Impact of Different Elution Profiles on Viral Filtration Step
As mentioned above, the CEX step is followed by a viral filtration using a normal flow parvovirus (NFP) filter is subsequently
used in the downstream step. During the development of this process step, it was determined that controlling the concentration
of NFP load to approximately 8.5 mg/mL and diluting the concentrated cation exchange product with a lower conductivity cation
exchange equilibration buffer to reduce the conductivity resulted in the optimal performance of this viral filtration step.
However, a consequence of having a wider than usual elution peak is a lower product concentration and a higher elution volume,
allowing little or no dilution with the equilibration buffer because of volumetric constraints in a manufacturing process.
This could result in a higher conductivity load for the NFP step. A higher conductivity load has the potential to impact the
flux across the filtration process, as shown in Figure 8.
Figure 8. Effect of conductivity on volumetric flux across an NFP filter
Process Development and Manufacturing Considerations
It may be possible to overcome the problem of lot-to-lot variability in resin particle size so that the cation exchange step
and subsequent steps are not affected. A suitable resin defining process could potentially remove the smaller particles, making
the resin lots much more uniform. Although such a process would likely produce a more normal elution profile, it could be
expensive in terms of the volume of resin discarded.
In this study, significant visual differences in elution profile and product volume were observed for the resin lots that
had > 10% of particles smaller than 40 μm. Concomitantly, there is an increase in product volume on elution with an increase
in the percentage of smaller particles. This increase in product volume generally parallels the increase in the asymmetry
factor measured with 1 M NaCl as the analyte. Studies with higher strength elution buffer and acetone rule out differences
in column packing as possible causes of the variable elution profiles. Instead, the unusual elution profile is likely caused
by higher accessibility of the antibody to the charged sites inside the pores. This higher accessibility is probably caused
by the shorter diffusional length inside the pores of smaller particles. An effect of this is probably a stronger interaction
and more resolution of the charged isoforms of the antibody, resulting in a broader elution profile. Despite these differences,
performance of the cation exchange step was not affected. No differences in yield, clearance of the impurities, or the distribution
of charged isoforms were observed for these lots. However, an increase in the cation exchange pool conductivity was observed
and this could impact the volumetric flux across the subsequent viral filtration step, especially at higher volumetric loadings.
Jane Wahome is a former development engineer from PDL BioPharma, Brooklyn Park, MN,
Weichang Zhou, PhD, is the senior director, bioprocess engineering, technology development, at Genzyme, Framingham, MA, and Amitava Kundu, PhD, is the associate director, process development at GenMab, Brooklyn Park, MN.