Prep Symposium | Poster July, 2013
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 (NH 4 ) 2 SO 4 • 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) ∆G es = Gibbs free energies for retention by electrostatic interactions ∆G hΦ = 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(c salt ) + C(c salt ) where c salt is the mobile phase salt concentration in molar units and A, B and C are constants Retention factor determined by: k’ = (t r – t m ) /t m t m = time for mobile phase to pass through column t r = 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 2.102.202.302.402.502.602.702.80 -0.20 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 mAb1 Log [NaCl] Log k' baseline 1M urea 5% ethylene glycol 50mM arginine 1.50 1.70 1.90 2.10 2.30 2.50 -0.40 -0.20 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 RNase Log [NaCl] Log k' 2.60 2.80 3.00 3.20 3.40 3.60 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 Lysozyme Log [NaCl] Log k' 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 Elution Volume (mL) % Elution Buffer mAb3, pH 8.0, 500mM NaCl gradient 38.9% B 357 312 221 249 202 0 RNase Elution [NaCl] (mM) 2,217 1,862 1,766 1,981 1,248 2,500 Lysozyme Elution [NaCl] (mM) ∞ 1,482 1,602 847 962 916 1,800 mAb4 Elution [NaCl] (mM) ∞ 300 296 198 136 235 400 mAb3 Elution [NaCl] (mM) ∞ 314 304 209 132 237 400 mAb2 Elution [NaCl] (mM) ∞ 1,219 1,145 824 795 809 2,500 mAb1 Elution [NaCl] (mM) ∞ ∞ = 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 80 100 120 140 160 0 500 1000 1500 2000 2500 3000 0 20 40 60 80 100 mAb1 Capto MMC Baseline Polish Chromatogram Volume (mL) Absorbance (mAu) % B Intermediate Wash Buffer Conditions Tested for Capto MMC Capture Intermediate Wash buffer Recove ry Normalized HCP Intermediate Wash Buffer Recove ry 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 0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0% 0.50 0.70 0.90 1.10 1.30 1.50 HCP vs. Recovery after Intermediate Wash for Capto MMC Capture Recovery Normalized HCP baseline Intermediate Wash Buffer Conditions Tested for Capto MMC Polishing Intermediate Wash buffer Recove ry Normaliz ed HCP Intermediate Wash Buffer Recove ry Normaliz ed 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 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0% 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 HCP vs. Recovery after Intermediate Wash for Capto MMC Polishing Recovery Normalized HCP 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 pH 6. 0 5% ethylene glyco l, pH 6.0 50mM argi ni ne, pH 6.0 50mM sodium thioc yanate, pH 6. 0 1M urea, pH 6.0 1M ammoni um sulfa te, pH 6 .0 pH 7.0 5% ethyle ne glyco l, pH 7. 0 50m M argini ne, pH 7.0 50m M sodium thiocyanat e, pH 7. 0 1M u r ea, pH 7.0 1M ammonium sulfa te, pH 7.0 pH 8. 0 5% et hylene glycol, pH 8.0 50m M argi ni ne, pH 8.0 50m M sodium thioc yanate, p H 8. 0 1M urea, pH 8.0 1M ammoni um sulfa te, pH 8 .0 0 500 1000 1500 2000 2500 [NaCl] for Capto MMC Elution mAb1 mAb2 mAb3 mAb4 Lysozyme RNase [NaCl] (mM) 6.0 7.0 8.0 0 500 1000 1500 2000 2500 Lysozyme No modulator 5% ethylene glycol 50mM arginine 50mM sodium thiocyanate 1M urea 1M ammonium sulfate pH Elution [NaCl] (mM) 6.0 7.0 8.0 0 100 200 300 400 RNase No modulator 5% ethylene glycol 50mM arginine 50mM sodium thiocyanate 1M urea 1M ammonium sulfate pH Elution [NaCl] (mM) ∞ 6.0 7.0 8.0 0 500 1000 1500 mAb1 No modulator 5% ethylene glycol 50mM arginine 50mM sodium thiocyanate 1M urea 1M ammonium sulfate pH Elution [NaCl] (mM) ∞ 6.0 7.0 8.0 0 200 400 600 mAb3 No modulator 5% ethylene glycol 50mM arginine 50mM sodium thiocyanate 1M urea 1M ammonium sulfate pH Elution [NaCl] (mM) ∞ 6.0 7.0 8.0 0 200 400 600 mAb2 No modulator 5% ethylene glycol 50mM arginine 50mM sodium thiocyanate 1M urea 1M ammonium sulfate pH Elution [NaCl] (mM) ∞ 6.0 7.0 8.0 0 500 1000 1500 2000 mAb4 No modulator 5% ethylene glycol 50mM arginine 50mM sodium thiocyanate 1M urea 1M ammonium sulfate pH Elution [NaCl] (mM) ∞ ∞ *For capture, harvest supernatant was u
-
Upload
kbi-biopharma -
Category
Health & Medicine
-
view
24 -
download
2
Transcript of Prep Symposium | Poster July, 2013
Characterization of Protein Interactions on Mixed Mode Chromatography and Augmenting Selectivity Using Selective Mobile Phase Modulators
Leslie S. Wolfe and Abhinav A. ShuklaProcess Development and Manufacturing, KBI Biopharma, Durham, NC
