Biomedical Engineering Reference
In-Depth Information
to give a final concentration of approximately 1.0M and the product is loaded onto the
HIC column. The column is subjected to an intermediate wash step containing 1.0M
ammonium sulfate, and the product is then recovered with an elution buffer containing a
reduced ammonium sulfate concentration. The HIC step reduces the level of host-cell
related impurities, aggregated FP, and the level of a slightly more hydrophobic
C-terminus variant (CTV).
Relatively narrow release specifications were established for the level of the CTV
allowed in the purified drug substance on the basis of previous clinical exposure and a
limited amount of manufacturing experience. As additional manufacturing experience
was gained, however, variability in the amount of the CTV produced during cell culture
was observed. This resulted in variability in the amount of the CTV in the HIC column
load and eluate, which resulted in the occasional failure of the release specification for
the CTV in the drug substance.
The reduction of the CTV form of FP on the HIC column is primarily controlled
by column loading and the concentration of ammonium sulfate in the elution buffer
(Fig. 7.4a). As anticipated, increased ammonium sulfate concentration in the elution
buffer increases the retention of the more hydrophobic CTV, which results in improved
clearance. Loading the column to lower levels of protein also improves the removal of the
CTV. These two variables interact such that the effect of ammonium sulfate concentra-
tion on clearance of CTV is more pronounced at a lower column loading. As expected,
increased ammonium sulfate concentration in the elution buffer decreases the overall
recovery of the product. This effect is dramatic at low column loadings andminimized by
operating at the higher end of the column loading range (Fig. 7.4b). Consequently, the
preferred design space would entail a flexible ammonium sulfate concentration in the
elution buffer, with column loading maintained at the higher end of the range (
7.5 g
protein/L resin). The opportunity to vary the ammonium sulfate concentration in the
elution buffer from 0.75 to 0.95M would allow the column to be operated under
conditions that optimize the trade-off between CTV removal and step yield as needed.
It should be mentioned that viral clearance has been shown to be comparable across the
proposed range of elution buffer concentrations. Ideally, implementation of the HIC
design spacewould be supported by process analytical technologies. Specifically, a rapid
measurement of the level of the CTV in the bioreactor or after the rPA capture column
could be used to prompt a decision as to the concentration of ammonium sulfate to be
used in the HIC column elution buffer.
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7.7 ANION EXCHANGE CHROMATOGRAPHY
This final example pertains to the purification of a humanized monoclonal antibody
(mAb3) by anion-exchange (AEX) chromatography. The product is loaded onto the
column and the column is subjected to an intermediate wash step with slightly elevated
conductivity. The intermediate wash step was developed to remove low levels of more
basic charged isoforms produced during cell culture. The basic isoforms, however, are
not well resolved and even under fully optimal conditions some of the desired product
(
4%) is removed along with the undesired charged isoforms. Further increase in losses
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