Boubeva Ralitza, Reichert Christian, Handrick René, Müller Claudia, Hannemann Jürgen, Borcharda Gerrit
School of Pharmaceutical Sciences University of Geneva, University of Lausanne 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
Chimia (Aarau). 2012;66(5):281-5. doi: 10.2533/chimia.2012.281.
Human recombinant granulocyte colony stimulating factor (rhG-CSF) is widely used in hematology and oncology for the treatment of neutropenia, for the restoration of neutrophil production after bone marrow transplantation, for myelodysplastic syndromes, and aplastic anemia. The E. coli expression system is commonly used for fast recombinant production of rhG-CSF at a large scale. We have applied a novel autoinduction method for the batch expression of rhG-CSF to study whether this new system would increase cell mass and target-protein yield compared to conventional E. coli cell culture and induction with isopropyl β-D-thiogalactopyranoside (IPTG). We could demonstrate 3-fold higher culture densities and a 5-fold higher protein yield compared to IPTG induction without the need to monitor cell growth in a shortened 24 h expression procedure. rhG-CSF expressed in autoinduction media was successfully extracted from E. coli inclusion bodies and refolded by dialysis. After size exclusion chromatography (SEC) purification, rhG-CSF showed similar conformation, biological activity and aggregation profile compared to the commercially available biosimilar TEVAgrastim(®) (TEVA Pharma AG). Expression by autoinduction is suggested as a cost- and time-effective method for rhG-CSF production.
重组人粒细胞集落刺激因子（rhG-CSF）在血液学和肿瘤学领域被广泛用于治疗中性粒细胞减少症、骨髓移植后中性粒细胞生成的恢复、骨髓增生异常综合征以及再生障碍性贫血。大肠杆菌表达系统通常用于大规模快速重组生产rhG-CSF。我们应用了一种新型自诱导方法进行rhG-CSF的分批表达，以研究与传统大肠杆菌细胞培养及用异丙基-β-D-硫代半乳糖苷（IPTG）诱导相比，这种新系统是否会增加细胞量和目标蛋白产量。与IPTG诱导相比，在缩短至24小时的表达过程中，无需监测细胞生长，我们能够证明培养密度提高了3倍，蛋白产量提高了5倍。在自诱导培养基中表达的rhG-CSF成功地从大肠杆菌包涵体中提取出来，并通过透析进行复性。经过尺寸排阻色谱（SEC）纯化后，与市售生物类似药TEVAgrastim®（梯瓦制药公司）相比，rhG-CSF显示出相似的构象、生物活性和聚集情况。自诱导表达被认为是一种生产rhG-CSF的经济高效方法。
