Strategies for Chromatographic Capture

Principals and applications of:

• Mixed-Mode Hydrophobic Interaction Chromatography

• Enhanced Diffusion Ion Exchange Chromatography

Warren Schwartz, Ph.D.Senior Technical DirectorPall Life Sciences

Office: (978) 635-1670Email: warren_schwartz@pall.com

© Pall Corporation

Conventional Ion Exchange: Feedstock conductivity is too high for direct capture.

Conduct diafiltration in advance of chromatography.

Dilute the feedstock.

Conventional HIC: Would require significant addition of salt

Costs to purchase salt and dispose of waste-salt can be significant.

Addition of salt can lead to precipitation.

Product may be recovered in buffer containing significant concentration of salt.

Chromatographic Capture: Considerations & Challenges

Evaluate Mixed-Mode HIC: Binding accomplished at “physiological” ionic strength.

Evaluate Enhanced Diffusion IEX: Binding accomplished at “moderate” ionic strength.

Evaluate Mixed-Mode HIC: Binding accomplished at “physiological” ionic strength.

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New Sorbents for Mixed-Mode HIC: PPA and HEA HyperCel

New members of a family of Pall BioSepra sorbents carrying mixed-mode ligands.

MEP HyperCel™

PPA HyperCel™

HEA HyperCel™

Shared features of mechanism & function:

Binding principally by hydrophobic &/or affinity interaction.

Binding principally by hydrophobic interaction.

This approach facilitates design of sorbents that provide efficient hydrophobic binding at low-to-moderate salt concentration.

Desorption: Driven principally by electrostatic charge repulsion.

Binding: Driven principally by hydrophobic or affinity interaction.

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PPA and HEA HyperCelThe sorbents carry ligands based on aromatic & aliphatic amines

WorkingpKa ~ 8.0

(both amines)

Binding (Typically at pH 7 – 8): Ligands can function as anion exchangers or by hydrophobic interaction.

At salt concentrations recommended for binding, ion exchange is not the dominant mode of binding. (Typically ~150 mM NaCl. Increase as needed.)

Binding is principally by hydrophobic interaction and is achieved at saltconcentrations considerably less than those used during traditional HIC.

HEA HyperCel: The amine includes an n-hexyl substituent CH2-(CH2)4-CH3

PPA HyperCel: The amine includes a phenylpropyl substituent

CH2-CH2-CH2-

NH +:

Binding of very basic proteins may require increased pH.

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PPA and HEA HyperCelDesorption (Typically at pH 3 – 5): As pH is reduced to values below the pI of the protein -&- below the pKa of the ligand:

Electrostatic charge repulsion will develop, increasing as pH is reduced.

Basic proteins will desorb earlier in the pH gradient or step-elution sequence

Acidic proteins will desorb later in the pH gradient or step-elution sequence

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Comparison of PPA & HEA HyperCel with Conventional AIEX

Sorbent Recovery (%)

Bound (mg)

FT&Wash(mg)

Load (mg)

Protein Eluted (mg)

PPA

DEAE

20BSA

20BSA

200

020

98None Bound

HEA 20BSA 200 9318.5

19.5

0

PPA

DEAE

HEA

20Ovalbumin 70

20Ovalbumin 200 8316.6

14200

None Bound002020Ovalbumin

(pI ≈ 4.9)

(pI ≈ 4.8)

Binding in Phosphate Buffered Saline, pH 7.4

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Comparison of PPA & HEA HyperCel with Conventional HIC

PPA

HEA

Phenyl HIC

Hexyl HIC

PPA

HEA

Hexyl HIC

Phenyl HIC

PPA

HEA

Hexyl HIC

Phenyl HIC

20BSA 020 None Bound

20BSA 5.214.8 00

0

20BSA

20BSA

200

200

93

98

18.5

19.5

20Ovalbumin 200 8316.6

20Ovalbumin 7014200

20Ovalbumin 020 None Bound0

20Ovalbumin 020 None Bound0

20α-Chy’gen 100

20α-Chy’gen 3.216.8 100+3.8

20200

20α-Chy’gen 020 None Bound0

20α-Chy’gen 020 None Bound0

Sorbent Recovery (%)

Bound (mg)

FT&Wash(mg)

Load (mg)

Protein Eluted (mg)

(pI ≈ 4.9)

(pI ≈ 4.8)

(pI ≈ 8.8–9.6)

Binding in Phosphate Buffered Saline, pH 7.4

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Chromatography of Model Proteins on PPA & HEA HyperCel

Equilibration, Load & Wash:

PBS, pH 7.4. (10 mM sodium phosphate containing 140 mM NaCl + 30 mM KCl)20 mg of each protein was applied to a 1.1 cm ID x 2 cm column.

