制样品阳离子交换各组成分析比对结果
实施例8
使用沃特世2695高效液相色谱仪2489紫外多波长检测器,波长为275nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒,10μm粒径。流动相A是10mM的2-(N-吗啉代)乙磺酸,含0.5mM EDTA,用NaOH溶液调pH值至4;流动相B是10mM的2-(N-吗啉代)乙磺酸,含5mM EDTA,含2M的NaCl,用NaOH溶液调pH值至7,流动相A和B的比例依据表7进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量15μL,流速设为1mL/min,柱温采用25℃。某一样品色谱分析图见附图2-2
实施例9
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒,5μm粒径。流动相A是50mM的2-(N-吗啉代)乙磺酸,含1mM EDTA,用NaOH溶液调pH值至6;流动相B是50mM的2-(N-吗啉代)乙磺酸,含2mM EDTA,2M的NaCl,用NaOH溶液调pH值至5,流动相A和B的比例依据下表7进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约10mg的溶液,供测试。进样量50μL,流速设为1mL/min,柱温采用30℃。某一样品色谱分析图见附图2-3。
实施例10
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒,5μm粒径。流动相A是20mM的2-(N-吗啉代)乙磺酸,含1mM EDTA,用NaOH溶液调pH值至5.2;流动相B是20mM的2-(N-吗啉代)乙磺酸,含2mM EDTA,2M的NaCl,用NaOH溶液调pH值至5.8,流动相A和B的比例依据下表9进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为1mL/min,柱温采用30℃。某一样品色谱分析图见附图2-4,可见,在该洗脱条件下,不能对样品的组分进行有效分离。
表9实施例10流动相A与B的比例
实施例11
使用沃特世2695高效液相色谱仪2489紫外多波长检测器,波长为275nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为叔铵基基键合聚苯乙烯-二乙烯苯颗粒,5μm粒径。流动相A是30mM的2-(N-吗啉代)乙磺酸,含3mM EDTA,用NaOH溶液调pH值至5;流动相B是10mM的2-(N-吗啉代)乙磺酸,含2mM EDTA,含2M的NaCl,用NaOH溶液调pH值至6,流动相A和B的比例依据下表10进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为1mL/min,柱温采用35℃。其各组分比例见表11。
表10是实施例11流动相A与B的比例
表11是3批对照品与3批试制样品阳离子交换各组成分析比对结果
反相液相色谱案例(实施例12-17)
实施例12
使用安捷伦1260高效液相色谱仪紫外多波长检测器,检测波长设为275nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为硅胶基体键合C18配基颗粒,3μm粒径。流动相A是乙腈;流动相B是50mM硫酸铵溶液,用磷酸溶液调pH值至2.5,流动相A与B的比例按下表12进行设置。分别取对照品和试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为1mL/min,柱温采用30℃。其对照品与试制样品个组分比对分析见下表13,某一样品色谱分析图见附图3-1。
表12是实施例12流动相A与B的比例:
表13是3批对照品与3批试制样品反相各组分分析比对结果
实施例13
使用安捷伦1260高效液相色谱仪紫外多波长检测器,检测波长设为275nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为硅胶基体键合C8配基颗粒,10μm粒径。流动相A是乙腈;流动相B是0.1%三氟乙酸溶液,动相A与B的比例按表12进行设置。分别取对照品和试制样品加流动相溶解并制成每1mL含约10mg的溶液,供测试。进样量50μL,流速设为1mL/min,柱温采用50℃。其某一样品色谱分析图见附图3-2。
实施例14
使用安捷伦1260高效液相色谱仪紫外多波长检测器,检测波长设为275nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为硅胶基体键合C4配基颗粒,3μm粒径。流动相A是乙腈;流动相B是30mM硫酸铵溶液,用磷酸溶液调pH值至2,流动相A与B的比例按表12进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量15μL,流速设为0.5mL/min,柱温采用25℃。某一样品色谱分析图见附图3-3。
