A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
Leslie S. Wolfe
Eric J. Suda
Carnley L. Norman
Sigma Mostafa
Abhinav A. Shukla
Process Development & ManufacturingKBI Biopharma, Durham, NC
Overview
● Background○ Mixed Mode Chromatography○ Mixed Mode Resin characterization
● Comparison of Mixed Mode Resins○ High throughput method for identifying optimal
operating ranges for mixed mode resins○ Chromatography experiments to characterize HCP
and HMW removal● Conclusions
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Mixed Mode Chromatography
● Takes advantage of more than one type of interaction ○ i.e. ionic, hydrophobic, hydrogen bonding
● Provides enhanced selectivity, “pseudo-affinity”● Can reduce process steps● Several mixed mode resins have recently been developed with:
○ Increased loading capacities○ Higher ionic strength tolerance
GE Healthcare, CaptoTM MMC ligand
Ionic interactions
Hydrophobic interactions
Hydrophobic interactions
GE Healthcare, CaptoTM Adhere ligand
Ionic interactions
Capto is a registered trademark of GE Healthcare
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Mixed Mode Ligand Examples
Pall Corporation
NuviaTM cPrimeTM
Bio-Rad Laboratories
Different mixed mode resins are available on the market and each couldpotentially result in molecule specific performance (yield and selectivity)
EshmunoTM HCXEMD Millipore
CaptoTM AdhereGE Healthcare
MX-Trp-650MTosoh
CaptoTM MMCGE Healthcare
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Capto is a registered trademark of GE Healthcare Capto is a registered trademark of GE Healthcare Nuvia and cPrime are registered trademarks of Bio-Rad
TOYOPEARL is a registered trademark of Tosoh corporation Eshmuno is a registered trademark of Merck KGaA
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Mixed Mode Chromatography potential based on previous work at KBI● Demonstrated that mixed mode chromatography has the potential of
achieving superior selectivity by effectively exploiting multiple properties of a target protein
● Previous work at KBI work focused on characterizing Capto MMC resin and the use of mobile phase modulators to further enhance selectivity○ Inclusion of Capto MMC into a mAb process was compared with and
without selective washes○ Inclusion of a selective wash on Capto MMC resulted in higher product
purity
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Wolfe, L., Barringer, C., Mostafa, S., Shukla, A. Multimodal chromatography: characterization of protein binding and selectivity enhancement through mobile phase modulators, Journal of Chromatography A, 1340, 151-156, 2014.
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Effects of Modulators on Different Proteins● Resin: Capto MMC● Modulator added to equilibration, wash and elution
buffers● Products eluted with a linear NaCl gradient● Impact of modulator on retention was determined by the
NaCl concentration at peak maxima
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Modulator Modulator EffectMgCl2, NaSCN, KI Decrease hydrophobic interactions
Ethanol, Methanol, Isopropanol Decrease hydrophobic interactions (used in low concentrations)
Urea, Sodium Thiocyanate Weakens hydrogen bonding, denaturantGlycerol Weakens hydrophobic interactionsEthylene Glycol Weakens hydrophobic interactions and hydrogen bonding
Arginine Weakens hydrophobic interactions, induces protein unfolding, disrupts electrostatic interactions
Ammonium Sulfate Strengthens hydrophobic interactions
Model Protein
RNase (pI 8.9)
Lysozyme (pI 9.6)
mAb1
mAb2
mAb3
mAb4
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
7Effect of pH on Retention
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Log k’ vs. Log [NaCl]: Theory
log k’ = A – Blog(csalt) + C(csalt)
Melander, W.; El Rassi, Z.; Horvath, Cs. Journal of Chromatography, 469, 3-27, 1989.
