Development of an Alternative Monoclonal Antibody Polishing Step - The authors describe a mAb polishing step using salt tolerant interaction membrane chromatography. - BioPharm International
Breakthrough curves were used to determine the DLC of antibodies on 1 mL STIC Nano membrane adsorber. When the low-pH- treated
protein A eluate of Mab-T was neutralized to the pH value (defined in the previous Condition Screening and Optimization section)
and clarified, the conductivity fell into the optimal operating window. This conditioned protein A eluate was then directly
applied into the STIC Nano in flowthrough mode. The Mab-T chromatogram is shown in Figure 3a. As expected, the sharp rising
shape of the breakthrough curve during the load and sharp decreasing UV trace in the wash step suggest that mass transfer
in STIC membrane adsorber is convective flow, and not limited by diffusion as in the case of porous chromatography resins.
This finding is consistent with the results of previous works on other membrane adsorbers (15–17, 24). In addition, compared
with Q column chromatography, the product pool was not diluted significantly by the wash, as the load volume was 150 MV while
the wash volume was only 5 MV (see Figure 3a). Thus, STIC might provide the benefit of a lower dilution factor because of
the smaller volume of buffer required in the wash step, which is extremely valuable when there is a limit on tank capacity
in manufacturing.
Figure 4
We examined the HCP breakthrough by collecting different flowthrough fractions and determining the HCP level in each fraction.
As shown in Figure 3b, Mab-T dynamic loading capacity was 0.5 g Mab-T/mL-STIC at 10 ppm HCP breakthrough or 0.9 g Mab-T/mL-STIC
at 20 ppm breakthrough, which was higher than that achieved from Q column chromatography in a flowthrough mode (data not shown).
Table II
The same screening and optimization procedures using 96-well plates were also applied to three other antibodies, Mab-S, Mab-D
and Mab-K. The DLC was determined under the conditions defined by screening and optimization experiments. For Mab-S, an equilibration
buffer at pH 7.0 and 9.53 mS/cm, equivalent to 60 mM NaCl, was used in the experiment. Again, different flowthrough fractions
were collected and HCP was determined. With increasing load of Mab-S, HCP in the flowthrough remained at a background level
up to 2.5 g mAb/mL-STIC (see Figure 4). After that HCP started to increase gradually and reached 10 ppm at 3.0 g mAb/mL-STIC.
By contrast, only 0.5 g /mL-STIC DLC was observed for Mab-T. The significant difference in the capacity might be due to the
initial HCP level (578 ppm for Mab-T vs. 212 ppm for Mab-S), as well as the initial HMW level (2.0% for Mab-T vs. 0.9% for
Mab-S). Similarly, the DLC for Mab-D and Mab-K was 3.5 and 3.7 g-mAb/mL-STIC, respectively (see Table II), under the tailored
operating conditions developed on 96-well plates. Mab-D and Mab-K thus showed a reasonably high process capacity on the STIC
membrane adsorber.
