کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1200985 | 1493612 | 2013 | 11 صفحه PDF | دانلود رایگان |

• Single step purification platform for E. coli derived proteins.
• Novel combination of combined pH and salt based elution gradients used to enable separation of the different product related variants.
• Adequate removal of host cell proteins and nucleic acids demonstrated.
• Analytical similarity to commercial product established along with bioactivity.
Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pH < pKa of the HCIC ligand, selective removal of various product variants using the combined pH and salt based elution gradient and removal of the host cell impurities in flow-through are the key novel features of the developed multimodal chromatographic purification step.
Journal: Journal of Chromatography A - Volume 1314, 1 November 2013, Pages 188–198