Strategies for Chromatographic CaptureStrategies for Chromatographic Capture Principals and...
Transcript of Strategies for Chromatographic CaptureStrategies for Chromatographic Capture Principals and...
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: [email protected]
© 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.
© Pall Corporation
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.
© Pall Corporation
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.
© Pall Corporation
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
© Pall Corporation
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
© Pall Corporation
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
© Pall Corporation
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
© Pall Corporation
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%
© Pall Corporation
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
© Pall Corporation
Hydrophobic Charge Induction Chromatography on MEP HyperCel
S
N
pKa = 4.8Hydrophobicinteraction
Adsorption at near-neutral pH
Hydrophobic& Affinity
Interaction
© Pall Corporation
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
© Pall Corporation
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.
© Pall Corporation
Enhanced Diffusion Ion Exchange Chromatography
Chromatography on Ceramic HyperD® Sorbents
© Pall Corporation
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.
© Pall Corporation
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.
© Pall Corporation
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
© Pall Corporation
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
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Charge Densityμeq/ml μeq/ml sorbent hydrogel
(packed bed) (in situ)
CM >250 >860Q >200 >690DEAE >180 >620S >150 >515
© Pall Corporation
Enhanced Diffusion in the Gel-Filled Pore
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++++++
+++
++
+++ +
++
+ + +
++
+++
++++
+
+++
+ ++
++
+
- -
Also referred to as “Hyperdiffusion”
© Pall Corporation
BlackCeramicBackbone
White-GrayHydrogel
Gray spotsGold-labeledalbumin
Scanning EM of Cross-Section Through Q Ceramic HyperD Sorbent Bead
© Pall Corporation
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
© Pall Corporation
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.
© Pall Corporation
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%.”
© Pall Corporation
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
© Pall Corporation
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.