摘要
现代生物技术,几乎使人们可以获得任何需要的蛋白,用于蛋白质结构和功能研究。然而,要利用这一技术获得大量的能够满足产业化需要、质量符合临床要求的药用蛋白,却面临巨大的挑战。蛋白质的复性和纯化,作为生物工程下游技术的重要部分,往往是以大肠杆菌为表达系统,生产特定蛋白药物的瓶颈。白细胞介素-1受体拮抗剂(IL-1ra)为白细胞介素-1(IL-1)家族的一员,能特异性地与IL-1受体结合,从而拮抗IL-1的各种生物学效应。在临床上广泛用于治疗IL-1参与病理变化过程的一些相关疾病。IL-1ra临床用量很大,每剂量高达100mg。但目前为止,国内还没有IL-1ra产业化生产的报道。因此本研究对重组IL-1ra中试生产工艺进行研究。首次成功地在中试规模下,将复性原理应用于IL-1ra包涵体复性,实现复性纯化同步完成,复性率达到70%,产物得率达到65%,比国内外最好产率提高3.7倍。这对于国内重组蛋白药物的产业化工艺研究具有重要的参考价值。
本文主要研究结果如下:
1.根据GenBank中人类IL-1ra基因序列(GenBank登录号: EF140714),兼顾稀有密码子和大肠杆菌密码子偏好性,改造编码序列,人工合成hIL-1ra成熟蛋白cDNA编码序列。构建原核表达载体pLY-hIL-1ra。通过对表达量和遗传稳定性的筛选,确定BL21为最适宿主菌,并成功构建工程菌株BL21/pLY-hIL-1ra。
2.在揺瓶条件下对工程菌株BL21/pLY-hIL-1ra发酵条件进行初步摸索,确定最佳诱导时机为生长中期,诱导温度42℃,诱导时间为4h。在此基础上,进一步用3.7L发酵罐,对实验室条件下发酵工艺条件进行优化。确定的工艺条件为:①用半合成培养基,以甘油作为碳源,采用溶氧反馈分批补料培养方法进行发酵;②25%浓氨水调节pH维持在7.0左右,30℃培养5小时后,开始以脉冲方式流加补料液,通过调节转速、进气量、罐压、补料速度控制溶氧大于30%;③接种10小时后,升温到42℃,诱导4小时,菌体湿重达到58 g/L,表达量38%。将此工艺放大到30L发酵罐,菌体湿重可达60 g/L,重组蛋白质的表达量为34%。表明该工艺条件适合工程菌株的高密度发酵,达到了中试规模要求。
3.在实验室规模下,对重组IL-1ra包涵体释放、洗涤和溶解条件进行初步研究。以获得的参数作为参考,放大到中试规模。确定中试规模条件为:
①菌体破碎。高压匀浆破碎菌体,菌体(g):缓冲液(ml)=1:10,压力40M Pa,循环4次;破菌率可达96%,一次处理湿菌体量1 000g,可获得粗包涵体约250g;缓冲液组成:20 mmol/L Tris-HCl,5 mmol/L EDTA,pH 8.0。
②包涵体洗涤。洗涤分4步进行,洗涤缓冲液(ml):粗包涵体(g)=10:1,在搅拌罐内混悬,转速控制在200 rpm,每个洗涤步骤洗涤30min,目标蛋白的纯度可达72%。洗涤缓冲液组成:第一次为20 mM Tris-HCl,5mM EDTA, pH8.0;第二次为20 mM Tris-HCl,5mM EDTA,100 mM NaCl,1%TritonX-100,1M尿素,pH8.0;第三次为20 mM Tris-HCl,5mM EDTA,1%TritonX-100,100 mM NaCl,pH8.0;第四次为20 mM Tris-HCl,5 mM EDTA,pH8.0。
③包涵体溶解。溶解缓冲液(ml):包涵体(g)=20:1,溶解时间6h,温度为室温,同时进行搅拌,转速为200 rpm。溶解缓冲液组成:20 mM Tris-HCl,5 mM EDTA,5 mM DTT,6M尿素,pH8.0。
4.在实验室规模条件下,对IL-1ra包涵体色谱复性及纯化条件进行研究,实现复性和纯化同步完成。条件为:选择Q-Sepharose Fast Flow阴离子色谱;复性温度10℃,pH8.0;尿素浓度梯度为6mol/L降到2.1mol/L时,复性缓冲液用量4倍柱体积;线性流速为0.565cm/min;负载量为200ml,含目标蛋白约1 024mg;pH6.5条件下梯度洗脱,流速控制在6.0 ml/min,NaCl浓度由0到0.4M的时间控制在50min。在此条件下,蛋白收率约70%,样品生物比活性达到10万单位/mg,与Sigma公司销售的对照品一致,纯度达98%以上。以实验室条件作为参照,将此工艺成功放大到中试规模。选择BPGTMΦ70mm×L 500mm色谱柱,流速控制在0.754 cm/min,在复性温度、pH等与实验室规模一致的条件下,负载量增加到2 000ml包涵体溶液,含目标蛋白约10240mg,目的蛋白回收率65%,纯度98%,生物比活性达到10万单位/mg,与对照品一致,每升发酵液纯品产率为890mg。表明此工艺到达中试规模要求。
5.内毒素去除。选用SP-Spharose Fast Flow强阳离子交换色谱,让内毒素直接穿出色谱介质,而样品仍保留在色谱介质上。通过进一步改变pH,让目标蛋白解吸下来,达到去除内毒素的目的,同时去除复性纯化样品中残留的尿素。在实验室条件基础上,将此工艺成功放大到中试规模,单次处理量达到6.7g目的蛋白,内毒素由200EU/mg-260EU/mg降低到小于0.5EU/mg,满足了安全用药的要求,回收率为93%。
6.初步建立了原液的稳定性条件。20℃条件下,通过研究pH和保护剂浓度等因素对目的蛋白稳定性的影响,确定了保存目的蛋白活性的适宜条件为:pH6.5,蔗糖浓度为1.8%。
7.对原液的质量进行了鉴定和检测。①理化鉴定:用MADLI-TOF MS法,进行分子量的测定,复性纯化后的rhIL-1ra分子量为17.224kD,与理论分子量一致;肽图分析和West-Blotting免疫特异性分析及N端15个氨基酸残基序列分析结果显示,复性纯化后的rhIL-1ra与对照品一致,基本证实复性纯化后的rhIL-1ra理化特性与理论一致。②纯度、活性检测:用RP-HPLC检测纯度,rhIL-1ra原液纯度大于98%,满足临床用药纯度要求;用小鼠胸腺细胞-MTT法测定活性,rhIL-1ra原液比活力10万U/mg,与对照品一致。③对部分安全性指标的检测结果显示,安全性指标满足2005版中国药典相关要求。
With the development of biotechnology, large-scale production of recombinant protein is possible.