AbstractMixed-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 TypeCapto MMC Multimodal weak cation exchangerCapto Adhere Multimodal strong anion exchangerNuvia cPrime Hydrophobic cation exchangerEshmuno HCX Multi-mode cation exchangerToyopearl MX-Trp-650M Multimodal weak cation exchanger
Ionic interactions
Hydrophobic interactions
Capto MMC ligand
Modulator Modulator EffectMgCl2, NaSCN, KI Decrease hydrophobic interactionsEthanol, Methanol, Isopropanol
Decrease hydrophobic interactions (used in low concentrations)
Urea Weakens hydrogen bonding, denaturantGlycerol Weakens hydrophobic interactionsEthylene Glycol Weakens hydrophobic interactions and hydrogen bondingArginine Weakens hydrophobic interactions, induces protein unfoldingAmmonium 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] PlotsProtein 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’ = ΦKwhere 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 columntr = target retention timeGiven 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
2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
mAb1
Log [NaCl]
Log
k'
baseline 1M urea 5% ethylene glycol 50mM arginine
1.50 1.70 1.90 2.10 2.30 2.50
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
RNase
Log [NaCl]
Log
k'
2.60 2.80 3.00 3.20 3.40 3.600.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Lysozyme
Log [NaCl]
Log
k'
0 20 40 60 80 100 120 140 160 180 2000
20
40
60
80
100
Elution Volume (mL)
% E
luti
on B
uffer
mAb3, pH 8.0, 500mM NaCl gradient
38.9% B
357 312 221 249 2020
RNase
Elu
tion
[NaC
l] (m
M)
2,217 1,862 1,766 1,981 1,2482,500
LysozymeE
lutio
n [N
aCl]
(mM
)
∞
1,482 1,602847 962 916
1,800mAb4
Elu
tion
[NaC
l] (m
M)
∞300 296 198 136 235400
mAb3
Elu
tion
[NaC
l] (m
M)
∞
314 304 209 132 237400
mAb2
Elu
tion
[NaC
l] (m
M)
∞1,219 1,145 824 795 8092,500
mAb1
Elu
tion
[NaC
l] (m
M)
∞
∞ = 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 80 100 120 140 1600
500
1000
1500
2000
2500
3000
0102030405060708090100
mAb1 Capto MMC Baseline Polish Chromatogram
Volume (mL)
Abso
rban
ce (m
Au)
% B
Intermediate Wash Buffer Conditions Tested for Capto MMC CaptureIntermediate 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
0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%0.50
0.70
0.90
1.10
1.30
1.50HCP vs. Recovery after Intermediate Wash for Capto MMC Capture
Recovery
Nor
mal
ized
HCP
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% 1.03All buffers contain 25mM Tris, pH 7.0
20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10HCP vs. Recovery after Intermediate Wash for Capto MMC Polishing
Recovery
Nor
mal
ized
HCP
baseline
Conclusions
ReferencesMelander, 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 CVSanitization 0.5M NaOH 5Equilibration 25mM Tris pH 7.0 5Sample Load ProA eluate, pH adjusted to 7.0* 10g/L load
Wash 1 25mM Tris pH 7.0 2Intermediate Wash 25mM Tris pH 7.0 + mobile phase modulators 5
Wash 2 25mM Tris pH 7.0 3Elution 25mM Tris, 500mM NaCl, pH 9.0 5
Strip 2M NaCl 3Sanitization 0.5M NaOH 3
Baseline Capto MMC process overview for polishing
pH 6.0
5% et
hylen
e glyc
ol, pH
6.0
50mM ar
ginine
, pH 6.0
50mM so
dium th
iocyan
ate, p
H 6.0
1M ur
ea, pH 6.
0
1M am
monium
sulfa
te, pH
6.0pH
7.0
5% et
hylen
e glyc
ol, pH
7.0
50mM ar
ginine, p
H 7.0
50mM so
dium th
iocyan
ate, p
H 7.0
1M ur
ea, pH 7.
0
1M am
monium su
lfate,
pH 7.0
pH 8.
0
5% et
hylen
e glyc
ol, pH
8.0
50mM ar
ginine, p
H 8.0
50mM so
dium th
iocyan
ate, p
H 8.0
1M ur
ea, pH 8.
0
1M am
monium
sulfa
te, pH
8.00
500
1000
1500
2000
2500
[NaCl] for Capto MMC Elution
mAb1
mAb2
mAb3
mAb4
Lysozyme
RNase
[NaC
l] (m
M)
6.0 7.0 8.00
500
1000
1500
2000
2500
Lysozyme No modulator
5% ethylene glycol
50mM arginine
50mM sodium thiocyanate
1M urea
1M ammonium sul-fate
pH
Elu
tion
[NaC
l] (m
M)
6.0 7.0 8.00
50100150200250300350400
RNase No modulator
5% ethylene glycol
50mM arginine
50mM sodium thiocyanate
1M urea
1M ammonium sul-fate
pH
Elu
tion
[NaC
l] (m
M)
∞
6.0 7.0 8.00
500
1000
1500
mAb1 No modulator
5% ethylene glycol
50mM arginine
50mM sodium thiocyanate
1M urea
1M ammonium sul-fate
pH
Elu
tion
[NaC
l] (m
M)
∞
6.0 7.0 8.00
100200300400500600
mAb3No modulator
5% ethylene glycol
50mM arginine
50mM sodium thiocyanate
1M urea
1M ammonium sul-fatepH
Elu
tion
[NaC
l] (m
M)
∞
6.0 7.0 8.00
100200300400500600
mAb2 No modulator
5% ethylene glycol
50mM arginine
50mM sodium thiocyanate
1M urea
1M ammonium sul-fate
pH
Elu
tion
[NaC
l] (m
M)
∞
6.0 7.0 8.00
500
1000
1500
2000
mAb4No modulator
5% ethylene glycol
50mM arginine
50mM sodium thiocyanate
1M urea
1M ammonium sul-fatepH
Elu
tion
[NaC
l] (m
M)
∞
∞
*For capture, harvest supernatant was used
