Column Evaluation with NaCl
Table 1 shows that the column asymmetries, evaluated with 1 M NaCl, varied from 1.0 to 1.7.4 The table also points to a correlation between column asymmetry, as evaluated with NaCl and elution volume. Generally, as
asymmetry increases, column volume also increases. This suggests that the columns may have been poorly packed, resulting in
varying flow patterns. To determine if this was the case, the probable correlation was investigated using two methods.
Table 1. Column asymmetries
Column evaluation with acetone. In the first method, ionic interactions between the analyte and the resin were eliminated by using a 3% v/v acetone injection.
Acetone should not interact ionically with the cation exchange resin. Column asymmetries when evaluated with acetone were
almost identical (Figure 5). Acetone evaluation showed that in the absence of ionic interactions, all four columns showed
equivalent flow characteristics.
Figure 5. Peaks from acetone injections
Elution with high conductivity buffer. The second method of investigation used a high conductivity buffer to elute the bound antibody off the columns. Columns containing
cation exchange resin lots A and D were used because they represented the extremes of asymmetry and column volume. The purpose
of this experiment was to see if the different shapes of the elution peaks were caused by poor column packing or if they were
because of different selectivity of the resin for the charged isoforms of the antibody. The use of a much stronger elution
buffer would significantly reduce the selectivity for all the isoforms so that if column packing was predominantly responsible
for the different peak shapes, these differences would still be observed.5 The chromatograms are shown in Figure 6.
Figure 6. Elution with high conductivity buffer
The solid lines show the widely varying elution profiles and volumes that were produced when the normal elution buffer was
used. The dashed lines show that with the high conductivity buffer, the elution peaks were almost identical. The fact that
no difference in the elution profile was observed under high conductivity conditions indicates that the variation in the elution
under normal conditions is largely because of the different lots of resin having a difference in selectivity for the charged
isoforms of the antibody. Thus, this variation is not likely to be related to column packing. To further understand the reason
for this different selectivity, we examined the ionic capacity and the particle size distribution of the four resin lots.
Certificates of analysis from the manufacturer contained the ionic capacity of each lot of resin. Table 2 lists the ionic
capacities of each of the four lots. They are very similar and no trends are apparent. Ionic capacity of the resin lots did
not cause the lot-to-lot variability in peak shape and elution volume that we had observed.
Table 2. No variation in ionic capacity