Biomedical Engineering Reference
In-Depth Information
product during the intermediate wash step. Further investigation indicated that the
lot-to-lot variation in the AEX chromatography resin was the root cause of the yield
variation. As shown in Fig. 7.5, the lot-to-lot variability in the ionic capacity of the resin is
correlated to the loss in yield, with higher ionic capacity resin more prone to yield loss
during the intermediate wash step. While the reason for this has not been investigated,
ligand overcrowding and steric effects have been shown to reduce binding capacity on a
number of adsorbent types [18].
The ionic capacity of each lot of AEX resin is reported on the vendor certificate of
analysis and is predictive of resin performance (Fig. 7.5). The operating conditions for
the AEX step can therefore be modified to modulate product losses during the wash step
according to ionic capacity for the specific lot of resin to be used. The amount of protein
removed during the intermediate wash step is dependent on the interaction of three
factors—the pH and ionic strength of the wash buffer and the level of protein loaded onto
the column. As one would predict, lower pH, and higher conductivity favor higher losses
of product during the wash step and more protein is lost during the wash step at higher
column loading. From a design space perspective, column loading was selected as the
simplest means of compensating for the variability in resin ionic capacity. As shown in
Fig. 7.5, the dependency of yield loss on ionic capacity during the wash step drops off
dramatically at column loadings below 20 g protein/L resin. For resin lots with higher
ionic capacity (e.g.,
>
150
m
eq/mL), column loading can be easily maintained below
20 g/L resin by increasing the number of chromatography cycles used to process all of the
product from 2 to 3 or 4 cycles per batch. When using resins of low ionic capacity (e.g.,
<
eq/mL) the preference would be to process all of the material in two AEX cycles.
The stability of the product in the AEX column load and eluate is adequate to allow for
150
m
35.0
Load 32 mg/mL resin
30.0
Load
22 mg/mL resin
25.0
≤
20.0
15.0
10.0
5.0
0.0
95
115
135
155
175
195
215
235
Resin ionic capacity (µeg/mL)
Figure
7.5. Anion exchange chromatography: lot-to-lot variability in resin ionic capacity can
result in variable amounts of product loss during the intermediate wash step. At decreased
column loading the effect of resin ionic capacity was negligible.
