Knowledge Management System Of Institute of process engineering,CAS
|Thesis Advisor||刘永东 ; 苏志国|
|Place of Conferral||北京|
|Keyword||药物半衰期 长效策略 聚乙二醇 巯基选择性修饰 白蛋白结合肽|
为解决生物技术药物半衰期短的问题，各种长效化策略相继开发并成功应用于多种蛋白药物。聚乙二醇（PEG）修饰药物蛋白，能够显著延长半衰期，降低免疫原性，同时提高药物水溶性和稳定性。偶联PEG分子，可增大药物蛋白的水力学半径，从而减缓肾小球滤出速率，延长药物半衰期；另外一种方法是借助新生儿Fc受体（FcRn）介导的再循环途径，延长药物蛋白的半衰期。本论文针对这两种策略，开发了一种新型的巯基定点的PEG修饰试剂；拓展了白蛋白结合肽（ABD）融合表达的长效策略，并对两种重要的长效策略进行详细比较。巯基定点修饰对于实现高度选择性修饰、保证批次间重复性有着重要价值，已成为PEG修饰的重要方法。然而现有的巯基修饰试剂存在稳定性差、反应活性低、偶联产物易发生PEG脱落等问题。本论文利用邻苯二酚类化合物与巯基官能团的特异性反应，将邻苯二酚与PEG偶联制备一种新型的巯基定点修饰试剂（PEG-DAQ）。首先，利用有无游离巯基的同种突变蛋白，及巯基是否被N-乙基马来酰亚胺封闭的同一种蛋白确认了PEG-DAQ的巯基定点选择性；通过对睫状神经营养因子（CNTF）、鞭毛蛋白突变体（CBLB502-Cys）和牛血清白蛋白（BSA）三种含巯基蛋白进行修饰反应，证实了该修饰剂的普适性；通过对比PEG-DAQ与常用的巯基修饰试剂PEG-MAL、PEG-VS的修饰效率，发现PEG-DAQ具有反应活性高的优点，其修饰率与PEG-MAL相近，且远高于PEG-VS；考察修饰试剂PEG-DAQ在不同pH条件下反应，发现在pH5.5-8.5之间均可以进行修饰反应，pH越高，修饰效率也越高。进一步，考察了修饰剂的稳定性，当PEG-DAQ在水溶液放置96h后，其修饰能力保持了几乎100%；与之对照的PEG-MAL，修饰能力仅有最初的70%。以CNTF作为模型蛋白，对修饰剂的应用做进一步研究。经PEG-DAQ修饰后分离纯化得到单修饰样品，经过圆二色光谱和荧光光谱确认，PEG-DAQ修饰的CNTF与原蛋白的二三级结构保持一致；考察修饰产物稳定性，发现PEG-DAQ修饰产物在37℃放置2周，单修饰产物存留率达93.5%；而PEG-MAL修饰产物仅保留71.2%。TF-1. CN5a. l细胞增殖实验证实了PEG的亲水柔性链使CNTF的体外活性降低，但两种修饰试剂PEG-DAQ和PEG-MAL的修饰产物细胞活性没有显著差异，说明了PGE-DAQ不存在明显的细胞毒性； SD大鼠实验证实了PEG-DAQ-CNTF半衰期延长为原蛋白的3倍。因此，本论文的研究提供了一种新型巯基定点修饰试剂PEG-DAQ，这一修饰剂有着广泛的应用前景。人血清白蛋白（HSA）是血浆中含量最丰富的蛋白。HSA可以与FcRn受体结合，并借助其介导的再循环途径避免被胞内溶酶体水解，半衰期可达19天。治疗性蛋白可与HSA融合表达以此延长药物蛋白的半衰期。这种融合表达策略无需化学修饰及修饰后的纯化步骤，工艺简单，且产品均一性好。然而，与大分子量的白蛋白融合，一些治疗性的融合蛋白往往难以在原核表达系统中表达，需要真核表达系统，存在表达量低，成本高的问题。而小分子量的白蛋白结合肽（ABD）可以特异性的与白蛋白结合，借助白蛋白的稳定性和长效性，同样可以实现延长药物半衰期的目的。本论文利用这一原理，构建ABD与人睫状神经营养因子（CNTF）融合蛋白，通过原核体系表达制备ABD-CNTF，并与不同分子量的PEG修饰CNTF做对比，比较其药物作用的半衰期变化。结果发现，融合蛋白ABD-CNTF为可溶表达，表达量为90-100 mg/L发酵液；经过两步层析纯化，纯度为95%以上。SEC实验了ABD-CNTF保留了ABD与HSA结合的能力，且结合可在数分钟（<10 min）内完成；圆二色光谱和荧光光谱表明ABD-CNTF保留了原蛋白二、三级结构。TF-1.CN5a.l细胞增殖实验证明ABD-CNTF保留了90%以上的原蛋白细胞活性；与之对照的是，PEG修饰CNTF损失了绝大部分细胞活性，PEG-40k-CNTF的细胞活性小于原蛋白的10%。最后，SD大鼠实验证实了ABD-CNTF半衰期延长14倍，延长效果介于PEG-20k与PEG-40k之间。研究结果表明，ABD融合药用蛋白是一种低廉有效的制备长效蛋白药物的新方向。
Various half-life extension strategies have been developed and applied on biotherapeutic proteins to improve their pharmacokinetics. PEGylation of drug protein is able to extend the protein’s half-life, to reduce their innate immunogenicity and improve the physiochemical stability and water-solubility. Coupling polyethylene glycol with biotherapeutics increases proteins’ hydraulic radius and thus reduce glomerular filtration to improve their pharmacokinetics. Another widely-used approach is based on neonatal Fc receptor (FcRn) mediated cell recycling. Here in this dissertation, we developed a new thiol-selective PEGylation reagent and, extended the application of albumin-binding domain fusion strategy on CNTF, as well as compared the two strategies.Thiol-specific PEGylation of clinically used protein via free cysteine residue has been widely applied for a homogeneous product. However, limitations have been reported to market-available reagents, such as the succinimide ring is susceptible to ring-opening hydrolysis, lower reactivity or de-conjugation of the protein from the PEG. Herein, we developed a new thiol-specific PEGylation reagent based on catechol-derived reactive quinone species. Catechol-derived polyethylene glycol (PEG) was synthesized by coupling linear PEG N-hydroxysuccinimide to dopamine and then oxidized to quinone. First, PEG-dopaquinone (PEG-DAQ) mostly modifying the free cysteines of three model proteins of truncated flagellin (CBLB502) and recombined human ciliary neurotrophic factor (CNTF), BSA evidenced its thiol-reactivity