Step-Elution Sequence (20 mM sodium acetate buffer at the specified pH)

1) pH 5.02) pH 4.03) pH 3.0

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Binding & Elution Behavior on HEA & PPA HyperCelHEA HyperCel

Load / WashFlowthru

Elution, pH 5.0

Elution,pH 4.0

Elution, pH 3.0

pH 3.0pH 4.0pH 5.0LWFT

0% 0%

93%

0%

Mass Balance = 93%

0% 0%

69%

14%

Mass Balance = 83%

84%

19%0%0%

Mass Balance = 103%

87%

12% 0%0%

Mass Bal. = 99%

BSA(pI ≈ 4.9)

Ovalbumin(pI ≈ 4.8)

Chymo-trypsinogen A(pI ≈ 8.8 – 9.6)

Bovine IgG(pI ≈ 6.0 – 7.5)

PPA HyperCel

Load / WashFlowthru

Elution, pH 5.0

Elution,pH 4.0

Elution, pH 3.0

pH 3.0pH 4.0pH 5.0LWFT

0% 0%58%

31%

Mass Balance = 89%

98%

0% 0%0%

Mass Balance = 98%

0% 0% 12%58%

Mass Balance = 70%

88%

0%0% 18%

Mass Bal. = 106%

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Separation of a protein mixture on PPA HyperCel & HEA HyperCel

pH 10.0

pH 5.4

pH 2.6

pH 7.0

pH 7.4

pH 7.0

pH 5.4

pH 2.6

Lysozyme

Lysozyme

α-Chymotrypsinogen A

α-Chymotrypsinogen A

BSA

Ovalbumin

Ovalbumin

BSA

HEA HyperCel

PPA HyperCel

Equilibration and Load: pH 10.0

Equilibration and Load: pH 7.4

pHpH

Phosphate buffered saline

25 mM sodium carbonate buffer containing 150mM NaCl.The protein mixture was applied

to 1.1 cm ID x 7 cm columns of PPA HyperCel and HEA HyperCel.

A step-elution sequence was conducted followed by gradient elution from pH 5.4 to 2.6, all conducted using sodium phosphate / citrate buffers.

BSA pI ≈ 4.9

Ovalbumin pI ≈ 4.8

α-Chymo’gen pI ≈ 8.8 – 9.6

Lysozyme pI ≈ 10 – 11

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Hydrophobic Charge Induction Chromatography on MEP HyperCel

S

N

pKa = 4.8Hydrophobicinteraction

Adsorption at near-neutral pH

Hydrophobic& Affinity

Interaction

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Hydrophobic Charge Induction Chromatography on MEP HyperCel

Desorption at pH 4

H

S

N

++

+++

+

Electrostatic Repulsion

pH % in (+)Form

4.8 50%

5.8 10%

Desorption at pH 4.0 – 5.8

S

N

pKa = 4.8Hydrophobicinteraction

Adsorption at near-neutral pH

Hydrophobic& Affinity

Interaction

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Summary: Mixed-Mode Hydrophobic Interaction Chromatography

Achieve binding from feedstocks that can not be directly applied to traditional ion exchange or HIC sorbents:

Conductivity is too high for traditional ion exchange sorbents

Would require significant addition of salt for traditional HIC.

Chromatographic behavior is based on a combination of electrostatic & hydrophobic properties of the protein and ligand:

Chromatography can be controlled and optimized based on pH.

Can provide unique selectivities not accessible with traditional IEX or HIC.

The differing retentivities & seletivities of PPA HyperCel, HEA HyperCel and MEP HyperCel can be screened to facilitate process development.

Unlike traditional HIC, the target protein is typically recovered in dilute buffer, reducing the need for, or extent of, intermediate diafiltration, etc.

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Enhanced Diffusion Ion Exchange Chromatography

Chromatography on Ceramic HyperD® Sorbents

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Enhanced Diffusion: A Mechanism of Mass-Transfer

Also known as “solid diffusion” or “hyperdiffusion”.

Much faster than classical pore diffusion observed with traditional macroporous ion exchangers.

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Enhanced Diffusion: A Mechanism of Mass-Transfer

Reduced dependence of dynamic binding capacity (DBC) on linear velocity.