实施例15
使用安捷伦1260高效液相色谱仪紫外多波长检测器,检测波长设为275nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为硅胶基体键合C4配基颗粒,3μm粒径。流动相A是乙腈;流动相B是80mM硫酸铵溶液,用磷酸溶液调pH值至3,流动相A与B的比例按表12进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量25μL,流速设为0.5mL/min,柱温采用50℃。某一样品色谱分析图见附图3-4。
实施案16
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为硅胶基体键合C18配基颗粒,3μm粒径。流动相A是乙腈;流动相B是0.1%三氟乙酸溶液,动相A与B的比例按下表14进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量50μL,流速设为1mL/min,柱温采用30℃。某一样品色谱分析图见附图3-5,可见,常规方法完全无法分离试制样本中的各个成分。
表14实施例16流动相A与B的比例
实施案17
使用沃特世2695高效液相色谱仪2475荧光多波长检测器,激发波长(Ex)为230nm,发射波长(Em)为300nm,色谱柱尺寸250mm长,4.6mm直径,色谱柱填料为硅胶基体键合C18配基颗粒,3μm粒径。流动相A是乙腈;流动相B是0.1%三氟乙酸溶液,动相A与B的比例按下表15进行设置。取对照品或试制样品加流动相溶解并制成每1mL含约20mg的溶液,供测试。进样量50μL,流速设为1.5mL/min,柱温采用30℃。其各组分比例见表16。
表15是实施例17流动相A与B的比例
时间(min) | 流动相A(%) | 流动相B(%) |
0 | 100 | 0 |
15 | 100 | 0 |
15.1 | 90 | 10 |
30 | 90 | 10 |
30.1 | 80 | 20 |
45 | 80 | 20 |
45.1 | 70 | 30 |
60 | 70 | 30 |
60.1 | 60 | 40 |
75 | 60 | 40 |
75.1 | 55 | 45 |
85 | 55 | 45 |
85.1 | 50 | 50 |
100 | 50 | 50 |
100.1 | 100 | 0 |
120 | 100 | 0 |
表16是3批对照品与3批试制样品反相各组分分析比对结果
由以上实施例可知,本发明用于分析醋酸格拉替雷样本中各个组分的含量的方法是有效的,可行的,可以用于醋酸格拉替雷的对比分析与质量一致性研究。
最后需要说明的是,以上实施例仅用于帮助本领域技术人员理解本发明的实质,而不用做对本发明保护范围的限制。
Claims (3)
1.一种采用高效液相色谱法对醋酸格拉替雷进行线性或阶梯式梯度洗脱,并分析该共聚物各组分含量是否合格的方法,其特征在于,该方法包括如下步骤,
步骤(1)配置待测醋酸格拉替雷溶液;
步骤(2)以阳离子交换液相色谱法,对待测样本进行梯度洗脱,
步骤(3)检测分析共聚物各组分对应峰面积,通过与对照品比对,确定待测样本各组分含量范围是否合格。
2.根据权利要求1所述的方法,其特征在于,
步骤(2)所述采用阳离子交换液相色谱法的色谱条件为:
采用叔铵基键合聚苯乙烯-二乙烯苯颗粒为填料的色谱柱,所述的色谱柱填料颗粒粒径为1.7-10μm;
以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA溶液为流动相A,用NaOH溶液调pH值至4-6;
以含10-50mM的2-(N-吗啉代)乙磺酸、0.5-5mM的EDTA、1-2.5M的NaCl的溶液为流动相B,用NaOH溶液调pH至5-7;
进样量5-50uL,样品浓度1-20mg/ml,
流速0.5-1.5mL/min,洗脱时间50-250min;
洗脱梯度为:
总洗脱时间等分为N(2≤N≤20)步依次进行梯度洗脱,前N-1步梯度中流动相A的比例由100%渐次降至0%,流动相B的比例由0渐次升至100%,第N步时流动相A比例为90%,流动相B为10%;
优选的,5≤N≤15,更优选的,8≤N≤12,最优选的,N=10;
柱温25-50℃。
3.根据权利要求1-2任一所述的方法,其特征在于,
步骤(3)所述的检测分析采用紫外检测器检测波长260-280nm;
采用荧光检测器时,其激发波长为230nm,发射波长为300nm。
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