A presentation from 2015 by KBI Biopharma on: 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 & HMW removal.

1. 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 & Manufacturing
KBI Biopharma, Durham, NC

2. 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
2

3. 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
3

4. 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 could
potentially result in molecule specific performance (yield and selectivity)
EshmunoTM
HCX
EMD Millipore
CaptoTM
Adhere
GE Healthcare
MX-Trp-650M
Tosoh
CaptoTM
MMC
GE Healthcare
4
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

5. 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
5
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.

6. 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
6
Modulator Modulator Effect
MgCl2
, NaSCN, KI Decrease hydrophobic interactions
Ethanol, Methanol, Isopropanol Decrease hydrophobic interactions (used in low concentrations)
Urea, Sodium Thiocyanate Weakens hydrogen bonding, denaturant
Glycerol Weakens hydrophobic interactions
Ethylene 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

7. A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
7
Effect of pH on Retention

8. 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
)/tm
tm
= time for mobile phase to pass through column
tr
= 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
8

9. 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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10. 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
10

11. 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

12. 12
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

13. 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
13
DS
Process
Platform
Cell Line
Diversity
Media/feed
type diversity
HCP level
variability
Cell Density
variability
HMW level
variability

14. 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
14

15. A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
Mixed-mode cation exchange/hydrophobic interaction resins
15
NuviaTM
cPrimeTM
Bio-Rad Laboratories
MX-Trp-650M
Tosoh
EshmunoTM
HCX
EMD Millipore
CaptoTM
MMC
GE Healthcare
TM

16. 16
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

17. A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
High Throughput Screening: Plate Experimental Design
Variable 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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18. A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
18
● 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

19. 19
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

20. 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
20
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

21. 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

22. A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
Cumulative Yield vs. Cumulative HMW
mAb 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

23. 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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24. A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
Summary Results – Optimization Factor
Resin
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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25. 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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26. A Comparison of Multimodal Chromatographic Resins:
Protein Binding & Selectivity
Best resins for each measure & overall performance based
on the chromatography experiment results
26
● 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.
Molecule
Best resins based on
yield
Best resins based on
HMW reduction
Best resins based on
HCP reduction
Overall best resin based on
optimization factor
mAb1
Capto MMC
Tosoh MX-Trp
Nuvia cPrime
Capto MMC
Eshmuno HCX
Capto MMC
Nuvia cPrime
Capto MMC
mAb2
Capto MMC
Tosoh MX-Trp
Eshmuno HCX
Nuvia cPrime
Eshmuno HCX
Capto MMC
Eshmuno HCX
Nuvia cPrime
mAb3
Tosoh MX-Trp
Nuvia cPrime
Capto MMC
Eshmuno HCX
Capto MMC
Tosoh MX-Trp
Capto MMC
Nuvia cPrime

27. 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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28. 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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29. www.kbibiopharma.com
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