The retention factor under isocratic conditions is represented by:
k’ = (tr – tm)/tmtm = time for mobile phase to pass through columntr = target protein retention time
Melander et. al described the dependency of the linear retention factor on a mixed mode sorbent as a function of salt concentration as:
● Electrostatic interactions predominate: a linear relationship is expected between log k’ vs log[NaCl]● Hydrophobic interactions predominate: a linear relationship is expected until a minimum is reached
at which point further increases in salt result in increased retention
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Log k’ vs. Log [NaCl]
RNase
● electrostatic interactions
● No effect from urea or ethylene glycol
All experiments performed at
pH 7.0
Lysozyme
● hydrophobic and electrostatic interactions
● urea has largest effect
mAb1
● driven by electrostatic interactions, hydrophobic contribution
● Urea and arginine have the largest effect
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Taking Advantage of Modulators
● Incorporation of modulators into process can help increase selectivity and purity of product
● Combinations of modulators can further enhance process step
● Goal: Utilize mobile phase modulators to decrease HCP levels during Capto MMC process step for antibody purification
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Incorporation of a process step utilizing a modulator wash can improve overall process HCP clearance
baseline
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Overview
● Background○ Mixed Mode Chromatography○ Mixed Mode Resin characterization
● Comparison of Mixed Mode Resins○ High throughput method for identifying optimal
operating ranges for mixed mode resins○ Chromatography experiments to characterize HCP
and HMW removal● Conclusions
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Mixed Mode Chromatography & KBI’s mAB Purification Process Platform● Mixed Mode resins are ideal for KBI’s mAb platform process since by the nature of our business
there is a significant diversity in mammalian cell clones/cell culture harvest and mAbs● Mixed mode resins can provide added robustness to a mAb platform to reduce both HMW and HCP
impurities● Given the complexity of mixed mode resin interactions, optimizing a mixed mode chromatography
step can be time consuming and potentially material limiting
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DS Process Platform
Cell Line Diversity
Media/feed type diversity
HCP level variability
Cell Density variability
HMW level variability
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Objectives● Determine if there is at least one mixed mode cation exchange/hydrophobic
interaction polishing step resin that performs well for multiple mAbs○ Ideal candidates for a mAb process platform
● Present a high throughput, low material requirement method for identifying optimal operating ranges for multiple mixed mode resins in a single set of experiments○ Show how the method can be use to identify optimal chromatography
conditions
● Show the relationship between the high throughput screening data and chromatography performance for different mAbs
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Mixed-mode cation exchange/hydrophobic interaction resins15
NuviaTM cPrimeTM
Bio-Rad LaboratoriesMX-Trp-650M
Tosoh
EshmunoTM HCXEMD Millipore
CaptoTM MMCGE Healthcare
TM
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Overview
● Background○ Mixed Mode Chromatography○ Mixed Mode Resin characterization
● Comparison of Mixed Mode Resins○ High throughput method for identifying optimal
operating ranges for mixed mode resins○ Chromatography experiments to characterize HCP
and HMW removal● Conclusions
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
High Throughput Screening: Plate Experimental DesignVariable Conditions Evaluated
Load pH 4, 5, 6, 7, 8
Elution pH 4, 5, 6, 7, 8
Elution [NaCl] (M) 0.20, 0.65, 1.10, 1.55, 2.00
Three mAbs evaluated under a series of conditions
● One plate executed per load pH
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
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● The low yield process condition zone size varies significantly with respect to resin, mAbs and process conditions and are easily identifiable
● In general, Capto MMC has slightly larger low yield zones for the mAbs and test conditions within our typical operating pH range .
● In general, each resin has a characteristic surface response map profile
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Overview
● Background○ Mixed Mode Chromatography○ Mixed Mode Resin characterization
● Comparison of Mixed Mode Resins○ High throughput method for identifying optimal
operating ranges for mixed mode resins○ Chromatography experiments to characterize HCP
and HMW removal● Conclusions
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Chromatography Experiments ● Goal: Compare resin ability to reduce HCP and HMW levels at multiple pHs● Linear gradient studies executed at pH 5, 6 and 7
○ Product eluted from 0-2M NaCl○ Each 1/8th CV fractions collected ○ Analyzed fractions by SEC-HPLC for high molecular weight species (HMW)
