Characterization of Protein Interactions on Mixed Mode Chromatography and Augmenting Selectivity Using Selective Mobile Phase Modulators

1. Characterization of Protein Interactions on Mixed Mode Chromatography
and Augmenting Selectivity Using Selective Mobile Phase Modulators
Leslie S. Wolfe and Abhinav A. Shukla
Process Development and Manufacturing, KBI Biopharma, Durham, NC
Abstract
Mixed-mode chromatography resins are increasingly being
incorporated into preparative purification processes. Mixed-mode
resins have the ability to interact with target proteins through multiple
types of interactions including electrostatic interactions, hydrophobic
interactions and hydrogen bonding depending on the ligand. Further
selectivity can be obtained through the use of modulators in buffers
during the product load, wash or elution phase of the process step.
Here, the mixed-mode Capto MMC resin is described in terms of its
protein adsorption characteristics by using a set of four monoclonal
antibodies and two non-IgG model proteins. Linear gradient elution
experiments were executed to compare the elution conditions
required for each antibody and non-IgG model protein at three pHs in
the presence of several mobile phase modulators. For each protein,
experiments performed at pHs farther from the protein isoelectric
point resulted in tighter protein:resin interactions. Additionally, in the
presence of modulating agents, varying binding and retention were
observed that were in-line with the known abilities of these
modulators to reduce different kinds of interactions (electrostatic,
hydrophobic, hydrogen bonding etc.). The influence of mobile phase
modulators on linear retention was characterized by the use of log k’
vs. log (salt concentration) plots in the presence of various agents.
These studies enabled a classification of the key interactions that
drove retention for different types of proteins.
The results of these studies characterizing fundamental interactions on
this mode of chromatography were employed to develop highly
selective wash steps. The use of combinations of some of these mobile
phase modulators in a wash step were found to augment HCP
clearance by > 5 fold in comparison with a conventional wash step. The
results presented in this study can significantly enhance selectivity that
can be obtained during protein separations on this mode of
chromatography and offer the enticing possibility of creating a pseudo-
affinity separation using a non-affinity chromatographic stationary
phase.
Mobile Phase Modulators
Mixed Mode Chromatography
• Mobile phase modulators: additives incorporated into
process buffers to alter protein-ligand interactions
• Modulators can enhance resin selectivity and eluate purity
when incorporated into load, wash and/or elution process
steps
• Addition of a combination of modulators can further
improve selectivity
• Takes advantage of more than one type of interaction
• i.e. ionic, hydrophobic, hydrogen bonding
• Provides enhanced selectivity, “pseudo-affinity” over
conventional single mechanism based stationary phases
such as ion-exchange or hydrophobic interaction
chromatography
• Can potentially reduce process steps
• Proteins typically eluted with pH change or with salt
increase
• Several mixed mode resins have recently been developed
with:
• Increased loading capacities
• Higher ionic strength tolerance
Resin Type
Capto MMC Multimodal weak cation exchanger
Capto Adhere Multimodal strong anion exchanger
Nuvia cPrime Hydrophobic cation exchanger
Eshmuno HCX Multi-mode cation exchanger
Toyopearl MX-Trp-650M Multimodal weak cation exchanger
Ionic interactions
Hydrophobic interactions
Capto MMC ligand
Modulator Modulator Effect
MgCl2, NaSCN, KI Decrease hydrophobic interactions
Ethanol, Methanol,
Isopropanol
Decrease hydrophobic interactions (used in low
concentrations)
Urea Weakens hydrogen bonding, denaturant
Glycerol Weakens hydrophobic interactions
Ethylene Glycol Weakens hydrophobic interactions and hydrogen bonding
Arginine Weakens hydrophobic interactions, induces protein unfolding
Ammonium Sulfate Strengthens hydrophobic interactions
Linear Gradient Elution
Studies
• Experimental aim: Determine [NaCl] required to elute six
different basic proteins from Capto MMC resin in the
presence of a modulator
• [NaCl] determined by the %B buffer at peak maxima
• Modulator added to equilibration, wash and elution buffers
• Product eluted with increasing NaCl gradient
Effect of pH on Retention
Modulator Effects at pH 7.0
• Antibodies behave differently
• mAb1, mAb4 behave similarly
• mAb2, mAb3 behave similarly
• Modulator with largest effect differs for different proteins
• Lysozyme requires much higher [NaCl] for elution
• 1M (NH4)2SO4
• RNase does not bind
• Lysozyme, mAb1, mAb2, mAb3, mAb4 do not elute
• RNase
• Driven by electrostatic interactions
• No effect seen with 1M urea or 5% ethylene glycol
log k’ vs. log [NaCl] Plots
Protein retention under linear loading conditions is dependent
on the thermodynamics of the interaction between the protein
and the stationary phase represented by:
log K = (- ΔG°es / 2.3RT) + (-ΔG°hΦ / 2.3RT)
∆Ges = Gibbs free energies for retention by electrostatic interactions
∆GhΦ = Gibbs free energies for retention by hydrophobic interactions
T = the absolute temperature
R = the universal gas constant
Retention factor (k’) relates to K by,
k’ = ΦK
where Φ is the ratio of stationary and mobile phase volumes
This relationship was further described by Melander et. al to
express the dependency of the linear retention factor on a mixed
mode sorbent as a function of salt concentration.
