CAS OpenIR
新型膜色谱介质的简易和绿色制备及其在蛋白纯化中的应用
Alternative TitleFacile and green fabrication of novel membrane adsorbers and its application in protein purification
MUHAMMAD KAMRAN KHAN
Thesis Advisor万印华
2018-06
Degree Grantor中国科学院大学
Degree Name博士
Degree Discipline生物化工
Keyword生物分离,蛋白纯化,膜色谱,氧化海藻酸钠,膜色谱介质
Abstract

高效的生物加工技术是生产生物药物的关键。随着生物药专利保护过期、仿制药生产飞速发展,亟需降低生物药物的生产成本以提高其市场竞争力。由于上游生产工艺已取得巨大突破,生物分离成本将成为生物药物生产的主要成本,因此,亟需建立高效的生物分离技术。膜色谱技术由于结合了膜分离和柱层析的优势,使得高通量、高选择性的生物加工过程成为可能,是一种极具潜力的生物分离技术。本论文通过简单、绿色的合成工艺制备了新型膜色谱介质并研究了其在蛋白纯化中的应用。首先,利用氧化海藻酸钠(ADA)对尼龙微滤膜进行功能化改性(ADA上的醛基与尼龙膜上的氨基反应),ADA链上丰富的羧基及其残余的醛基可进一步接枝其他配基,从而使其成为一个多样化的膜色谱介质制备平台,同时该功能化过程不使用任何有机溶剂。以ADA-涂覆的尼龙膜为平台,可制备阴离子交换、金属螯合以及亲和膜色谱介质。通过对比尼龙膜和膜色谱介质的物化性质(Zeta电位、元素分析、接触角、跨膜压力和表面形态),表明不同的配基被成功接枝到尼龙膜表面。所制备的阴离子交换、金属螯合、氨基酸亲和以及多肽亲和膜色谱介质对免疫球蛋白G/人血清白蛋白(IgG/HSA)具有较高的分离选择性,产品回收率和纯度分别为100%和94-99%,同时获得了较高的吸附容量,分别为30, 24, 21 和 28 mg/mL (膜体积),均高于其他报道的膜色谱介质的吸附容量。另外,由于ADA涂覆提高了尼龙膜的亲水性,从而消除了蛋白质在膜色谱介质上的非特异性吸附。常用的Protein A凝胶成本较高且有配基降解脱落的风险,因此它在IgG纯化上的应用受到挑战。以ADA涂覆的尼龙膜为平台,通过进一步接枝多肽DWHW,制备了具有协同作用(离子交换和多肽亲和)的膜色谱介质,优化了制备参数以及操作pH,并将其用于纯化人血浆中的IgG。通过尼龙膜和ADA上的羧基以及多肽配基的协同作用, IgG的回收率和纯度分别为99%和98.6%。另外,所制备的膜色谱介质的动态吸附容量(DBC)为38.7 mg/mL,高于Protein A凝胶的DBC(24.4 mg/g)。通常膜色谱介质对分子量较低的蛋白吸附容量要低于柱层析。为了进一步提升膜色谱介质的吸附容量,以ADA涂覆的尼龙膜为制备平台,在ADA链上引入磺酸基团(水相反应,无需有机溶剂),制备了具有双离子交换配基的膜色谱介质。首先研究了ADA涂覆时间、溶液pH值、离子强度以及初始溶菌酶浓度对溶菌酶最大吸附量的影响,获得了最佳的溶菌酶纯化条件。由于膜表面和孔内具有丰富的磺酸基和羧基,使得新制备的膜色谱介质对溶菌酶有超高的吸附容量(286 mg/mL),且可以在温和的条件下简单再生(蛋白完全洗脱),同时可至少重复使用5次。另外,新制备的膜色谱介质可从鸡蛋清中纯化溶菌酶,其纯度和回收率分别为100%和98%。纯化所得的溶菌酶比活力与商品化产品接近。;Efficient bio-processing technology is the key requirement in the manufacture of biopharmaceuticals. The increasing need to reduce biopharmaceutical cost is driven by the business challenges such as the emerging bio-generics. The great advances in upstream technologies make bio-separation the major cost in bioprocessing. Developing efficient bio-separation technologies is therefore strongly desired. Membrane chromatography is a promising bio-separation technology which combines the advantages of membrane technology and chromatography, thus making high-throughput and high resolution bioprocesses feasible. This thesis focuses on the production of novel membrane adsorbers via a facile and green fabrication mechanism and its application for the purification of proteins. A versatile platform was created by functionalizing nylon membrane via a non-immunogenic bio-adhesive polymer, Alginate dialdehyde (ADA), without any organic solvent usage. Cation exchange (CEX), metal-affinity (M.A), histidine-affinity (His-Affinity) and peptide-affinity (Pep-affinity) membrane adsorbers were fabricated based on ADA-coated nylon membrane platform. By comparing the physiochemical properties of the nylon membrane and obtained membrane adsorbers, it was confirmed that different ligands were grafted on the membranes. All the membrane adsorbers exhibited a high selectivity in fractionation of IgG (immunoglobulin)/HSA (human serum albumin) mixture. Along with high purity and recovery of 100% and 94-99% respectively, a