亲和仿生层析及在抗体纯化中的应用

亲和仿生层析是一种新型的生物分离技术,可用于生物活性物质分离,尤其在抗体纯化中表现出良好的应用前景,具有价格低廉、结构稳定、特异性较高等优点。亲和仿生层析的核心是具有特定结构的仿生配基,主要包括化学合成配基和短肽配基两种,通常通过合理的手段进行筛选和设计。理性设计是一种行之有效的方法,针对一个特定的目标蛋白,随机地去选择配基是不可能的,必须采用理性设计构建一个合理的配基库,并通过高通量的筛选技术,才能得到合适的仿生配基。本文根据近年来国内外亲和仿生层析的研究进展,着重介绍了亲和仿生配基的筛选和设计以及在抗体纯化中的应用。     

染料配基亲和色谱

一、前言染料配基亲和色谱是七十年代以来迅速发展起来的一种新型纯化生物活性物质的分离技术。亲和色谱是利用生物大分子的生物特异性和生物分子间所具有的专一性亲和力而设计的层析技术。生物体内的生物分子间,如酶与底物,酶与抑制剂,酶与辅酶,抗原与抗体等生物大分子和生物小分子配体间有一种特殊的亲和力,在一定条件下它们能紧密地结合成可逆的复合物。利用这种可逆化合物的结合与解离的原理而建立起来的新型纯化分离技术的     

接枝聚合物配基的蛋白质吸附层析

以离子交换、亲和结合和疏水性吸附为主的蛋白质吸附层析是药用蛋白质生产过程的核心技术,开发新技术和提高蛋白质吸附层析操作的分离效率(如选择性和动态吸附容量等)是该领域的主要研究目标。近年来聚合物配基接枝的层析介质由于同时具有较高的吸附容量和传质速率,得到产学界的广泛关注。本文针对聚合物配基接枝修饰的蛋白质吸附层析介质的配基化学特征、吸附和传质特性、层析分离应用和设计等方面进行评述。首先介绍不同种类的聚合物接枝介质,然后系统阐述聚合物配基化学特性对介质吸附和传质性能的影响机制,并分析上述性质对聚合物配基接枝层析介质分离特性的影响机理和应用,最后讨论和展望了高效聚合物配基接枝介质设计、开发和应用的前景。     

金属螯合双水相亲和分配技术分离纳豆激酶的研究

利用金属螯合亲和双水相分配技术对纳豆激酶的分离纯化进行了研究。考察了双水相系统、聚合物的分子量和浓度、亲和配基加入量、pH值、相比以及生物质加入量等因素对亲和分配的影响。结果表明,双聚合物系统比聚合物／无机盐系统更有利于纳豆激酶亲和分配;pH值和亲和配基加入量是影响分配的关键因素。优化的分配条件为：2．6％聚乙二醇,20．2％羟丙基淀粉,5％亲和配基PEG-IDA—Cu(Ⅱ),相比12,pH8．2,发酵液加入量15％。分配系统放大到100g,仍保持一致的酶活收率(90％)和纯化因子(2.0)。设计了两次分配分离流程,纯化因子达到3．52,总收率为81％。     

抗体药物分离纯化中的层析技术及进展

抗体,特别是单克隆抗体是一类重要的生物技术药物,具有巨大的市场前景。目前抗体药物生产的瓶颈已由抗体表达转移到抗体的分离纯化中,其中层析是最关键的技术。典型的抗体纯化过程首先通过蛋白A亲和层析对抗体进行捕获,然后进一步精制,主要方法包括离子交换层析、疏水相互作用层析以及羟基磷灰石层析等,这些传统的层析方法不可避免存在一定的局限性。为此,研究者致力于开发合适的新方法,如疏水性电荷诱导层析和短肽仿生层析等,很好地弥补了传统层析方法的不足。本文根据近年来国内外在单抗药物分离纯化方面所取得的研究进展,着重介绍了在单抗分离纯化中的常用的层析技术及研究进展。     

抗体药物分离纯化中的层析技术及进展

抗体,特别是单克隆抗体是一类重要的生物技术药物,具有巨大的市场前景。目前抗体药物生产的瓶颈已由抗体表达转移到抗体的分离纯化中,其中层析是最关键的技术。典型的抗体纯化过程首先通过蛋白A亲和层析对抗体进行捕获,然后进一步精制,主要方法包括离子交换层析、疏水相互作用层析以及羟基磷灰石层析等,这些传统的层析方法不可避免存在一定的局限性。为此,研究者致力于开发合适的新方法,如疏水性电荷诱导层析和短肽仿生层析等,很好地弥补了传统层析方法的不足。本文根据近年来国内外在单抗药物分离纯化方面所取得的研究进展,着重介绍了在单抗分离纯化中的常用的层析技术及研究进展。     

