Gabrielson John P, Brader Mark L, Pekar Allen H, Mathis Kathrin B, Winter Gerhard, Carpenter John F, Randolph Theodore W
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, USA.
J Pharm Sci. 2007 Feb;96(2):268-79. doi: 10.1002/jps.20760.
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.
尺寸排阻高效液相色谱法（SE-HPLC，即SEC）一直是生物制药行业用于定量可溶性蛋白质聚集体的标准方法。最近，沉降速度分析超离心法（SV-AUC）已成为一种可与SEC互补用于定量可溶性聚集体的技术。此外，不对称流场流分馏法（AF4）在定量可溶性和不溶性蛋白质聚集体方面已初显成效。我们报告了通过SEDFIT/c(s)分析的SEC、AF4和SV-AUC测量的重组人源化单克隆抗体酸性应激和非应激样品中的可溶性聚集体数量。在等效抗体样品中，SV-AUC和AF4检测到的总聚集体水平明显高于SEC。此外，SEC未能检测到SV-AUC和AF4分析中明显存在的较高分子量可溶性聚集体。对含有可溶性二聚体聚集体的合并馏分进行纯化，并通过SV-AUC和SEC重新分析。将纯化的二聚体重新注入SEC柱会诱导形成可检测量的单体和三聚体。所有样品类型通过SEC柱都显示出具有统计学意义（p值<0.01）的抗体损失。SEC中这种不完全的质量回收表明抗体可能物理吸附到凝胶过滤介质上。对高分子量组分沉降行为的分析表明，随着分子量增加，分子非球形度增加。我们提出了一种基于单体亚基几乎线性端对端组装的聚集模型，该模型与SV-AUC、SEC、AF4和动态光散射（DLS）结果一致。
