Efficient protein purification methods are an important element for on-demand production of protein biologics. In this study, the rapid in vitro expression of a protein was accomplished using lyophilized cell-free extracts derived from Chinese hamster ovary (CHO) cells. A CHO-based expression system was chosen because most biologics produced at the industrial scale utilize CHO cells. The purification of proteins produced in a cell-free expression system was performed using a versatile purification platform consisting of ion exchange chromatography or immobilized metal affinity chromatography (IMAC) as an initial capture step. As proof-of-principle, granulocyte colony-stimulating factor (G-CSF) was selected as a model case because it is a well characterized biologic and was the first approved biosimilar introduced in the market. The specific conditions employed for the capture step, such as the volume of the column and the compositions and flow rates of the wash and elution buffers, were optimized to achieve the maximum product yield and purity. In order to more easily develop the purification platform, and also to measure the quality of the final product, high-sensitivity silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with quantitative image analysis was employed to detect impurities down to 10 ppm in the purified product. This method was validated using other analytical methods, such as enzyme-linked immunosorbent assay (ELISA), size exclusion chromatography (SEC) and the Bradford (660 nm) assay. Results of this study indicate that the optimized platform that was investigated achieved &gt; 90% purity of target proteins in only one step. Furthermore, the high-sensitivity silver-stained SDS-PAGE method used provided accurate measurements of the purity and yield after each purification step. Overall, the data presented in this study show the potential of the validated purification methods under investigation when is applied to therapeutic proteins produced in a CHO-based cell-free system.

Development and validation of a purification platform for proteins expressed in a CHO cell-free system

255th National Meeting (2018)

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