摘要
本文围绕如何在大肠杆菌中生产药用蛋白的关键技术问题包括目的蛋白的高效表达、包涵体蛋白的复性、蛋白质的大规模分离纯化以及蛋白质药物的稳定性提高等开展研究,并以处于不同开发阶段的三个重组蛋白药物为研究对象,发展了满足实验室阶段、中试生产和已有成熟市场等特定时期迫切需要解决问题的关键技术。
促红细胞生成素模拟肽(EMP1)是一种由20个氨基酸组成的环肽,氨基酸序列与EPO不存在同源性但其生物功能与EPO基本相同。本文首先通过二种不同的连接肽将两分子的EMP1串联构成了二串体,代替化学法的二聚体,然后与硫氧还蛋白或DsbA等融合在大肠杆菌中实现了可溶性高效表达。在最优诱导温度和IPTG浓度下,二串体融合蛋白的可溶性表达水平可达到0.83g/L,并可采用金属螯合层析和疏水作用层析获得较高纯度的目标融合蛋白,纯化收率约为6.3-7.8%。融合蛋白再经过肠激酶切割和反相作用层析可以获得EMP1二串体,两种不同长度连接肽的EMP1二串体与EMP1单体相比,生物活性提高了10倍,连接肽的长短对活性没有显著影响。
重组人骨形态发生蛋白2(rhBMP-2)由114个氨基酸组成,其生物活性形式为同源二聚体或与BMP-7形成异二聚体,由于极强的疏水性,在大肠杆菌中包涵体形式表达的BMP-2复性率低下而且成本高,后分离困难。本文系统地研究了表达后处理包括包涵体的溶解条件和不同复性液组成对复性的影响,发现盐酸胍体系溶解包涵体更有效,目标蛋白回收率也更高。通过筛选获得了最佳复性液组成,建立了操作简便的可放大的稀释法复性方案,最佳条件下的复性率大于70%,复性液成本大大下降,复性液不需任何预处理就可以直接进行后续纯化。开发了新颖的利用疏水作用层析原理的纯化方法,筛选出N,N-二甲基甲酰胺(DMF)作为添加剂并优化了添加浓度,发现5%的DMF具有最好的分离度；通过不同盐浓度下的操作模式的比较,建立了两步法、DMF辅助的疏水作用层析分离工艺,最后分离达到的rhBMP-2同二聚体不仅纯度高,而且生物活性与商业化的CHO细胞来源的rhBMP-2相比ALP活力相当,与Wyeth公司的rhBMP-2制剂][NDUCTOSTM相比异位成骨活性更高,平均收率达到每克包涵体湿重产出80mgrhBMP-2同二聚体。DMF可以通过透析和冻干从rhBMP-2产品中完全去除,残留量未检出。这一套有效的复性和纯化工艺在中试生产车间进行了放大验证。另外,还针对大肠杆菌来源的rhBMP-2产品初步建立了反相高效液相色谱法和分子筛高效液相色谱法的纯度测定方案,并与毛细管电泳法和SDS-PAGE电泳法进行相互验证。
重组人粒细胞集落刺激因子(rhG-CSF)是一类重要的已经成功实现产业化的细胞因子类重组蛋白药物,如何提升该产品的稳定性和质量水平具有重要的现实意义。本文建立了可靠的基于C4柱的反相HPLC纯度测定方法,经过方法学验证,分离度大于现有药典标准方法；通过37℃加速和4℃长期稳定性试验研究了生产过程三个关键环节对产品稳定性的影响,筛选了来自不同厂家的三类常见药品包材-安瓿瓶、西林瓶和预装针,通过制剂工艺条件的优化、确立和新处方优化,确立了稳定性最佳的三种不同内包材类型的rhG-CSF制剂工艺,使商品rhG-CSF的纯度和稳定性获得了显著的提高。
总之,通过对处于不同研发和生产阶段的3种蛋白质药物的研究,不仅建立了多种蛋白质药物工业化生产的关键技术,而且实际上大大促进了这几种蛋白质药物从研究到大规模生产的转化,提升了生产效率和产品稳定性。
In the present work, several key technologies were developed to produce therapeutic proteins in recombinant Escherichia coli, such as high-efficient expression technology, protein refolding of inclusion body, separation and preparation of protein at pilot scale, and protein stability improvement, etc. Three kinds of recombinant proteins (EMP1, rhBMP-2and rhG-CSF) in different development stages were targeted in order to solve their specific and urgent problems at lab, at pilot plant and in mature markets respectively.
