Clark, William M.
This Major Qualifying Project (MQP) investigated the viability of using a two-phase electrophoresis separation method to purify a target protein from the mammalian cells that produced it. The aqueous two-phase system studied consisted of buffer, 6.0% Dextran 500, and 4.0% polyethylene glycol (PEG) 8,000. A small, 65 mL capacity batch electrophoresis device was used to apply the electric field to the two-phase system. SP2 mouse cells were the representative mammalian cells. The initial protein of interest, immunoglobulin (IgG) that can be found in sheep serum, partitioned almost entirely into the top phase of the two-phase system without the use of electrophoresis. The cells and cell debris favored the bottom phase. These results indicate that IgG could potentially be recovered from mammalian cells in a commercial process using aqueous two-phase extraction. Our second target protein, hemoglobin, partitioned approximately equally between the top and bottom phases of the PEG-Dextran system; thus, hemoglobin's partition coefficient was nearly one (K [approximately]= 1). Several electrophoresis experiments, with varying cell concentrations, voltages, and run times, resulted in the migration of positively charged hemoglobin from the bottom to the top phase. After electrophoresis, the effective partition coefficients, K[subscript]eff, ranged from 3 to 30, with K[subscript]eff generally increasing with longer run times. However, for longer experimental runs, hemoglobin bound to the mammalian cells and debris, and dragged these "fingers" of cells first to the interface, and eventually into the top phase. This presence of these cells in the top phase is detrimental to the overall value of this purification method. The addition of poly(ethylene imene), PEI, to the system eliminated the hemoglobin-cell binding, but its exact effect on the overall separation (hemoglobin yield) is still unclear. Performing the experiment using a two-phase system containing cell growth media diluted 10:1 with buffer, instead of pure puffer, resulted in overheating and boiling of the solution, and denaturing of the hemoglobin. We recommend that further attention and research be applied to this method of separating a target protein from mammalian cells, including the use of a larger, continuous-flow electrophoresis device. Other recommendations include: (1) replacing SP2 mouse cells with the universally utilized Chinese hamster ovary (CHO) cells, (2) replacing hemoglobin with a more biotechnologically valuable protein, (3) improving the current batch device, (4) investigating the use/effects of PEI, and (5) introducing a desalination process prior to electrophoresis, in order to reduce the cell media's ionic strength.
Worcester Polytechnic Institute
Major Qualifying Project
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