Multimodal Chromatography

Leslie S. Wolfe, Ph.D. Abhinav A. Shukla, Ph.D.

Process Development KBI Biopharma, Durham, NC

• Background • Mixed Mode Chromatography • Mobile Phase Modulators

• CharacterizaGon of Protein Binding •  Linear salt gradient eluGon studies •  log k’ vs. log salt concentraGon plots

• Enhancing purificaGon through modulator washes • During Capture step • During Polishing step •  Process impact

•  Takes advantage of more than one type of interacGon »  i.e. ionic, hydrophobic, hydrogen bonding

•  Provides enhanced selecGvity, “pseudo-‐affinity” •  Can reduce process steps •  Several mixed mode resins have recently been developed with:

»  Increased loading capaci4es »  Higher ionic strength tolerance

GE Healthcare, Capto MMC ligand

Ionic interactions

Hydrophobic interactions

Hydrophobic interactions

GE Healthcare, Capto Adhere ligand

Ionic interactions

•  Mobile phase modulators: addiGves incorporated into process buffers to alter protein-‐ligand interacGons

•  Modulators can enhance resin selecGvity and eluate purity when incorporated into load, wash and/or eluGon process steps

•  AddiGon of a combinaGon of modulators can further improve selecGvity

Modulator Modulator Effect

MgCl2, NaSCN, KI Decrease hydrophobic interacGons Ethanol, Methanol, Isopropanol Decrease hydrophobic interacGons (used in low concentraGons) Urea Weakens hydrogen bonding, denaturant Glycerol Weakens hydrophobic interacGons Ethylene Glycol Weakens hydrophobic interacGons and hydrogen bonding

Arginine Weakens hydrophobic interacGons, induces protein unfolding, disrupts electrostaGc interacGons

Ammonium Sulfate Strengthens hydrophobic interacGons


• Characteriza2on of Protein Binding •  Linear salt gradient elu2on studies •  log k’ vs. log salt concentraGon plots


•  Resin: Capto MMC •  Modulator added to equilibraGon, wash and eluGon buffers •  Products eluted with a linear NaCl gradient

•  Model proteins: •  RNase (pI 8.9) •  Lysozyme (pI 9.6) •  mAb1 •  mAb2 •  mAb3

•  mAb4

•  Mobile phase modifiers: •  Ethylene glycol •  Urea •  Arginine •  Sodium Thiocyanate •  Ammonium sulfate

357 312 221 249 202

0

No modulator

5% ethylene glycol

50mM arginine

50mM sodium

thiocyanate

1M urea 1M ammonium

sulfate

Elu

tion

[NaC

l] (m

M) 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

Elu

tion

[NaC

l] (m

M) 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

Elu

tion

[NaC

l] (m

M) mAb4

∞ 300 296 198 136 235

400

No modulator

5% ethylene glycol

50mM arginine

50mM sodium

thiocyanate

1M urea 1M ammonium

sulfate

Elu

tion

[NaC

l] (m

M) mAb3

∞

314 304 209 132 237

400

No modulator

5% ethylene glycol

50mM arginine

50mM sodium

thiocyanate

1M urea 1M ammonium

sulfate

Elu

tion

[NaC

l] (m

M) 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

Elu

tion

[NaC

l] (m

M) mAb1

∞

∞ = target protein did not elute during NaCl gradient

•  AnGbodies behave differently •  In absence of modulator

»  mAb2, mAb3 – low salt (~300mM) »  mAb1, mAb4 – high salt (~1.5M)

•  Modulator with largest effect »  mAb1, mAb2, mAb3 – sodium thiocyanate »  mAb4 – arginine

•  Lysozyme requires highest NaCl for eluGon •  RNase does not bind in the presence of 1M (NH4)2SO4

•  All other proteins tested irreversibly bind in 1M (NH4)2SO4

•  As pH approaches pI, retenGon decreases •  Lysozyme does not elute in absence of modulator at pH 6.0 •  AnGbodies behave more similarly as pH approaches pI


• Characteriza2on of Protein Binding •  Linear salt gradient eluGon studies •  log k’ vs. log salt concentra2on plots


•  Protein retenGon under linear loading condiGons is dependent on the thermodynamics of the interacGon between the protein and the staGonary phase

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

k’ = ΦK

Retention factor (k’) relates to K by,

where Φ is the ratio of stationary and mobile phase volumes

•  This relaGonship was further described by Melander et. al to describe the dependency of the linear retenGon factor on a mixed mode sorbent as a funcGon of salt concentraGon as:

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

The retenGon factor under isocraGc condiGons is represented by:

k’ = tr – tm /tm tm = Gme for mobile phase to pass through column tr = target protein retenGon Gme

Melander, W.; El Rassi, Z.; Horvath, Cs. Journal of Chromatography, 469, 3-27, 1989.