Erythropoietin mimetic peptide1(EMP1) is a20-amino acid cyclic peptide which are not found in the primary sequence of EPO, but acts as full agonists and can also stimulate erythropoiesis in mice. In this work, two repeats of EMP1linked by flexible peptide with different length were expressed in E. coli as fusion proteins with thioredoxin or DsbA. Under optimal temperature and IPTG concentration, the highest soluble expression level of fusion proteins was0.83g/L. The soluble part of fusion proteins could be purified by metal chelating affinity chromatography and hydrophobic interaction chromatography with enhanced purity, and the overall process yield was about6.3-7.8%. The two repeat peptide of EMP1was released by enterokinase cleavage of fusion proteins and obtained by followed reversed phase chromatography. The biological activities of the resulted products, EMP1-EMP1(S) and EMP1-EMP1(L) were10-fold greater than monomeric EMP1, the length of flexible linker has negligible effect on the biological activity.
The mature bone morphogenetic protein-2(BMP-2) consists of114amino acids and acts as a bioactive disulfide-linked homodimer, or a heterodimer linked with BMP-7. Due to its high hydrophobity, in vitro refolding of insoluble rhBMP-2was low-efficient, and subsequent purification of soluble rhBMP-2was still difficult and expensive due to the employment of heparin sepharose affinity chromatography. In order to solve the above problems, the post-expression processing procedure including inclusion bodies solubilization and in vitro refolding in different solutions were systematically investigated. The results indicated that Gdn-HCl solution (buffer A) was relatively more effective in solubilizing inclusion body, resulting in higher protein recovery and the optimal refolding solution was formulated. Under the optimized conditions, an effective and economic refolding process by simple dilution was established and successfully scaled up with yield higher than70%. The refolding mixture could be subjected to subsequent purification directly without any adjustment or pre-treatment. Then a novel purification process to produce active rhBMP-2homodimer from refolding mixture by hydrophobic interaction chromatography (HIC) was proposed. N, N-dimethylformamide (DMF) was adopted as additive and its concentration was optimized. It was found that5%(V/V) DMF can enhance the resolution of rhBMP-2homodimer most effectively. The rhBMP-2homodimer was purified to homogeneity through two HIC separations at different salt content, the purified rhBMP-2homodimer with high purity exhibited equivalent alkaline phosphatase (ALP) activity to commercial rhBMP-2produced from Chinese hamster ovary cell (CHO) and higher ectopic bone formation capacity. The average process yield was80mg rhBMP-2homodimer per g of wet inclusion bodies. DMF could be totally removed from the purified protein without any detectable trace after dialysis and lyophilization of final product. This efficient refolding and purification procedure was successfully scaled up in the pilot pharmaceutical plant. In the mean time, reversed phase chromatography and size exclusion chromatography were first used to measure the purity of rhBMP-2from E.coli which was validated with capillary electrophoresis and electrophoresis.
The recombinant human granulocyte colony stimulating factor (rhG-CSF) is an important therapeutic protein which has been produced at large scale in biotechnology industry. How to guarantee and improve the stability and quality of final product is a practical and important issue for the manufacturers. In this work, a reversed phase chromatography using C4column was applied to determine the purity of rhG-CSF samples with better resolution. Three stages of manufactering process were evaluated to decide their effects on the stability of product. The effects of different sources of ampoules, vials and prefilled syringes on the rhG-CSF stability were compared. After the optimization of preparation condition and the adoption of novel formulation, rhG-CSF products filled in selected ampoule, vial and prefilled syringe exhibited much higher quality and stability than the traditional products.
In conclusion, by targeting at three different recombinant proteins with E. coli, the present work not only developed several key technologies for their efficient production, but also pushed the transfer of these therapeutic proteins from research to large-scale production with enhanced productivity and improved stability.
引文
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