Interleukin-1 receptor antagonist (IL-1ra), a naturally occurring anti-inflammatory protein, competitively blocks the binding of IL-1αand IL-1βto their receptors. High dosage of IL-1ra are needed to relieve disease because even very low IL-1 can induce complete biological response. The dosage is 1-2 mg/kg/d when treating RA. The aim of the present study was to establish a method for large-scale producing IL-1ra by recombinant technology.
1. cDNA encoding of IL-1ra were synthesized, and the seed strain was constructed by transformation of PLY-hIL-1ra into host strain BL21, designated as BL21/PLY-4-hIL-1ra.
2. Fermentation parameters were investigated in shaking bottle. Inducetion point was exponentional phase stage of growth. The target protein synthesis was induced at 42°C for 4h. Then, fermentation was established in a 3 L and 30 L bioreactor was trialed, respectivlely.In 30 L bioreactor, cell weight was 60g/L, and the rate of recombinant protein was 34%.
3. The purification of rhIL-1ra was studied
i Rupturing cells and releasing inclusion body. High-pressure homogenization was used and the rate of cell rupture was 96%.
ii Washing inclusion body. The rate and purification of target protein was 89.6 %and 72 %, respectively.
iii Dissolving inclusion body. Dissolving buffer (ml): inclusion body (g) =20:1. Inclusion body is dissolved for 6h with stir (200 rpm). Recipe of dissolving buffer: 20 mM Tris-HCl, 5 mM EDTA, 5 mM DTT, 6M urea, pH8.0.
4. Protein refolding with chromatography. Anion exchange chromatography column (Q-Sepharose Fast Flow) was used. The precodure was perfomed at 10°C, pH 8.0. The concentration of urea was reduced from 6 M to 2.1 M. The volume of refolding buffer was four fold of column volume and the linear velocity was 0.565 cm/min. Two hundred milliliter sample solution with 1024 mg target protein was loaded onto chromatography column and eluted with linear gradient elution with a rate of 6.0 ml/min. The concentration sodium chloride varied from 0 M to 0.4 M within 50 min. The recovery rate was 70 %. Specific activities was 100 000 U/mg. The purity was 98%.These parameters were applied to a large-scale production and excellent results obtained.
5. Strong cation-exchange chromatography column (SP-Spharose Fast Flow) was used to remove endotoxin.With this technique did endotoxin flow through the column by adjusting pH but protein absorbed. The level of endotoxin was decreased to 0.5 EU/mg and the recovery rate was 93%.
6. Storage condtion. rhIL-1ra should kept in 1.8 % sucrose solution at 20°C, pH6.5.
7. Analysis of Specification. i. Molecular weight determined by MADLI-TOF MS was 17.25 kD. ii. Purity of original protein solution was not less than 98% by RP-HPLC. Specific bioactivity was 100 000 U/mg by thymus cell assay.
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