Demeule Barthélemy, Shire Steven J, Liu Jun
Late Stage Pharmaceutical and Processing Development, Genentech, South San Francisco, CA 94080, USA.
Anal Biochem. 2009 May 15;388(2):279-87. doi: 10.1016/j.ab.2009.03.012. Epub 2009 Mar 14.
During the development of protein therapeutics, characterization of the active pharmaceutical ingredient is performed extensively to ensure the stability, safety, and efficacy of the drug. Little is known, however, about the characteristics of protein drugs circulating in the blood. The recent availability of a fluorescence detection system (FDS) in analytical ultracentrifugation (AUC) instruments enables the characterization of fluorescently labeled proteins in biological fluids. AUC provides information about protein size, shape, self-association, and binding while avoiding many limitations associated with size exclusion chromatography. Furthermore, with the specificity and sensitivity of FDS, measurements can be performed at physiological concentrations directly in serum. In the current study, we used omalizumab, an anti-immunoglobulin E (IgE) monoclonal antibody, to demonstrate the potential of using AUC-FDS for the study of a monoclonal antibody and its complexes directly in human serum. Omalizumab properties were essentially unaltered after labeling with the fluorescent dye Alexa Fluor 488. In addition, omalizumab and IgE formed different complexes in serum than in phosphate-buffered saline in terms of both size and affinity.
在蛋白质治疗药物的研发过程中，会对活性药物成分进行广泛表征，以确保药物的稳定性、安全性和有效性。然而，对于血液中循环的蛋白质药物的特性却知之甚少。分析超速离心（AUC）仪器中最近可用的荧光检测系统（FDS）能够对生物流体中的荧光标记蛋白质进行表征。AUC可提供有关蛋白质大小、形状、自缔合和结合的信息，同时避免了与尺寸排阻色谱相关的许多局限性。此外，凭借FDS的特异性和灵敏度，可以直接在血清中的生理浓度下进行测量。在本研究中，我们使用抗免疫球蛋白E（IgE）单克隆抗体奥马珠单抗来证明使用AUC-FDS直接在人血清中研究单克隆抗体及其复合物的潜力。用荧光染料Alexa Fluor 488标记后，奥马珠单抗的性质基本未改变。此外，就大小和亲和力而言，奥马珠单抗和IgE在血清中形成的复合物与在磷酸盐缓冲盐水中形成的不同。