几种共同配基亲和层析系统的制备和应用

近年来,一种新的纯化生物高分子物质的方法——亲和层析法得到了飞速的发展。亲和层析是建立在具有亲和力的生物分子之间的可逆结合和解离的基础上的一种层析方法。酶与抑制剂、酶与底物、酶与辅酶、抗原与抗体、激素与激素受体、核糖核酸与其互补的脱氧核糖核酸等广泛地存在着亲和力。亲和层析法对含量极少的生物高分子物质的纯化特别有价值,目前在分子生物学的研究中已为广大实验室采用。共同配基亲和层析系统是将一些对同一类高分子物质具有作用的配基结合于固相载体上。由于该配基系统并非只对一种高分子     

用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能

以仿生多肽配基FYEILHC为亲和配基、以葡聚糖修饰的聚甲基丙烯酸缩水甘油酯[Dextran-poly(glycidyl methacrylate), Dextran-PGMA]超大孔微球为基质，制备用于单克隆抗体纯化的仿生多肽超大孔PGMA微球，在环氧氯丙烷中滴加2 mol/LNaOH使其表面衍生出环氧基，在表面修饰FYEILHC；用扫描电镜表征微球表面形貌，用AKTA蛋白纯化系统考察了Dextran-PGMA微球和琼脂糖微球对抗体的动态吸附量随线性流速的变化。结果表明，偶联FYEILHC后Dextran-PGMA微球仍能保持其大孔结构，在923 cm/h线性流速下，其对抗体的动态吸附量仅下降约8%，而琼脂糖微球的动态吸附量则迅速下降25%。表明在较高流速下，抗体在Dextran-PGMA微球上的传质性能较好。吸附?用0.1 mol/L NaOH原位清洗重复40次后，Dextran-PGMA微球对抗体的动态吸附量约为(21?1) mg/mL，表明微球具有良好的化学稳定性；血清中回收的抗体纯度为95.0%，表明仿生多肽亲和介质具有从复杂生物样品中纯化抗体的巨大潜力，可满足高流速、高通量抗体分离纯化需求。     

多糖类亲和分离材料的研究进展

亲和分离技术的核心是设计亲和分离材料,基于分子识别与亲和机制,针对多糖类高分子化合物的结构特点,探讨了其材料的设计原则,并综述了国内外多糖类亲和分离材料的制备技术及其应用研究进展。在此基础上提出了进一步改进多糖类高分子化合物制备技术的措施,并展望了此类材料的应用前景。     

疏水性电荷诱导层析——原理、性能及其应用

介绍了疏水性电荷诱导层析(hydrophobic charge induction chromatography,HCIC)的原理及特点:其功能基团结合了疏水作用和静电相互作用,是一种混合型的双模式层析方法。由于HCIC在蛋白质(特别是抗体)的分离纯化中表现出操作简便、选择性较好等优点,受到了学术界和工业界的关注。对HCIC的技术原理、发展历程、层析行为以及应用现状等进行综述,以期对HCIC的深入研究和应用开发提供指导。     

Protein A-based ligands for affinity chromatography of antibodies

Protein A chromatography is a key technology in the industrial production of antibodies, and a variety of commercial protein A adsorbents are available in shelf. High stability and binding capacity of a protein A adsorbent are two key issues for successful practice of protein A chromatography. Earlier versions of protein A adsorbents ever exhibited serious fragility to typical cleaning-in-place protocols (e.g. washing with sodium hydroxide solution), and suffered from low binding capacity, harsh elution, ligand leakage and other problems involved in industrial applications. During the last three decades, various techniques and approaches have been applied in the improvement of chemical stability and enhancement of binding capacity of protein A-based ligands and adsorbents for antibody purifications. This mini-review focuses on the technical explorations in protein A-based affinity adsorbents, especially protein A-based ligands, including the efforts to increase the chemical stability by site-directed mutations and to improve the binding capacity by ligand polymerization and site-directed immobilization. Moreover, the efforts to develop short peptide ligands based on the structure of protein A, including the biomimetic design strategies and the synthesis of peptide-mixed mode hybrid ligands are discussed. These peptide and peptidebased hybrid ligands exhibit high affinity and selectivity to antibodies, but noteworthy differences in the binding mechanism of antibody from protein A. As a result, bound antibody to the ligands could be effectively eluted under mild conditions. Perspectives for the development of the protein A-based peptide ligands have been extensively discussed, suggesting that the ligands represent a direction for technological development of antibody purification.     

