【摘要】： rhG-CSF是通過大腸桿菌發(fā)酵培養(yǎng),以分泌型表達生產(chǎn)的重組人粒細胞刺激因子,可以刺激多種造血干細胞的增殖和分化,主要用于各種原因引起的粒細胞減少癥。 為了獲得高純度、有活性的rhG-CSF,我們摸索并建立了以細菌破碎和蛋白質(zhì)鹽析、G-25脫鹽、CM-Sepharose層析和Sephacryl S-100 H.R層析組成的rhG-CSF純化工藝流程: 1、細菌破碎和碎片分離:在低溫條件下反復(fù)凍融,低溫高速離心(8000rpm ,4℃)去除細菌碎片; 2、蛋白質(zhì)鹽析:在裂解上清液中加入高濃度中性鹽,使蛋白質(zhì)沉淀; 3、G-25脫鹽:采用分子篩層析除去高濃度的中性鹽; 4、CM -Sepharose層析:采用了以濃度為0.5M和0.7M的洗脫緩沖液分別洗脫雜蛋白和rhG-CSF; 5、Sephacryl S-100 H.R層析:利用分子量的差別進行最后的精純。 我們發(fā)現(xiàn),將層析過程中涉及的緩沖液pH值降至4.0,可顯著抑制rhG-CSF的降解,并獲得純度大于95%具有生物學(xué)活性的rhG-CSF。 利用等線速度原理, rhG-CSF與CM -Sepharose介質(zhì)解離所需的離子強度范圍較廣的特點,通過調(diào)整rhG-CSF洗脫液濃度,我們將純化工藝逐步放大至生產(chǎn)規(guī)模。 采用上述工藝,我們能夠生產(chǎn)出充分滿足臨床需求的、合格rhG-CSF。
[Abstract]:RhG-CSF is a recombinant human granulocyte stimulating factor expressed in secretory form through E. coli fermentation. It can stimulate the proliferation and differentiation of various hematopoietic stem cells and is mainly used for granulocytopenia caused by various causes. In order to achieve high purity, We fumbled and established a rhG-CSF purification process consisting of bacterial breakup and protein salting-out, G-25 desalination, CM-Sepharose chromatography and Sephacryl S-100 H.R chromatography: 1, bacterial fragmentation and fragment separation: freezing and thawing at low temperature. Low temperature and high speed centrifugation (8000rpm, 4 鈩，

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