近年来,全球生物制药发展异常迅猛,新的生物药分析检测技术亦层出不穷,SPR技术作为其中一员,凭借着其突出的准确性、稳定性和高重复性,在2016年被正式收录到美国和日本的药典。小编怀着强烈的好奇心,在“SPR技术知识”的海洋中游走多天,终于捞到了一点点干货,给大家分享一下。
Q1:什么是SPR?
SPR(Surface plasmon resonance),被称为表面等离子体共振,又称表面等离激元共振,本质上是一种物理光学现象。
Q2:SPR技术检测原理是什么?
首先,回顾几个物理概念:
等离子体:通常指由密度相当高的自由正、负电荷组成的气体,其中正、负带电粒子数目几乎相等,对外呈现电中性,是物质存在的一种状态,与固态、液态和气态并列,成为物质的第四态,宇宙中大部分物质处于等离子状态。
金属表面等离子波:把金属的价电子看成是均匀正电荷背景下运动的电子气体,这实际上也是一种等离子体。因为金属中的价电子可以自由移动,入射光可能激起电子气的纵向振动,振动产生的电荷密度波,沿着金属和电介质的界面传播,形成表面等离子波。
消逝波:当以波动光学的角度来研究全反射时,人们发现当入射光到达界面时并不是直接产生反射光,而是先透过光疏介质约一个波长的深度,再沿界面流动约半个波长再返回光密介质,而光的总能量没有发生改变,则透过光疏介质的波被称为消逝波(见下图)。
现在,我们来看看SPR的检测原理:
当光源发出的P偏振光(电磁波)以一定的角度入射到棱镜中,在棱镜与金属的界面处将发生反射和折射,当入射角大于临界角时,光线将发生全内反射,在全内反射的情况下,电场在金属与棱镜的界面处并不立即消失,而是向金属介质中传输振幅呈指数衰减的消逝波,同时引发金属中的自由电子产生表面等离子波,当金属表面等离子波与消逝波发生共振时,检测到的反射光强度会大幅度地减弱,能量从光子转移到表面等离子,入射光的大部分能量被金属表面等离子波吸收,使得反射光的能量急剧减少。当入射光波长固定时,反射光强度是入射角的函数,其中反射光强度最低时所对应的入射角为共振角(Resonance Angle)。表面等离子共振(SPR)对附着在金属薄膜表面的介质折射率非常敏感,当表面介质的属性改变或者附着量改变时,共振角将不同。因此,SPR谱(共振角的变化VS时间)能够反映与金属膜表面接触的体系的变化。
上图中展示的是SPR芯片工作原理, 这里的SPR芯片指的是一个带有葡聚糖(Dextran)的金属表面(Mental Surface),而配体蛋白(Ligand)的氨基端可以与葡聚糖结合从而被固定到金属表面。下方光源发出的一个单波长激光束进入到棱镜(Prism)中,导致多角度的光线入射到金属表面,几乎所有入射的光线都会发生反射,但有一个例外,在入射角达到某一个角度时,光子的能量会被金属吸收转化成表面等离子体波,在这个角度没有光线被反射出来,而是以很小的强度被检测器检测到,这个角度被称为共振角。由于等离子体波会在金属表面传播,所以金属表面偶联的配体蛋白与任何物质发生相互作用都将导致共振角发生改变。
这两张图是对SPR芯片工作原理的进一步解释:A图中有一偶联了抗原的芯片,当样本流过芯片,抗体与抗原结合,导致样本通道(Sample Channel 2)的共振角发生改变,而对照通道(Reference Channel 1,是没有偶联抗原的对照表面)共振角不发生改变,此时利用这两个通道共振角之差(Channel 2-Channel 1)绘制出的共振单位RU(Resonance Units)随时间变化的曲线是一个向上的曲线,当样本被洗去且抗体开始从抗原结合处解离下来,此时出现的传感曲线是一个向下的曲线。B图显示的是结合多个蛋白的情况,当抗体结合到抗原上时出现的传感曲线是一个向上的曲线,之后如果有另一个包含抗体的受体的样本流过芯片,则会在上一个曲线基础上向上形成另一个曲线。
Q3:SPR技术的发展历程是怎样的?
1902年Wood在光学实验中发现SPR现象;
1941年Fano解释了SPR现象;
1971年Kretschmann为SPR传感器结构奠定了基础;
1983年Liedberg将SPR用于IgG与其抗原的反应测定;
1987年Knoll等人开始SPR成像研究;
1990年Biacore AB公司开发出首台商品化SPR仪器;
2016年SPR技术被正式收录到美国和日本药典。
Q4:SPR技术有哪些用途?
SPR技术可用于生物分子间结合特异性的分析、浓度定量、结合动力学和亲和力分析以及热力学分析,能够实时检测DNA与蛋白质之间、蛋白质分子之间、药物与蛋白之间、核酸与核酸之间、抗原与抗体之间以及受体与配体等生物分子之间的相互作用。
Q5:如何使用SPR技术?
2016年SPR技术被正式收录到美国和日本药典后,越来越多的被应用到生物药的研发和质控中。近期发表于Biomaterials上的一篇关于改造Fc片段作为蛋白药物载体从而优化蛋白药物给药途径的文章中,SPR技术被用于对比FcRn(Cat#FCM-H5286, ACROBiosystems, Newark,DE)与改造前Fc片段和改造后Fc片段结合的动力学差异,具体实验流程及结果如下:
器材:Biacore T100 & CM5传感器芯片(GE Healthcare, Pittsburgh, PA ).
