|For the K-100 column, 250 g of a bacteria cell paste was thawed at 23° C and washed by resuspending in 1 L of 0.5 M sodium chloride and 20 mM tris-Cl at pH 8.0. The cells were harvested at 13,000 RPM in a GSA rotor using an RC5B centrifuge. Lysis of the cells was achieved by resuspending in 1 L of 20 mM tris-Cl, pH 8.0, with the addition of 0.5 g of hen egg white lysozyme and 0.05% Tween 80. One microgram/ml of DNAse was added and the sample was incubated for 2 hours at 4° C. Cell debris was removed by centrifugation as described above and the 1.1 L of supernatant was desalted by dialysis versus 80 L 20 mM tris-Cl pH 8.0.
The desalted crude extract was fractionated on a 5 X 10 cm Pharmacia K-100 column packed with the highly cross linked resin DEAE-Sepharose®* fast flow equilibrated with the above buffer. The sample was loaded and the column ran at a flow rate of 12 ml/min. Fractionation was achieved with a linear 500 ml gradient of 0 to 0.75 NaCl with the above buffer. Enzyme activity eluted in a single peak. After sample loading, the time required to perform gradient elution was 60 minutes and a total run time of 175 minutes.
For the Superflo® 1200 column, 1.3 Kg of a bacterial
cell paste was thawed, washed, lysed and cell debris was removed in essentially an identical manner as described above, except that the sample was several fold more concentrated having a total volume of 1.88 L. Desalting was performed as described above prior to fractionation.
Fractionation was performed on a Superflo® 1200 packed with DEAE-Sepharose®* fast flow equilibrated with the above buffer and operated at a flow rate of 250 ml/min. Elution of enzymatic activity occurred in a single sharp peak with a linear 5.0 L gradient of the above elution buffer. After sample loading, the time required to perform gradient elution was 24 minutes and total operation time was 80 minutes.
No noticeable backpressure was observed with either column. In a subsequent column, run flow rates of 350 ml/min with no significant pressure and essentially identical elution profile were achieved. This suggests higher flow rates are possible with no significant loss in performance.
Differences in sharpness of peaks are probably not significant because of the precision of the assay.
As seen from the chromatograms, exactly the same separation was obtained on both the development 200ml Axial Column and the production 1200 ml Radial Flow Column. Thus, a process can be developed on AFC and transferred to RFC in production. What should also be noted is that a higher binding of protein per ml of resin was observed using the Radial Flow Column. The flow rates were higher in the Radial Flow Column with no pressure build up, which happened in the axial column. This data suggests that processes can be converted from Axial columns to Radial Flow Columns with no optimization required. Bed height does not seem to play a role in this kind of on-off separation.
Faster Flow Rate on Radial Flow Column
Easy Scale-Up from Axial to Radial
Data courtesy of Dr. Brian Lawlis, Genencor Intl. (currently at Covance)