•  This relaGonship was further described by Melander et. al to describe the dependency of the linear retenGon factor on a mixed mode sorbent as a funcGon of salt concentraGon as:

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

The retenGon factor under isocraGc condiGons is represented by:

k’ = tr – tm /tm tm = Gme for mobile phase to pass through column tr = target retenGon Gme

Melander, W.; El Rassi, Z.; Horvath, Cs. Journal of Chromatography, 469, 3-27, 1989.

•  ElectrostaGc interacGons predominate: a linear relaGonship is expected between log k’ vs log[NaCl]

•  Hydrophobic interacGons predominate: a linear relaGonship is expected unGl a minimum is reached at which point further increases in salt result in increased retenGon

•  RetenGon factors (k’) were determined for »  mAb1 »  RNase »  Lysozyme

•  Mobile phase modulators tested »  No modulator »  1M Urea »  50mM Arginine »  5% Ethylene Glycol

•  RNase »  electrostatic interactions »  No effect from urea or ethylene glycol

•  Lysozyme »  hydrophobic and electrostatic interactions »  urea has largest effect

•  mAb1 »  driven by electrostatic interactions, hydrophobic contribution »  Urea and arginine have the largest effect

-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

Log

k'

Log [NaCl]

mAb1

All experiments performed at pH 7.0

-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

Log

k'

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 L

og k

'

Log [NaCl]

Lysozyme

¿ baseline � 1M urea ¢ 5% ethylene glycol p 50mM arginine



• Enhancing purifica2on through modulator washes • During Capture step • During Polishing step •  Process impact

•  IncorporaGon of modulators into process can help increase selecGvity and purity of product

• CombinaGons of modulators can further enhance process step

• Goal: UGlize mobile phase modulators to decrease HCP levels during Capto MMC process step for anGbody purificaGon

•  Case Study: »  Target molecule: E. coli derived

recombinant protein »  Process step: Phenyl Sepharose FF »  Ini4al product yield: 88.8% »  Result:

Individual modulators showed some selecGvity enhancement but also product loss

A combinaGon of urea, sodium thiocyanate and glycerol in the wash step increased product purity to >95%

Shukla AA, et al., 2002. Biotechnol Prog 18: 556–564.




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%

Normalized

HCP

Recovery

HCP vs. Recovery aFer Intermediate Wash for Capto MMC Capture

baseline




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%

Normalized

HCP

Recovery

HCP vs. Recovery aFer Intermediate Wash for Capto MMC Polishing

baseline




•  Inclusion of an intermediate wash using Tris, 0.1M NaCl, 50mM arginine, 5% ethylene glycol, pH 7.0 resulted in 2-‐fold lower HCP levels when compared to process where a modulator was not uGlized

•  In depth studies have afforded us an understanding of how modulators affect different molecules •  Despite similar class of molecules, anGbodies behave differently with Capto MMC ligand

• We have uGlized mixed mode chromatography to improve product purity and maintain process step yield •  IncorporaGon of a process step uGlizing a modulator wash can improve overall process HCP clearance

•  At KBI Biopharma we have developed several downstream processes and manufactured biopharmaceuGcal products where modulator washes on a mixed mode resin have significantly contributed to process HCP clearance

»  Effec4ve for both molecules derived from mammalian and microbial cell culture processes

Pre-Clinical Phase I Phase II Phase III

FIH Process •  Deliver clinical process

quickly •  Platform process •  Clinical Supply

Submission & Approval

Lifecycle management

Commercial Process •  Deliver manufacturing process for

registrational trials and market •  Design keeping large-scale manufacturing

in mind •  Improve productivity, efficiency, robustness,

manufacturability, COGs •  Analytical characterization and method

development

Process Characterization and Validation •  Develop IPC strategy through understanding of process inputs and

outputs (design space) •  Scale-down characterization and validation studies •  Large-scale process validation to demonstrate process consistency •  BLA preparation •  Supporting documents for licensure inspections •  Post-commercial process improvements (CI) •  Post-commercial process monitoring

FIH process Commercial process

Gottschalk U., Konstantinov K., Shukla A. Nature Biotechnology, 30(6), 489-491, 2012

Process Development

Process Validation

Process Monitoring &

Improvement

BLA &

PAI

Manufacturing for Tox

Clinical Manufacturing

Commercial Process

Development

Process Characterization

Protein: protein interactions P

rote

in: r

esin

inte

ract

ions

Modulator washes can augment protein:protein interactions and/or protein:resin interactions

• Mixed mode chromatography is advantageous because of its increased selecGvity by exploiGng mulGple chemical properGes of target protein

•  Abhinav Shukla, Ph.D. •  Sigma Mostafa, Ph.D. •  Cartney Barringer •  KBI Process Development Team

Work available online ahead of print in Journal of Chromatography A Wolfe, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.02.086

Health & Medicine

Multimodal Chromatography