Finally, the correlation was developed from a curve fit analysis of the expression with the experimental data. The reliability
of the model developed at a 100-mm scale was challenged by comparing the model with large-scale column experimental data (Figure
5) with promising results.
Figure 5. Large-scale experimental data versus model
Process parameters could be predicted at given conditions providing insights into the stability of the packed bed and settings
to achieve optimum performance. Significant influence of the interstitial bed porosity and particle size on pressure drop
(Figure 6) shows that a nonhomogeneous packed column will yield a larger pressure drop than expected from a homogeneous pack
in which the interstitial porosity or void fraction is smaller. Therefore, a typical pressure drop may be an indicator of
column bed deterioration. Increased interstitial porosity suggests an unstable pack because of partial motility of the beads
in the column. Decreased bead size or particle diameter for unstable packs increases the pressure drop substantially at any
given flow rate.
Figure 6. Pressure drop of packed beds of different interstitial bed porosities
The bench-scale results confirmed headspace formation at process flow rate with previous resin compression parameter thus
marking a relationship of resin amount to the pack instability and performance variability seen on large scale. To optimize
the chromatography operation, it was considered to pack at higher compression to increase resin stability at process flow
rates. The use of more resin rather than lowering the bed height to compress further the resin or decreasing flow rates to
minimize head space was avoided to prevent resolution loss and additional changes to regulatory filing.
The pressure-flow model predicted that incrementing compression to 1.18 (as recommended by manufacturer) will require a minimum
flow velocity of 191.6 cm/h to achieve a bed height of 12 cm. For large-scale columns using flow-packing techniques, in which
the resin is compressed by increasing the mobile phase velocity, this flow is not enough because of the resin expansion experienced
once the flow is stopped to lower the top plate adaptor to the desired height of 12 cm, and therefore, a higher flow rate
It was determined from the pressure-flow simulation data that, for an optimum packing flow (85% of u
CRI), the required flow rate to flow-pack the resin to 1.18 compression is close to 226 cm/h. For the existing 1,000-mm column
hardware, this represented a limitation because the expected pressure drop of the resin plus the pressure drop of the hardware
exceeded the maximum allowable working pressure of the column. Axial compression was introduced to the packing procedure to
compensate for system limitations of the flow-pack technique at higher compression.
LARGE-SCALE PACKING STUDIES
Packing studies were performed at large scale (1,000 mm) to understand the influence of column hardware, flow cell geometry
effects, and variable resin amount on the pack stability. The studies consisted of a series of experiments that include dye
and cleaning studies, and evaluation of various packing approaches.5
Dye studies using phenol red revealed equipment limitations of flow distribution under the top slurry nozzle through inspection
of bed cross-sectional cuts at various angles, indicating that the flow velocity under the slurry nozzle is lower than the
average velocity of the sample bands (~2.5 CV) or dye injection. A low compression pack revealed low stability underneath
mobile phase inlet across bed diameter. By increasing the resin amount and thus the compression, the sample band distribution
was more homogeneous throughout the bed under the top slurry nozzle.
A cleaning study evaluated the column cleaning using phenol red as a visual indicator for cleaning aptness. The packed column
was buffer exchanged from packing buffer into a 0.1 g/L phenol red in packing buffer for 2 CV duration. The equilibration
followed a five-hour static hold to allow diffusion toward stagnant zones at the edge of the packed bed. Once hold was completed,
the bed was washed with packing buffer at a 3 CV duration followed by a 1.5 static hold and storage. These set of conditions
were selected as a stress-case scenario when compared to current hold parameters. Inspection of cross-sectional cuts of the
packed bed revealed no traces of phenol red. This study provided cleaning capability of the column as retention volume basis
(it is not an evaluation of impurity or carryover clearance that could bind into the resin during the chromatography step).