and thiol-specificity. Furthermore, PEG-DAQ coupling efficiency were compared with another two available reagents PEG-MAL or PEG-VS. The yield of PEG-DAQ-CNTF under neutral condition was 87.5%, compared with PEG-maleimide of 92.3% and PEG-vinylsulfone of 17.6%, respectively. About 95% reactive capacity for PEG-DAQ was maintained after incubation in aqueous solvent for 96 hours, indicating that the reagent was superiorly hydro-stable; whereas, approximately 70% of the initial reactivity of PEG-MAL was remained.Furthermore, the application of PEG-DAQ reagents was investigated using CNTF as model protein. Mono-PEGylated CNTF has been prepared through bioconjugation and further purification. Moreover, the stability of the PEGylated species via PEG-MAL and PEG-DAQ were compared. It was found that PEGylation reaction with excess cysteine quenching was necessary to produce more stable conjugates. The resulted PEG-DAQ-CNTF remained about 93.5% of the initial products after storage for 14 days. As a comparison, PEG-MAL-CNTF was remained only 71.2 %. Finally, the CCK-8 analysis of cell viability demonstrated its low cell toxicity and the pharmacokinetic test suggested PEG-DAQ could extend CNTF’s half-life by three times. Our results revealed that PEG-dopaquinone had properties of high selectivity and reactivity with cysteine thiol, and superior stability for both the reagent itself and its conjugate, showing great promise for developing as an alternative reagent for thiol-selective modification. Human serum albumin (HSA) is one of most abundant protein in the serum. HAS generally exhibit a circulatory half-life of 19 days due to its large molecular size and neonatal Fc receptor (FcRn) mediated cell recycling. Fusion with endogenous serum albumin as a drug carrier is another increasingly attractive strategy to extend the circulatory time for small protein and peptide. This fusion strategy avoiding chemical modification and further purification. However, production of these fused large proteins generally requires mammalian cells, a relatively complicated and expensive host system, always leading to lower expression and higher cost. Albumin-binding domain (ABD) is a small peptide, which can bind with endogenous HSA and further utilize the naturally long-half-life property of HSA. Herein we constructed a new long-acting CNTF by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker, and compared the ABD fusion strategy with the established PEGylation approach. This fused protein ABD-CNTF was expressed in E. coli mainly in the soluble form and purified. The in-vitro binding ability of ABD-CNTF with HSA was verified by incubation of the two components together followed by HP-SEC analysis. ABD-fused CNTF showed similar secondary and tertiary structure as the native protein. It retained approximately 94.1% of the native bioactivity, whereas PEGylated CNTF suffered severe activity loss, as demonstrated via CCK-8 cell viability assay analysis. In-vivo studies in SD rats were performed and the terminal half-life of ABD-CNTF was determined to be 483.89 min, which is about 14 folds longer than that of native CNTF (34.28min) and is comparable with 20k-40 kDa PEGylated CNTFs. The new constructed ABD-CNTF represents a potential therapeutic modality. The results suggested the ABD-fusion is a cost effective and efficient strategy for long-lasting biopharmaceutics’ design.
|徐龙福. PEG定点修饰及白蛋白结合肽融合的长效策略[D]. 北京. 中国科学院研究生院,2017.|
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