■ Fractions containing cumulative yield 20%-80% pooled to reduce sample numbers
■ Fractions containing high %LMW were excluded from the cumulative yield vs. cumulative purity analysis
● Fractions containing 0 – 80% product yield after SEC-HPLC analysis were pooled for host cell protein (HCP) analysis
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Neutralized Protein A pools used for experiments
mAb Feed %HMW Feed HCP Level (ppm)
mAb1 ~1% 4,395
mAb2 ~4% 135
mAb3 ~8% 3,689
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Chromatography Experiments: Elution [NaCl] & elution peak volume data 21
● In general, Capto MMC and Eshmuno HCX resulted in higher elution volumes
● Nuvia cPrime elution volume changes significantly when pH increases from pH 5.0 to 6.0
∞ = target protein did not elute during NaCl gradient
● As expected, elution [NaCl] decreases with increasing pH
● Capto MMC and Eshmuno HCX require higher elution [NaCl] at each elution pH
● Tosoh MX-Trp had the lowest elution [NaCl] at each pH and did not vary as much with respect to pH
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Cumulative Yield vs. Cumulative HMWmAb Feed %HMW
mAb1 ~1%
mAb2 ~4%
mAb3 ~8%
mAb1, pH 5.0
mAb2, pH 5.0
mAb3, pH 5.0
mAb1, pH 6.0
mAb2, pH 6.0
mAb3, pH 6.0
mAb1, pH 7.0
mAb2, pH 7.0
mAb3, pH 7.0
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
HCP Reduction● Not all experiments yielded 80%
product in eluate fractions (*).○ Capto MMC at pH 5.0 did not
yield any product for the 3 mAbs tested
○ mAb 3 only achieved 80% yield in 6 of the 12 conditions tested
● HCP clearance is mAb specific with conditions tested.○ The greatest HCP reduction
consistently achieved across the experiments is with mAb 1
mAb Feed HCP Level (ppm)
mAb1 4,395
mAb2 135
mAb3 3,689
** * ** ** *
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Summary Results – Optimization FactorResin mAb 1 Optimization Factor
(Max. Yield * %HMW Red. * %HCP Red.)mAb 2 Optimization Factor
(Max. Yield * %HMW Red. * %HCP Red.)mAb 3 Optimization Factor
(Max. Yield * %HMW Red. * %HCP Red.)
pH 5.0
Capto MMC N/A* N/A* N/A*
Nuvia cPrime 0.81 0.32 N/A**
Eshmuno 0.39 N/A** N/A**
MX-Trp 0.08 N/A** N/A**
pH 6.0
Capto MMC 0.44 0.04 0.39
Nuvia cPrime 0.51 0.26 0.33
Eshmuno 0.08 -0.15 N/A**
MX-Trp -0.21 0.11 0.07
pH 7.0
Capto MMC 0.47 0.33 0.31
Nuvia cPrime 0.36 0.22 0.08
Eshmuno 0.29 0.65 N/A**
MX-Trp -0.03 0.01 0.14
*mAb did not elute from Capto MMC at pH 5.0 in up to 2M NaCl**Not tested as 80% yield was not achieved
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Findings regarding the relationship between HT filter plate & chromatography data
● High throughput filter plate data can be used to quickly limit the focus of a chromatography evaluation using minimal amounts of the target protein○ pH and NaCl conditions not favorable for high yields
are easily identifiable● There is a correlation between yield variability for each
condition (load pH and elution pH) and monomer selectivity○ more variability leads to better selectivity
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Best resins for each measure & overall performance based on the chromatography experiment results
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● There is not one best resin for all mAbs and conditions evaluated● For each mAb, the best resin for each performance measure varies● Nuvia cPrime provides good overall performance for all mAbs tested● Tosoh MX-Trp consistently had low “optimization factor” scores for all of the mAbs and
conditions tested*Conclusion will change based on the relative importance of each factor.
MoleculeBest resins based on
yieldBest resins based on
HMW reductionBest resins based on
HCP reductionOverall best resin based on
optimization factor
mAb1Capto MMC
Tosoh MX-TrpNuvia cPrimeCapto MMC
Eshmuno HCXCapto MMC
Nuvia cPrimeCapto MMC
mAb2Capto MMC
Tosoh MX-TrpEshmuno HCXNuvia cPrime
Eshmuno HCXCapto MMC
Eshmuno HCXNuvia cPrime
mAb3Tosoh MX-TrpNuvia cPrime
Capto MMCEshmuno HCX
Capto MMCTosoh MX-Trp
Capto MMCNuvia cPrime
A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Conclusions● Demonstrated that a high throughput filter plate method can be used as an
efficient approach in screening chromatography resins and identifying their optimal process conditions○ Can assess within a short time period if a resin is suitable for purifying a
specific molecule using minimal protein○ Narrows the focus of chromatography experimental conditions
● Each resin evaluated has its strengths● Overall performance is based on selecting optimal operating conditions for each
resin and mAb combination○ Nuvia cPrime provides good overall performance for all mAbs tested○ Backup options will be needed to meet the unique challenges of each mAb
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A Comparison of Multimodal Chromatographic Resins:Protein Binding & Selectivity
Acknowledgments
• Eric Suda
• Abhinav Shukla, Ph.D.
• Sigma Mostafa, Ph.D.
• Carnley Norman, Ph.D.
• KBI Process Development Team
• KBI Analytical Development Team
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