重组Ⅲ型人源化胶原蛋白关键质量参数分析方法建立

Establishment of Critical Quality Attributes Analysis Method for Recombinant Human CollagenⅢ

目的针对重组Ⅲ型人源化胶原蛋白的关键质量参数进行检测方法研究。方法采用基质辅助激光解吸/飞行时间质谱(matrix-assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF MS)及尺寸排阻色谱与多角度光散射联用(size exclusion chromatography with multi-angle light scattering,SEC-MALS)法测定相对分子质量及其分布;采用毛细管凝胶电泳(capillary electrophoresis sodium dodecyl sulfate,CE-SDS)和分子排阻高效液相色谱(size exclusion high performance liquid chromatography,SE-HPLC)分析纯度;采用离子交换法和毛细管区带电泳(capillary zone electrophoresis,CZE)分离分析电荷异构体;采用“双”酶切肽图法进行一级结构鉴别试验和序列覆盖率分析。结果MALDI-TOF MS测得相对分子质量45.01×10^(3);SEC-MALS得到其单体相对分子质量为45.17×10^(3)(±0.226%);两种相对分子质量测定方法结果一致。CE-SDS(非还原)纯度为98.77%,其他成分1.23%;SE-HPLC纯度为98.07%,其他成分1.93%;两种纯度测定方法结果一致。强阳离子交换共鉴定到14种电荷异构体组分,其中酸性峰占比52.08%,主峰占比26.22%,碱性峰占比21.70%;CZE鉴定到8种电荷异构体组分,其中酸性峰占比52.10%,主峰占比25.27%,碱性峰占比22.63%;两种电荷异构体鉴别方法,酸性组分、主峰与碱性组分,分布规律一致。“双”酶切肽图法实现重组Ⅲ型人源化胶原蛋白的序列100%覆盖,实测序列与预期序列一致。结论建立了重组Ⅲ型人源化胶原蛋白关键质量参数分析方法,该方法具有保证产品安全、有效、质量可控的特点,为我国重组Ⅲ型人源化胶原蛋白的质量控制提供了参考依据。

OBJECTIVE To establish critical quality attributes analysis method for recombinant human collagenⅢ.METHODS The recombinant human collagenⅢmolecular weight(MW)distribution was determined by matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS)and size exclusion chromatography with multi-angle light scattering(SEC-MALS).Capillary electrophoresis-sodium dodecyl sulfate(CE-SDS)and size exclusion high performance liquid chromatography(SE-HPLC)were used for purity analysis.Ion exchange chromatography and capillary zone electrophoresis(CZE)were used to separate and analyze charge isomers.The“double”enzyme digestion peptide mapping method was used for identification and sequence coverage analysis.RESULTS The recombinant human collagenⅢMW distribution measured by MALDI-TOF was 45.01×10^(3).The MW of monomer recombinant human collagenⅢdetermined by SEC-MALS was 45.17×10^(3)(±0.226%).CE-SDS analysis showed that the purity of recombinant human collagenⅢwas 98.77%and that of other ingredients was 1.23%.The SE-HPLC purity was 98.07%,and that of other component was 1.93%(dimer).A total of 14 charge isomers were identified through strong cation exchange,and the acidic peak was 52.08%,the main peak was 26.22%,and the basic peak was 21.70%.Eight charge isomers were identified by CZE,including 52.10%acidic peak,25.27%main peak,and 22.63%basic peak.Two identification methods for charge isomers have consistent distribution patterns.The“double”enzyme digestion peptide mapping method was used for identification and sequence coverage analysis,achieving 100%coverage of the recombinant human collagenⅢsequence.CONCLUSION A series of critical quality attributes analysis methods for recombinant human collagenⅢhave been established.These methods provide a reference basis for the quality control of recombinant human collagenⅢin China.