a comparison of multimodal chromatographic resin: protein binding & selectivity

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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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


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.


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


7Effect of pH on Retention


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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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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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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Incorporation of a process step utilizing a modulator wash can improve overall process HCP clearance

baseline

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Overview






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


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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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






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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● 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






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


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


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


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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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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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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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


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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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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Tel: +1 919 479 9898

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a comparison of multimodal chromatographic resin: protein binding & selectivity

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