Separation and purification of recombinant proteins by using ultrafiltration memranes

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2017
Akcan, Begüm
Membrane based processes have an increasing role in separation processes and used in many industrial areas like water treatment, food industry and biotechnology. Ultrafiltration can be an alternative to conventional techniques such as crystallization and chromatography due to superior advantages such as having low energy and chemical consumption and easy scale up in the field of bioseparation. Within the scope of the separation of the recombinant proteins from fermentation environment by using ultrafiltration membranes, two separate production media were considered. First production media contained recombinant human growth hormone (rhGH). In size based separation with cellulose membranes, it was observed that desired product, hGH, was specifically retained by the membrane. High retention of the hGH could arise from the agglomeration of the hGH molecule. Furthermore, larger molecular weight proteins passed to the permeate side whereas hGH was highly retained. In this environment, hGH was produced extracellularly so, all metabolites including salts were present in the feed solution. Presence of the salts may have made the permeation of the proteins easier since they decreased the electrical double layer thickness of the proteins. To improve separation performance, pH change and modification on membrane surface were performed. As a result of these experiments, changing pH of the feed solution did not affect the separation significantly but the positively modified membrane increased the retention of all proteins. For this production medium, diafiltration was also conducted to investigate the effect on separation behavior. Although separation performance increased, high rejection of hGH was observed from first set of the filtration. In addition to that, PES membranes were tried for filtrations due to suspected damage of cellulose membranes at the end of the diafiltration. However, there was still high retention of hGH and unexpected permeation of the larger molecular weight proteins. Second production medium containing recombinant granulocyte-colony stimulating factor (rGCSF) which was produced intracellularly, was used as feed solutions in ultrafiltration experiments after centrifugation and washing step with urea solution. In both 100 kDa and 30 kDa MWCO membrane filtration case, size based separation was achieved to some extent. For further separation of the desired protein, GCSF, pH of the feed solution was adjusted to different pH values and it was observed that the best result was achieved when the pH was 5.2 and permeate samples of the 100 kDa membrane filtration were used as feed solution for 30 kDa membrane filtration. Consequently, in hGH environment, hGH was selectively rejected by the membrane in any case. On the other hand, in GCSF environment, GCSF had selectivity over the other proteins in the pH range 4-6.5 especially in the case of pH was equal to 5.2.

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Citation Formats
B. Akcan, “Separation and purification of recombinant proteins by using ultrafiltration memranes,” M.S. - Master of Science, Middle East Technical University, 2017.