Analytical techniques
Antibody concentrations in purified solutions were determined by the absorbance at 280 nm, using the NanoDrop spectrophotometer
(Thermo Fisher Scientific, Wilmington, DE). Size exclusion high performance liquid chromatography (SE–HPLC) was used to monitor
the size heterogeneity of mAbs under native conditions on Agilent HPLC system using ChemStation as the controlling software
(Santa Clara, CA). A TSK-Gel G3000SWXL column (Tosoh Bioscience, Montgomeryville, PA) was utilized to separate HMW species, monomers, and fragments. The mobile
phase was phosphate buffer saline (without Ca2+ and Mg2+ ), pH 7.2 (Life Technologies, Carlsbad, CA).
A CHO host cell protein (CHOP) kit (Cygnus Technologies, Southport, NC) was used to determine the residual HCP level in purification
in-process samples and purified mAb product during the screening stage of experiments according to the manufacturer's protocol.
The HCP level in antibodies purified on STIC Nano was also measured by electrochemiluminescence (ECL) technology (Meso Scale
Discovery or MSD, Gaithersburg, MD) developed at ImClone. Briefly, 25 µL of 3 µg/mL in-house purified anti-CHOP capturing
antibodies were immobilized overnight on a 96-well MSD plate. The plate was blocked for 1 h with 3% BSA at room temperature.
25 µL of mAbs in 2-fold serial dilutions and HCP standards were added into the plate and incubated for 2 h at room temperature.
The bound HCPs were detected by addition of 25 µL of biotinylated anti-CHOP probe at 3 µg/mL, which was then detected by the
addition of 25 µL of streptavidin conjugated sulfo-Tag at 3 µg/mL. After the completion of reaction, 150 µL of MSD buffer
was added and the plate was read with MSD SECTOR Imager 2400 for relative electrochemiluminescence units (ECLU). The intensity
of the ECLU was proportional to the amount of residual HCP present in antibodies by extrapolation from the standard curve
with a quantification limit of 16 ng/mL. All HCP results were normalized to the in-house CHOP standards.
The leached MabSelect SuRe ligand in antibodies was determined using the RepliGen's protein A ELISA kit (Waltham, MA) with
a detection limit of 0.1 ng/mL according to the manufacturer's protocol. Residual CHO DNA in antibodies was measured by quantitative
PCR (qPCR) using the resDNASEQ quantitative CHO Kit (Life Technologies, Carlsbad, CA), combining high-recovery PrepSEQ sample
preparation and TaqMan based-quantitation. The assay was developed at ImClone using in-house CHO DNA standards. The quantification
limit of the assay was 0.1 pg/mL.
RESULTS AND DISCUSSION
Condition screening and optimization using 96-well plates
Table I
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Using protein A column chromatography under our platform operating conditions, we first prepared four antibodies, which served
as model proteins to evaluate STIC as an alternative antibody polishing platform to AEX chromatography. These partially purified
proteins and their properties are shown in Table I. Among them, Mab-D and Mab-S showed poor solubility at low ionic strength
solution conditions (< 5 mS/cm), which posed challenges to our current purification platform process. Mab-T was considered
as the worst-case scenario material in terms of levels of residual HCP and HMW impurities. Thus it was used here to illustrate
the procedure of condition screening and optimization. Process yield, HCP, and HMW were evaluated during the condition screening
and optimization. Although the study described here focused on HCP and HMW, a similar method could be applied for other impurities.
Figure 1
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The STIC equilibration buffer conditions were first screened using a Sartorius Sartobind STIC 96-well plate in a full factorial
experimental design, as described in the Materials and Methods section. The load eluate (or flowthrough) and wash from each
well, representing the purified product from an experimental run, were collected and evaluated for yield, HCP, and HMW. More
than 90% process yield was achieved in all 30 experimental runs. The residual HCP and HMW levels in the STIC purified Mab-T
were summarized in Figure 1. The residual HCP was <50 ppm at all tested conditions. Higher HCP removal was achieved when the
operating conditions moved to the center of pH-NaCl contour plot (see Figure 1a). In most cases, for a given NaCl concentration,
with increasing pH, HCP removal efficiency increased to the highest point and then started to decrease. This finding suggests
that the optimal pH operating window for Mab-T is at pH 7.0–7.5.
The presence of an optimal operating pH window is consistent with the amine protonation hypothesis reported previously (21,
23). As pH increases from 6.5 to 8.5, amine groups are less protonated. Thus, positive charges on the ligands available to
bind impurities decrease (21). Meanwhile, with increasing pH, there is an increase in the net negative charge of host cell
proteins, which results in more efficient binding to the positively charged ligands on the membrane adsorber. The presence
of an optimal pH operating window is due to the combination of amine protonation on the STIC membrane adsorber and changes
in protein surface charges.
In addition, in the pH range of 6.5–8.0, HCP removal was not dramatically affected by NaCl concentration, supporting the salt
tolerant nature of the STIC membrane adsorber. Through this quick, full factorial DOE study using 96-well plates, optimal
buffer conditions for HCP removal were identified.
Figure 2
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We next examined the impact of equilibration buffer conditions on the removal of HMW from the partially purified Mab-T. With
the understanding that in most cases, HMW level can be controlled to below 2.0% through pre-polishing steps, the goal of HMW
removal in this study was to reduce HMW species from 5.0% in the load to 3.0% in the flowthrough. When operating pH was increased
from 6.5 to 8.5, the HMW in the purified Mab-T increased from 2.7% to 4.0% as shown in Figure 1b. A concomitant decrease in
the IgG monomer was observed, suggesting that the HMW removal was less efficient as the pH increased. By contrast, HMW removal
was not sensitive to NaCl concentration, particularly in the range of 20–120 mM NaCl. These findings further suggest that
the process performance of Sartobind STIC is a result of its salt tolerant nature, supporting a wide design space of solution
ionic strength or NaCl concentration. In order to reduce the HMW in the final product to 3.0% and HCP to less than 30 ppm,
pH 7.0–7.5 and 25–75 mM NaCl were selected for further condition optimization.
Figure 3a
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The initial buffer conditions developed in the screening experiments were further optimized through 12 additional experimental
runs on a STIC 96-well plate via a central composite design (pH: 7.0–7.5, and NaCl concentration: 25–75 mM). The STIC response
surfaces of process yield, residual HCP, and HMW level, were defined based on these runs. Again, each well in a STIC 96-well
plate represented one unique combination of experimental conditions. As expected, >94% process yield was achieved in all experimental
runs. The sweet spot of the equilibration buffer conditions is illustrated as a pH-NaCl contour plot (see Figure 2). When
STIC was operated in the window of pH 7.2–7.3, and 30–60 mM NaCl, HCP was reduced to a lower level (< 20 ppm) and HMW to 3.0%.
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