|
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.
Summary:
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.
Conclusions:
• Faster Flow Rate on
Radial Flow Column
• Higher Yield
• Easy Scale-Up from Axial to Radial
• Higher Productivity
Data
courtesy of Dr. Brian Lawlis, Genencor Intl. (currently at Covance)
|
Column:
10 ml (1 X 10 cm) Axial Column
Column:
20 L (1 X 10 cm) Radial Column
Column:
K-100 Axial Column
Column:
Superflo® 1200 Radial Flow Column
Flow
Rate:
250 ml/min (24 cv/hr)