Reduced dependence of DBC on feedstock concentration.

With CM HyperD achieve efficient capture from feedstocks of moderate ionic strength.

Achieve high DBC at high linear velocity.

With enhanced diffusion, observe:

Reduce or eliminate need for preliminary diafiltration or dilution.

Reduce or eliminate need for preliminary concentration.

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Structure of Ceramic HyperD IEX Sorbents

In situ polymerization

Monomer intrusion

Porous, non-compressible ceramic bead>0.2 μm (2000 Å) ‘pores’In situ polymerization to form hydrogel beadCM, Q, S, DEAE IEX sorbents

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Ceramic HyperD IEX Sorbents: Gel Structure

Pores of ceramic shell are completely filled with hydrogel25-50 Å spacing between adjacent polymer chains~15 Å spacing between adjacent charged groups

++++

++++++++++++

++++++++

++++

++++

++++

++++

++++

++++

++++

++++

++++

Charge Densityμeq/ml μeq/ml sorbent hydrogel

(packed bed) (in situ)

CM >250 >860Q >200 >690DEAE >180 >620S >150 >515

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Enhanced Diffusion in the Gel-Filled Pore

++++

++++++

+++

++

+++ +

++

+ + +

++

+++

++++

+

+++

+ ++

++

+

- -

Also referred to as “Hyperdiffusion”

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BlackCeramicBackbone

White-GrayHydrogel

Gray spotsGold-labeledalbumin

Scanning EM of Cross-Section Through Q Ceramic HyperD Sorbent Bead

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0

20

40

60

80

0 100 200 300 400 500 600 700Linear flow rate (cm/h)

Bin

ding

cap

acity

(mg/

m

10% Breakthrough

50% Breakthrough

Influence of Linear Velocity on DBCQ Ceramic HyperD Sorbent

Sorbent: Q Ceramic HyperD F Column: ID 6.6 x 16 cm Sample: BSA at 5 mg/ml in 50 mM Tris/HCl pH 8.6

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0 5 10 15 20 25 30 35

CM Ceramic HyperD

Whatman

Poros

ABX

S Ceramic HyperD

Macro-Prep high S

Carboxy-sulfon

SE Hicap

ToyoPearl

mg of IgG/ml of sorbent

pH = 5.0pH = 4.5

Courtesy of Dr. Harish Iyer, IDEC Pharmaceuticals - San Diego CA, USA

From batch binding studies. Feedstock diluted 1:1. Conductivity = 7.3 mS/cm.

Binding of MAb from Clarified Cell Culture Supernatant

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Very High Binding Capacity with Diafiltered FeedstocksChromatography on CM Ceramic HyperD

Multiple clients have reported binding capacity values of 100 – 120 mg/ ml.

Especially useful for capture from high-producing expression systems.

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High Binding Capacity During Capture of an MAb from a High-Titer Feedstock

“Current Challenges in Protein SeparationInnovative Chromatographic Applications”by Vicki Glaser

February 15, 2005

From

“Another Pall client relies on CM Ceramic HyperD to capture a monoclonal antibody from its high producing cell culture supernatant. Advances in cell culture processes have enabled the company to generate antibody titers in the range of 1 to 5 g/L. These expression levels are necessary to support cost efficient production of antibody products as they move into Phase III development. The capture chromatography step yielded 100 mg/mL dynamic binding capacity at 5% breakthrough, substantially higher than with conventional macroporous cation exchangers. Product purity was >90%.”

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Summary: Ceramic HyperD IEX Sorbents

Rigid sorbentsNo swelling and shrinking; maintain bed integrity.Achieve high linear velocity at moderate pressure.

Achieve high binding capacity at high linear velocity

Achieve efficient capture from dilute feedstocks

With CM Ceramic HyperD achieve efficient capture from feedstocks of moderate ionic strength

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Conclusions: Strategies for Chromatographic Capture

When chromatographic capture must be achieved from:

Feedstocks whose conductivity is too high for direct capture using conventional ion exchange sorbents, or

Feedstocks that would require significant addition of binding-promoting salt to support capture using conventional HIC.

Evaluate capture using:

Mixed-mode hydrophobic interaction chromatography on PPA HyperCel & HEA HyperCel or HCIC on MEP HyperCel sorbents.

Enhanced diffusion ion exchange chromatography on CM Ceramic HyperD.

Screen these sorbents for selectivity characteristics not provided by conventional HIC or IEX sorbents.

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Thank you.