Separation and purification of recombinant proteins by using ultrafiltration memranes

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.


Fabrication of helical polymeric hollow fiber membranes and characterization of their fouling behaviours
Yücel, Hazal; Çulfaz Emecen, Pınar Zeynep; Department of Chemical Engineering (2018)
Membranes are used in many separation processes such as gas separation, microfiltration, ultrafiltration and hemodialysis. Hollow fiber membranes are advantageous since they have a high surface area per volume and are easily backwashed which is an advantage for fouling removal. The most important factors that affect the performance of filtration membranes are concentration polarization and fouling. They increase operational cost and reduce membrane lifetime and permeate flux. One approach that can reduce co...
Separation of the protease enzymes of Bacillus licheniformis from the fermentation medium by crossflow ultrafiltration
Takac, S; Elmas, S; Çalık, Pınar; Ozdamar, TH (2000-06-01)
The separation from fermentation medium of extracellular serine alkaline protease (SAP) enzyme produced by Bacillus licheniformis was investigated using a crossflow ultrafiltration system. SAP was separated from the high molecular weight neutral protease (NP) and amylase (AMY) enzymes and from the low molecular weight organic acids and amino acids in a crossflow ultrafiltration system with 30000 Da and 10000 Da MWCO polysulfone membranes, respectively. The effects of transmembrane pressure (TMP), recirculat...
Investigation of activated sludge bioflocculation : influence of magnesium ions
Turtin, İpek; Sanin, Faika Dilek; Department of Environmental Engineering (2005)
Activated sludge systems are the most widely used biological wastewater treatment processes all over the world. The main working principles of an activated sludge system are the oxidation of biologically degradable wastes by microorganisms and the subsequent separation of the newly formed biomass from the treated effluent. Separation by settling is the most troublesome stage of an activated sludge process. A decrease in the efficiency of the separation of microbial biomass from the treated effluent causes a...
Separation of silkworm proteins in cocoon cooking wastewaters via nanofiltration: Effect of solution pH on enrichment of sericin
ÇAPAR, GÖKŞEN (2012-02-01)
Cocoon cooking wastewater contains waste silkworm proteins including sericin, which is a valuable raw material for many industries including cosmetics and pharmaceuticals. Sericin can be recovered via nanofiltration (NF) with high efficiency; however the process conditions need to be optimized to maximize separation. In this study, the effect of solution pH on NF performance was investigated for separation of silkworm proteins towards sericin enrichment. Solution pH (3.5-9.0) slightly influenced sericin rej...
Fabrication of polyethersulfone hollow fibers for ultrafiltration
Kaltalı, Gülçin; Çulfaz Emecen, Pınar Zeynep; Kalıpçılar, Halil; Department of Chemical Engineering (2014)
Hollow fiber membranes take an important part in membrane separation processes. They are used in many areas like gas separation, pervaporation, ultrafiltration and microfiltration processes due to their advantages like high membrane area per volume and easy backwashability. Poly(ethersulfone) (PES) is one of the most commonly used polymers in preparing hollow fiber ultrafiltration membranes. Due to its hydrophobic character, use of hydrophilic additives is usually necessary to make membranes resistant to fo...
Citation Formats
B. Akcan, “Separation and purification of recombinant proteins by using ultrafiltration memranes,” M.S. - Master of Science, Middle East Technical University, 2017.