high IgG binding capacity of 30, 24, 21 and 28 mg/mL (membrane volume) was achieved by CEX, M.A, His-affinity and Pep-affinity membrane adsorbers respectively, which is superior to the reported membrane adsorbers. Furthermore, via ADA coating, non-specific adsorption on the nylon membrane was completely suppressed. The currently used Protein A agarose for the purification of IgG is challenged due to ligand leakage and high cost. Based on the ADA-coated nylon platform, a synergetic membrane adsorber (ion exchange and peptide-affinity) was constructed via immobilizing a peptide DWHW for the purification of IgG from real human plasma solution. The ADA coating time during adsorber fabrication and the operating pH during IgG static adsorption were optimized. The ADA carboxylic groups along with the peptide captured IgG synergistically with high recovery and purity of 99 % and 98.6 % respectively. Furthermore, a dynamic binding capacity (DBC) of 38.7 mg/mL was achieved for the newly fabricated adsorber, which was quite higher than the Protein A agarose (having a DBC of 24.4 mg/g). Usually the binding capacity of membrane adsorbers is lower for proteins with lower molecular weights as compared to the columns. Keeping in view this point a highly potent dual cation exchange membrane adsorber was developed based on the pre-coated ADA-nylon by the addition of sulphonic groups without any organic solvent usage. The influence of ADA coating time, pH, ionic concentration and initial lysozyme concentration on maximum lysozyme adsorption were examined,and the optimal conditions were obtained for lysozyme purification. Taking advantage of the abundant dual CEX (carboxylic & sulphonic) groups on sulphonic-ADA (S-ADA) ligands, this novel S-ADA-nylon membrane adsorber showed an unprecedented static binding capicity of 286 mg/mL for lysozyme adsorption. Meanwhile, the prepared membrane adsorber could be easily regenerated (complete protein elution) under mild conditions and be reused at least for five times. Featured with a unique selectivity, the S-ADA-nylon membrane also captured lysozyme from chicken egg white solution with a high purity (100%) and a high recovery of 98%. The purified lysozyme showed similar specific activity as commercial product. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26942
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
MUHAMMAD KAMRAN KHAN. 新型膜色谱介质的简易和绿色制备及其在蛋白纯化中的应用[D]. 中国科学院大学,2018.
Files in This Item:
File Name/Size DocType Version Access License
新型膜色谱介质的简易和绿色制备及其在蛋白(3236KB)学位论文 限制开放CC BY-NC-SAApplication Full Text
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Google Scholar
Similar articles in Google Scholar
[MUHAMMAD KAMRAN KHAN]'s Articles
Baidu academic
Similar articles in Baidu academic
[MUHAMMAD KAMRAN KHAN]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[MUHAMMAD KAMRAN KHAN]'s Articles
Terms of Use
No data!
Social Bookmark/Share
All comments (0)
No comment.
 

Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.