PACKING PROCEDURE EVALUATION
The pressure flow model suggested that flow pack techniques were not sufficient to reach the bed height of 12 cm at the desired
compression because of high pressure drop and resulting hardware limitations. The packing procedure was simplified and optimized
based on predicted hydrodynamic properties and use of readily available hardware. Several packing modifications were tested
to keep the pack simple, reliable, and reproducible with the current hardware limitations and availability (i.e., flow meter,
pressure gauge, pump, and column).
The final procedure includes resin quantification through centrifugation. Exact resin amount is transferred into a primed
column chamber at a rate of 61 cm/h monitoring flow rate at the column top mobile phase port. The buffer from the column is
recycled back into the resin slurry-mixing tank to assure that all the resin is transferred. After the transfer is completed,
the resin is consolidated with a downward flow at 61 cm/h followed by lowering the top head-plate to 12 cm with the top port
open and bottom closed. The top head-plate descent rate was controlled indirectly by targeting a predetermined top mobile
port flow rate. This methodology allowed control over the top head-plate descent rate without expensive hardware upgrades
to the large-scale column. Gradual increments are made to hydraulic pressure on the column as packed bed compression increases.
This mechanical compression is then followed by a downward high flow conditioning to uniformly stabilize the effect of this
compression. Unnecessary steps that added to the time required to pack the column and increased the variability of the packs
were removed. Buffer consumption was reduced to a minimum through recycling and priming. During characterization, the procedure
was repeated various times and packs were qualified with modified method. Table 1 shows the elution volume, height equivalent
to a theoretical plate (HETP), and peak asymmetry values for the six experimental packs.
Table 1. Qualification of low and high compression packs
By definition, low HETP values are associated with increased separation or peak resolution. Reduction of HETP indicator from
the new qualification method suggested a better performance on highly compressed packs. Lower HETP results were observed with
the use of final packing equipment once implemented on the large-scale process.
Ultimately, the process-development data generated from pressure-flow and packing studies fed information for the design of
a packing skid, defining flow rates, pump sizing, pipe ID, instrumentation, and procedural logic.
PILOT-SCALE AND LARGE-SCALE CHROMATOGRAPHY RUNS
The purification process was scaled to fit pilot 450 mm and 1,000-mm column keeping column-loading ratio consistent and equal
linear velocities. The sole idea of performing an actual run at large scale as part of the process development strategy was
to account for geometrical differences found on flow-cell hardware between each scale that could affect performance or scalability
of results from bench scale. Table 2 shows the pertinent column information.
Table 2. Column information for chromatography runs
Each column pack was qualified with new pulse injection method and judged for acceptability based on the results of the packing
studies. All the resin slurries were buffer exchanged into packing buffer. The resin quantification method relied on a column-based
method to determine the percentage of resin. The pilot-plant chromatograms (Figure 9) reflected a good resolution but also
a shift in retention volume.6
Figure 9. Pilot plant run at high compression versus commercial run at low compression