log k’ = A – Blog(csalt) + C(csalt)
where csalt is the mobile phase salt concentration in molar units and A, B and C are
constants
Retention factor determined by:
k’ = (tr – tm) /tm
tm = time for mobile phase to pass through column
tr = target retention time
Given this relationship,
• when electrostatic interactions predominate a linear
relationship is expected between log k’ vs log[NaCl].
• when hydrophobic interactions predominate a linear
relationship is expected until a minimum is reached at which
point further increases in salt result in increased retention.
Enhancing Purification
through Modulator
Washes
Intermediate Modulator Wash for Polishing
Intermediate Modulator Wash for Capture
• In depth studies have afforded us an understanding of how
modulators affect different molecules
• We have utilized mixed mode chromatography to improve
product purity and maintain process step yield
• Mixed mode chromatography is advantageous because of its
increased selectivity by exploiting multiple chemical properties of
target protein
• The characteristics of the resin provide the opportunity to
create pseudo-affinity separation using a non-affinity
chromatographic stationary phase
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.10 2.30 2.50 2.70
Logk'
Log [NaCl]
mAb1
 baseline
 1M urea
 5% ethylene glycol
 50mM arginine
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.50 2.00 2.50
Logk'
Log [NaCl]
RNase
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.60 3.10 3.60
Logk'
Log [NaCl]
Lysozyme
0
20
40
60
80
100
0 50 100 150 200
%ElutionBuffer
Elution Volume (mL)
mAb3, pH 8.0, 500mM NaCl gradient
38.9% B
357 312
221 249 202
0
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
RNase
2,217 1,862 1,766 1,981
1,248
2,500
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
Lysozyme
∞
1,482 1,602
847 962 916
1,800
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb4
∞
300 296
198 136
235
400
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb3
∞
314 304
209 132
237
400
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb2
∞
1,219 1,145 824 795 809
2,500
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb1
∞
∞ = target protein did not elute during NaCl gradient
• Experiments performed at pH farther from the protein isoelectric
point resulted in tighter protein:resin interactions
• pH affects each protein differently from across pH range
• mAb1, mAb4 behave similarly
• mAb2, mAb3 behave similarly
• Antibodies behave more similar as the pH approaches the
isoelectric point (pH 8.0)
• Lysozyme does not elute at pH 6.0 in up to 4M NaCl
0
20
40
60
80
100
0
500
1000
1500
2000
2500
3000
0 50 100 150
%B
Absorbance(mAu)
Volume (mL)
mAb1 Capto MMC Baseline Polish
Chromatogram
Intermediate Wash Buffer Conditions Tested for Capto MMC Capture
Intermediate Wash buffer Recovery Normalized HCP Intermediate Wash Buffer Recovery Normalized HCP

25mM Tris pH 7.0
(baseline)
96.4% 1.00  50mM arginine, 0.1M NaCl 98.4% 0.65
 50mM arginine 95.5% 0.68 
50mM arginine, 5% ethylene
glycol
99.0% 0.68
 1M urea 96.9% 0.77 – 50mM arginine, 50mM NaSCN 98.6% 0.61
 0.1M NaCl 97.4% 0.72  1M urea, 0.1M NaCl 51.1% 1.06
 5% ethylene glycol 97.9% 0.67  1M urea, 5% ethylene glycol 99.3% 0.69
 50mM NaSCN 97.1% 0.65  1M urea, 50mM NaSCN 97.4% 0.68