主要试剂: soluble human Neonatal Fc-receptor (FcRn, Cat#FCM-H5286, ACROBiosystems, Newark, DE).
简易流程:
1. 芯片表面预处理:利用共价结合将FcRn蛋白偶联在羧基化处理的葡聚糖芯片(CM5)表面。偶联方法分很多种,包括直接偶联(氨基偶联、巯基偶联、醛偶联等)和捕获(亲和素—生物素捕获系统、NTA—6His捕获系统等)两大类。此实验选择氨基共价偶联:第一步芯片活化,pH 4.5-5条件下使用交联剂EDC/NHS使芯片表面酯化;第二步配体偶联(配体蛋白纯度要求在90%以上,用量以30-50KD蛋白为例,需准备蛋白浓度10-50ug/ml,1ml左右,随着蛋白性质的不同和分子量的变化可以调整),将购自ACROBiosystems的FcRn蛋白偶联到CM5芯片表面(酯化后的芯片表面酯基和FcRn蛋白上的氨基发生反应);第三步封闭,用1M乙醇胺盐酸盐(PH8.5)封闭芯片上多余的有活性的羧基。此次实验FcRn最终偶联水平为~9000RU(Response Units)。
对照表面的设计:CM5芯片分1,2,3,4四个Flow Cell通道,一般1,2通道配对使用(1通道作为参比),3,4通道配对使用(3通道作为参比),参比通道对照表面可设计成空白表面、活化-封闭无配体固定的表面或偶联了伪装配体的表面。此次实验参比通道对照表面选择活化-封闭无配体固定的方式来做削减对照,当对进样过程进行分析时,对照表面的削减可用来消除因样品溶解缓冲液与仪器运行缓冲液成分不同所造成的容积差及一些非特异性结合信号。
2. 进样:首先用运行缓冲液(50 mM phosphate, 100 mM sodium chloride, and 0.01% vol/vol Tween 20, pH 6.0 or 7.4)稀释样本,此实验样本有recombinant Fc (重组Fc蛋白), sc(Fc)2 (单链Fc二聚体), hGH-Fc(人生长激素—Fc融合蛋白) 和 hGH-sc(Fc)2 (人生长激素—单链Fc二聚体融合蛋白) 四种。进样时间为2min(重组Fc蛋白组为1min除外),进样流速20ul/min, 蛋白解离时间为2.5min。
3. 芯片再生:残留蛋白用再生缓冲液(10 mM HEPES, 150 mM NaCl, and 0.01% vol/vol Tween 20, pH 7.4)洗脱下来,时间为30sec,流速设为30ul/min。
4. 数据分析:Biacore T100评价软件(version2.0.2)分析生成传感图。
SPR传感图结果展示:
上图是PH6.0条件下,连续两倍稀释法稀释的四种待测样本分别与CM5芯片上偶联的FcRn(Cat#FCM-H5286, ACROBiosystems, Newark, DE)结合的实时动态分析结果图,图中对应样本分别是:(A) Fc (稀释梯度为400 nM to 6.25nM), (B) sc(Fc)2 (稀释梯度为3,200 nM to 6.25 nM), (C) hGH- sc(Fc)2(稀释梯度为6,400 nM to 50 nM), and (D)hGH-Fc(稀释梯度为6,400 nM to 6.25 nM)。
除了上文中提到的应用外,SPR技术在EMA和FDA批准上市的药物中也有着广泛的应用,比如西妥昔单抗(Cetuximab)药物质控中,SPR技术被用于药物分子的亲和力检测和血药浓度测定[6],再比如IL-17单抗(Ixekizumab)药物研发中,SPR技术被用于检测药物分子与靶点IL-17A的亲和力和动力学数据,用于对比药物分子与同源二聚体IL-17A和异源二聚体IL-17A/F结合的动力学差异[7]。现如今SPR技术的应用不胜枚举,尤其在新药和生物类似药研发的领域中得到广泛认可。
参考文献:
[1] Sabban, Sari. Development of an invitro model system for studying the interaction of Equus caballus IgE with itshigh- affinity FcεRI receptor (PhD thesis), The University of Sheffield, 2011.
[2] Liedberg, Bo, et al. Surface plasmonresonance for gas detection and biosensing. Sensors and Actuators. 4(1983):299–304, doi:10.1016/0250-6874(83)85036-7.
[3] Zhou L, et al. Single chain Fc-dimer-humangrowth hormone fusion protein for improved drug delivery, Biomaterials (2016),doi:10.1016/j.biomaterials.2016.11.051.
[4] A.R. Mezo, et al. Reduction of IgGin nonhumanprimates by apeptide antagonist of the neonatal Fc receptor FcRn, Proceedings of theNational Academy of Sciences, 105 (2008) 2337-2342.
[5] 程慧,黄朝峰。SPR生物传感器及其应用进展。中国生物工程杂志。2003,23(5):46-49.
[6] Erbitux, INN-Cetuximab – EuropeanMedicines Agency 2004 (http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000558/WC500029113.pdf)
[7] Ling liu, et al. Generation andcharacterization of ixekizumab,a humanized monoclonal antibody that neutralizesinterleukin-17A. J Inflamm Res. 2016; 9: 39-50. doi: 10.2147/JIR.S100940.
[8] 美国药典USP39 General Information/(1105)Immunological Test Methods- Surface Plasmon Resonance章节。
[9] 日本药典2016版JP17 General Information/ Biotechnological/Biological Products-Surface Plasmon Resonance,Page2474-2478页。
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이 경우 Xie Qian은 “명령에 복종합니다! “라고 계속 반대 할 것이 없습니다.
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