重组乳液复杂体系中同类蛋白质分离纯化的科学基础

Mammary gland bioreactor could express effectively recombinant pharmaceutical proteins with low cost and non-pollution, owing broad application prospects. However, there still are some challenges in purification of recombinant proteins. The recombinant proteins tend to aggregate with casein micelles in milk, which induce significant loss of itself in phase separation. Furthermore, transgenic milk contains not only recombinatnt protein but also its animal homologous counterpart, which are very difficult to separate from each other because of extremely resemblance in properties. This program focuses on these challenges. On one hand, taking four recombinant milks which contain, respectively, recombinant human α-lactalbumin, lactoferrin, CD20 antibody and antithrombin III as models, we'll study the mechanism of aggregation between recombinant proteins and casein micelles using dual polarization interferometry (DPI) and liquid chromatography combined with mass spectrum (LC-MS). The results could reveal the physical adsorption driving force and aggregation site, determinate the kinetic parameters and finally provide theoretical support for novel phase separation strategies. On the other hand, based on analysis of difference in properties and species-sequence between homologous proteins, we will develop advanced chromatographic theory and design novel chromatographic resin (including species affinity chromatography and code affinity chromatography), developing a foundation for separation of homologous proteins.

乳腺生物反应器可以高效表达重组蛋白药物，成本低，无污染，具有广阔的应用前景，然而重组蛋白的分离纯化还存在一些挑战。重组蛋白容易与乳液中的酪蛋白胶束发生聚集反应，导致自身在相分离过程中大量损失；此外，重组乳液中存在与重组蛋白同类的动物蛋白，两者性质极其接近，分离难度很大。本项目针对这些挑战进行了研究。一方面，以4种重组动物乳液（分别表达重组人乳清白蛋白、乳铁蛋白、CD20抗体和抗凝血酶III）为模型，通过双偏振极化干涉和液质联用等技术研究重组蛋白与酪蛋白胶束之间的聚集反应机理，揭示重组蛋白/酪蛋白之间的物理吸附驱动力，确定聚集反应位点，计算聚集反应动力学参数，为构建新型相分离技术提供理论支持；另一方面，基于同类蛋白理化性质和种属特征序列分析，创新层析理论，设计新型亲和层析介质（包括种属亲和层析和密码亲和层析），为同类蛋白的分离奠定理论基础。

乳腺生物反应器可以高效表达重组蛋白药物，成本低，无污染，具有广阔的应用前景，然而重组蛋白的分离纯化还存在一些挑战。重组蛋白容易与乳液中的酪蛋白胶束发生聚集反应，导致自身在相分离过程中大量损失；此外，重组乳液中存在与重组蛋白同类的动物蛋白，两者性质极其接近，分离难度很大。本项目针对这些挑战进行了研究。一方面，以4种重组动物乳液（分别表达重组人乳清白蛋白、乳铁蛋白、CD20抗体和抗凝血酶III）为模型，研究重组蛋白与酪蛋白胶束之间的聚集反应机理，发现重组蛋白在预处理过程中的损失主要是由于疏水性的协同沉淀造成的，根据目标蛋白的疏水性制定了选择去除酪蛋白的方法，目标蛋白回收率高于85%；另一方面，基于同类蛋白理化性质和种属特征序列分析，分别使用离子交换层析、疏水层析和亲和层析分离了三对同类蛋白质，即同类乳铁蛋白、同类乳清白蛋白和同类抗体，目标蛋白中同类蛋白杂质少于5%。通过本项目的实施，为乳腺生物反应器表达产物中相分离奠定了理论基础，开发了分离同类蛋白质的理论和技术，从而建立了从乳腺生物反应器表达产物中分离纯化重组蛋白药物的策略。