 0.5M ammonium sulfate 94.7% 1.53  0.1M NaCl, 5% ethylene glycol 97.6% 0.78
1M ammonium sulfate 97.8% 2.15  0.1M NaCl, 50mM NaSCN 98.0% 0.85
 50mM arginine, 1M urea 97.5% 0.64 – 50mM NaSCN, 5% ethylene glycol 98.5% 0.85
All buffers contain 25mM Tris, pH 7.0
0.50
0.70
0.90
1.10
1.30
1.50
0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%
NormalizedHCP
Recovery
HCP vs. Recovery after Intermediate Wash for Capto MMC Capture
baseline
Intermediate Wash Buffer Conditions Tested for Capto MMC Polishing
Intermediate Wash buffer Recovery
Normalized
HCP
Intermediate Wash Buffer Recovery
Normalized
HCP
 25mM Tris pH 7.0 (baseline) 87.2% 1.00 – 0.1M NaCl, 1M urea 95.4% 0.57
 5% ethylene glycol 93.8% 1.07  0.1M NaCl, 1M urea, 5% ethylene glycol 94.6% 0.52
 50mM arginine 94.2% 0.84  0.1M NaCl, 1M urea, 5% glycerol 95.1% 0.61
 50mM NaSCN 95.8% 0.89  0.1M NaCl, 50mM arginine 95.6% 0.56
 1M urea 96.4% 1.18  0.1M NaCl, 50mM arginine, 5% glycerol 94.6% 0.61
 0.1M NaCl 96.1% 1.07 
0.1M NaCl, 50mM arginine, 5% ethylene
glycol
96.4% 0.54
- 0.5M ammonium sulfate 96.3% 0.73  200mM arginine 30.5% 1.03
All buffers contain 25mM Tris, pH 7.0
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%
NormalizedHCP
Recovery
HCP vs. Recovery after Intermediate Wash for Capto MMC Polishing
baseline
Conclusions
References
Melander, W.; El Rassi, Z.; Horvath, Cs. Journal of Chromatography,
469, 3-27, 1989.
• Lysozyme
• Driven by hydrophobic interactions
• Varying effects observed with modulators
• mAb1
• Driven by electrostatic interactions with some contribution
from hydrophobic interactions
• 50mM arginine and 1M urea have the largest effect
 baseline
 1M urea
 5% ethylene glycol
 50mM arginine
 baseline
 1M urea
 5% ethylene glycol
 50mM arginine
Block Buffer CV
Sanitization 0.5M NaOH 5
Equilibration 25mM Tris pH 7.0 5
Sample Load ProA eluate, pH adjusted to 7.0* 10g/L load
Wash 1 25mM Tris pH 7.0 2
Intermediate Wash 25mM Tris pH 7.0 + mobile phase modulators 5
Wash 2 25mM Tris pH 7.0 3
Elution 25mM Tris, 500mM NaCl, pH 9.0 5
Strip 2M NaCl 3
Sanitization 0.5M NaOH 3
Baseline Capto MMC process overview for polishing
0
500
1000
1500
2000
2500
[NaCl](mM)
[NaCl] for Capto MMC Elution
mAb1
mAb2
mAb3
mAb4
Lysozyme
RNase
0
500
1000
1500
2000
2500
6.0 7.0 8.0
Elution[NaCl](mM)
pH
Lysozyme No modulator
5% ethylene glycol
50mM arginine
50mM sodium
thiocyanate
1M urea
1M ammonium
sulfate
0
50
100
150
200
250
300
350
400
6.0 7.0 8.0
Elution[NaCl](mM)
pH
RNase No modulator
5% ethylene glycol
50mM arginine
50mM sodium
thiocyanate
1M urea
1M ammonium
sulfate
∞
0
500
1000
1500
6.0 7.0 8.0
Elution[NaCl](mM)
pH
mAb1 No modulator
5% ethylene glycol
50mM arginine
50mM sodium
thiocyanate
1M urea
1M ammonium
sulfate
∞
0
100
200
300
400
500
600
6.0 7.0 8.0
Elution[NaCl](mM)
pH
mAb3
No modulator
5% ethylene glycol
50mM arginine
50mM sodium
thiocyanate
1M urea
1M ammonium
sulfate
∞
0
100
200
300
400
500
600
6.0 7.0 8.0
Elution[NaCl](mM)
pH
mAb2 No modulator
5% ethylene glycol
50mM arginine
50mM sodium
thiocyanate
1M urea
1M ammonium
sulfate
∞
0
500
1000
1500
2000
6.0 7.0 8.0
Elution[NaCl](mM)
pH
mAb4 No modulator
5% ethylene glycol
50mM arginine
50mM sodium
thiocyanate
1M urea
1M ammonium
sulfate
∞
∞
*For capture, harvest supernatant was used