Versatile Use of Mixed-Mode Sorbents for Polishing Monoclonal Antibodies Following Protein A Capture
Aleksandar Cvetkovic , Amitava Kundu and René Gantier - 1. Pall Life Sciences, 50 Bearfoot Road, Northborough, MA, USA, 2. Genmab Inc., MN, USA
1
introduction
Mixed-mode chromatography is a flexible and cost-effective method applicable to intermediate and polishing steps in
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1
Optimization of Mixed-Mode Sorbents for
Post-Protein A Step MAb Purification
Figure 3
Criteria for MAb yield of recovery limited to 60% since higher yield is expected for higher protein load on column.
Data Processing Approach Used to Determine Optimal Operation Conditions (MAb Yield of Recovery and Purity) Using HEA
`` MEP and HEA HyperCel sorbents: meet operational space requirements:
HyperCel Sorbent in FT Mode
•• Broad affinity for different proteins in HCP mix of load
•• High selectivity allowing MAb and aggregates separation
monoclonal antibody (MAb) purification.
We used various mixed-mode sorbents for single step polishing of MAbs following Protein A capture. High-throughput
Load: Post-Protein A eluate (MAb pI 7.6; MAb concentration 19.5 mg/mL, 1.9% aggregate and 235 ppm (ng/mg MAb) host cell
screening tools were employed to identify optimum conditions in both bind/elute and flow through modes, and conditions
proteins (HCP).
transferred to a chromatography column.
Sorbents: MEP, HEA, PPA HyperCel (Pall)
While bind/elute mode of operations was not proven very efficient, some mixed mode sorbents used in Flow-through mode
The high throughput optimization strategy (pH and conductivity screening) is shown in Figure 2.
(HEA HyperCel™, PALL) provided excellent contaminant (aggregates and HCPs) clearance with MAb loads up to 150 mg/mL of
•• More flexible pH and conductivity conditions on HEA HyperCel sorbent
`` PPA HyperCel sorbent: no sweet spot identified, does not combine good MAb recovery and efficient capture of aggregates
(probably due to higher hydrophobicity of the ligand).
Optimal Conditions for FT Processing of MAb Determined by HTE Study
Figure 2
Mixed Mode Chromatography Optimization Strategy
Mixed-Mode Chromatography: MEP, HEA
and PPA HyperCel Sorbents
``High Throughput Screening on AcroPrep 96-well plates filled with
chromatography sorbents allows fast optimization of MAb purification
conditions (mode of operation, optimum pH/conductivity)
``Pall mixed-mode sorbents used in flow through as post-Protein A step
provide excellent separation performance:
Table 3
sorbent. This study confirms the applicability of mixed mode chromatography steps for post-Protein A purification of MAbs.
Conclusions
Sorbent
pH
Conductivity, mS/cm
MEP HyperCel
4.5
5.1
PPA HyperCel
3.9
4.0
HEA HyperCel
4.4
29.3
••High yield of recovery
••Efficient contaminant removal (aggregate, HCP)
••High MAb load  reduced sorbent volume
Transfer of Optimized FT Mode Conditions to Column Chromatography
Mixed-mode chromatography exploits multiple, distinct protein-ligand interactions to adsorb target proteins or impurities
Optimum conditions obtained for the three mixed-mode sorbents in FT mode (Table 3) were transferred on pre-packed Pall
(see Figure 1 and Table 1).
PRC columns (1 mL, 0.5 cm I.D., 5 cm bed height, 4 minute residence time). A sample load up to 150 mg MAb per mL of
Mixed-mode chromatography offers new solutions to separations
sorbent was applied. Results are shown in Figure 6a (aggregate content for all three mixed-mode sorbents) and 6b (HCP
`` Alternative to traditional methods (ion exchange, HIC, affinity…).
content for HEA HyperCel sorbent only).
••Best performance obtained with specific MAb on HEA HyperCel sorbent
up to 150 mg/mL MAb load
––~ 95% MAb yield
––< 0.5% aggregate
––< 25 ppm HCP
`` Alternative to cation exchange when feedstock is directly loaded (no dilution or UF/DF).
`` Alternative/complement to conventional hydrophobic interaction (HIC) or hydroxyapatite.
``Use of mixed-mode sorbents in flow through mode is a good alternative to
high capacity IEX sorbents used in Bind/Elute mode
Figure 6
Figure 1
Transfer of Optimized HTE Conditions on Column
Identification of Sweet Spot pH and Conductivity in B/E Under High pH Load Conditions
High Throughput Experiment (HTE) for the Determination of Optimum pH and Conductivity
Conditions in Bind/Elute (B/E) and Flow Through (FT) Modes
An axial point response surface DoE (Minitab, USA) was used to determine optimum pH and conductivity conditions for FT and
binding steps. The MEP, HEA and PPA HyperCel sorbents, manually dispensed in Pall 96-well AcroPrep™ Advance 1 mL filter
plates, were loaded with 5 mg MAb per mL of sorbent.
The data collected during HTE study (Table 2) were processed as described in Figure 3 (data obtained on HEA HyperCel
Potential Protein/Ligand Interactions Using MEP, HEA and PPA HyperCel Sorbents
sorbent with low load pH in FT mode shown as example) and results for both FT and B/E modes are shown in Figures 4 and 5,
Hydrophobic Interaction
Ionic Interaction
Pi-Pi Interaction
Thiophilic Interaction
MEP HyperCel
Ethyl group
Amine (pKa = 4.8)
Pyridine group
Thio-ether group
HEA HyperCel
Hexyl group
Amine (pKa ~ 8)
PPA HyperCel
Propyl group
Amine (pKa ~ 8)
Phenyl group
-
The aggregate content was analyzed for MEP/HEA/PPA HyperCel sorbents as a function of MAb load (6a). The HCP content was analyzed
only for HEA HyperCel sorbent (6b).
Operation space criteria: (red) MAb binding yield > 95%; (green) MAb elution yield > 90%
The best performance for aggregate removal was obtained on HEA HyperCel sorbent (Figure 6a). The HCP content was
Good binding achieved at pH >6 on all sorbents but low MAb yield at elution (pH 3). Using mixed-mode in B/E mode at post-
therefore analyzed by ELISA only for this sorbent (Figure 6b). The final MAb yield and contaminant content (aggregate
protein A step is not adapted for the purification of this specific MAb.
and HCP) on HEA HyperCel sorbent at 150 mg MAb load per mL of sorbent presented in Table 4 show the good overall
performance of HEA HyperCel sorbent in FT mode.
Table 1
Sorbent
Aggregate content (C/C0), %
Figure 4
Aggregate content (C/C0), %
Ligand Structure of HyperCel Mixed-Mode Sorbents
respectively.
Figure 5
Identification of Sweet Spot pH and Conductivity in FT Under Low pH Load Conditions
Table 2
Table 4
Best Column Performance: HEA HyperCel Sorbent (150 mg/mL MAb Load)
Sorbent
pH
Conductivity(mS/cm)
MAb Yield (%)
Aggregate (%)
HCP (ppm)
HEA HyperCel
4.3
29.3
94.9
< 0.5
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Experimental Conditions Used in HTE Screening Study
Conditions Tested
Sorbent
MEP, HEA and PPA HyperCel
Protein load
Load pH
5 mg/mL of sorbent
Low pH 3 to 6
Load conductivity (mS/cm)
Elution pH
Elution conductivity (mS/cm)
High pH 6 to 9
4 to 30
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Operation space criteria: (red) MAb yield > 60%; (green) FT HCP content < 25 ppm; (blue) FT aggregate content <0.75%
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