A new ligand for immunoglobulin g subdomains by screening of a synthetic peptide library

By screening a synthetic peptide library of general formula (NH(2)-Cys1-X2-X3-X4)(2)-Lys-Gly-OH, a disulfide-bridged cyclic peptide, where X2-X3-X4 is the tripeptide Phe-His-His, has been selected as a ligand for immunoglobulin G (IgG). The peptide, after a preliminary chromatographic characterization, has proved useful as a new affinity ligand for the purification of polyclonal as well as monoclonal antibodies from biological fluids, with recovery yields of up to 90% (90% purity). The ligand is able to bind antibody fragments containing both Fab and Fc from different antibody isotypes, a fact suggesting the presence of at least two different antibody-binding sites. While the recognition site on Fab is unknown, comparative binding studies with Fc, in association with the striking similarities of the peptide (named Fc-receptor mimetic, FcRM) with a region of the human FcgammaRIII receptor, strongly indicate that the peptide could recognize a short amino acid stretch of the lower hinge region, which has a key role in autoimmune disease triggering. The unique properties make the ligand attractive for both the purification of antibody fragments and as a lead for the generation of Fc-receptor antagonists.     

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.     

Trastuzumab-Peptide Interactions: Mechanism and Application in Structure-Based Ligand Design

Understanding of protein-ligand interactions and its influences on protein stability is necessary in the research on all biological processes and correlative applications, for instance, the appropriate affinity ligand design for the purification of bio-drugs. In this study, computational methods were applied to identify binding site interaction details between trastuzumab and its natural receptor. Trastuzumab is an approved antibody used in the treatment of human breast cancer for patients whose tumors overexpress the HER2 (human epidermal growth factor receptor 2) protein. However, rational design of affinity ligands to keep the stability of protein during the binding process is still a challenge. Herein, molecular simulations and quantum mechanics were used on protein-ligand interaction analysis and protein ligand design. We analyzed the structure of the HER2-trastuzumab complex by molecular dynamics (MD) simulations. The interaction energies of the mutated peptides indicate that trastuzumab binds to ligand through electrostatic and hydrophobic interactions. Quantitative investigation of interactions shows that electrostatic interactions play the most important role in the binding of the peptide ligand. Prime/MM-GBSA calculations were carried out to predict the binding affinity of the designed peptide ligands. A high binding affinity and specificity peptide ligand is designed rationally with equivalent interaction energy to the wild-type octadecapeptide. The results offer new insights into affinity ligand design.     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

Hydrophobic charge-induction chromatographic resin with 5-aminobenzimidazol ligand: Effects of ligand density on protein adsorption

Hydrophobic charge-induction chromatography (HCIC) is a highly promising technology for antibody separation. HCIC resins ABI-B-6FF were prepared with 5-aminobenzimidazole (ABI) as the functional ligand. The effects of ligand density on the adsorption of human immunoglobulin G (hIgG) and bovine serum albumin were focused. It was found that the adsorption capacity and dynamic binding capacity (DBC) were improved with the increase of ligand density. The adsorption capacity and DBC of hIgG reached 128.07 mg/g gel and 67.63 mg/g gel. The results indicated that ABI-B-6FF resin has a promising potential for the application of antibody purification.     

Influences of Ligand Structure and pH on the Adsorption with Hydrophobic Charge Induction Adsorbents: A Case Study of Antibody IgY

The adsorption behaviors of hydrophobic charge induction chromatography (HCIC) adsorbents with different functional ligands were investigated with immunoglobulin of egg yolk (IgY) as a model antibody. The adsorption isotherm and retention behavior in the column were studied, and the influences of the ligand structure and the pH on the adsorption were discussed. The results indicated that the pI of the target protein and pKa of HCIC ligand are the important parameter to determine the maximum adsorption pH of HCIC adsorbent, and high adsorption of IgY was found at pH 5 for all five adsorbents tested. Some differences could be found for different HCIC adsorbents, and the ligand structure influenced pH effect on the binding/elution of target protein. 2-mercapto-1-methyl-imidazole (MMI) ligand with a sulfone group showed a high adsorption capacity and strong pH-sensitivity, which would be more suitable for antibody purification. Moreover, the retention experiments indicated that IgY could be efficiently eluted from the adsorbents with 4-mercapto-ethyl-pyridine (MEP) or MMI as the ligand at acid conditions, while 2-mercapto-benzimidazole (MBI) ligand showed some difficulties on the elution. The retention study would help in defining not only the effective pH of elution for a given protein but also the elution efficiency of a given adsorbent.