大小排阻色谱法
化学
色谱法
超离心机
分馏
分析超速离心
单克隆抗体
沉降平衡
高效液相色谱法
场流分馏
凝胶渗透色谱法
分子质量
沉积作用
抗体
聚合物
生物化学
生物
古生物学
有机化学
酶
免疫学
沉积物
作者
John P. Gabrielson,Mark L. Brader,Allen H. Pekar,Kathrin B. Mathis,Gerhard Winter,John F. Carpenter,Theodore W. Randolph
摘要
Size-exclusion high-performance liquid chromatography (SE-HPLC, SEC) is the long-standing biopharmaceutical industry standard for quantitation of soluble protein aggregates. Recently, sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged as a possible orthogonal technique to SEC for soluble aggregate quantitation. Moreover, asymmetrical flow field flow fractionation (AF4) has shown early promise in quantifying protein aggregates, both soluble and insoluble. We report soluble aggreg ate quantities measured by SEC, AF4, and SV-AUC analyzed by SEDFIT/c(s) for acid stressed and unstressed samples of a recombinant humanized monoclonal antibody. In equivalent antibody samples, SV-AUC, and AF4 detect markedly higher total aggregate levels than SEC. Furthermore, SEC fails to detect higher molecular weight soluble aggregates apparent in SV-AUC and AF4 analyses. Pooled fractions containing soluble dimeric aggregates were purified and re-analyzed by both SV-AUC and SEC. Reinjection of purified dimer onto the SEC column induces formation of detectable quantities of monomer and trimer. All sample types show statistically significant (p-values<0.01) antibody losses through the SEC column. This incomplete mass recovery from SEC indicates probable antibody physical adsorption to gel filtration media. Analysis of the sedimentation behavior of high molecular weight components suggests increased molecular asphericity with increasing molecular weight. We present an aggregation model based on nearly linear end-to-end assembly of monomeric subunits which is shown to be consistent with SV-AUC, SEC, AF4, and dynamic light scattering (DLS) results.
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