designing next generation chromatography media for modern...
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Designing Next Generation Chromatography Media for Modern High-Throughput Plasma ProcessesMats GruvegårdPPB 09Menorca, Spain
Introduction
Performance
Screening and optimization
Screening strategy
Conclusions
Outline
Process needs
Designing a base matrix
Novel Affinity and Multimodal media
Improved productivity in IgG process
Summary
Introduction
Performance
Screening and optimization
Screening strategy
Conclusions
Outline
Process needs
Designing a base matrix
Novel Affinity and Multimodal media
Improved productivity in IgG process
Summary
Robustness, High Productivity @ Low Cost
Process needs
• Modern Bioprocesses require:
Capacity, Throughput, Lifetime, SelectivityLow “batch to batch” variability, Security of supply
Bead size, Pore size, Construction material, Ligand, Ligand density, Particle size distribution, Surface modification,
Regulatory support, Specification limits, etc
Higher productivities New selectivitiesTo solve new and old separation challenges that customers arefacing now and in the future.
Improving the overall process economy in down stream purification
Next generation BioProcess™ media Key driving forces for new media development
What is chromatography media
Key components of the construction:
+ Ligand
Construction material
Pore size
Particle size
Particle and pore size distribution
Type of ligand
Ligand density
Surface modification
Mode of interaction
Introduction
Performance
Screening and optimization
Screening strategy
Conclusions
Outline
Process needs
Designing a base matrix
Novel Affinity and Multimodal media
Improved productivity in IgG process
Summary
Choice of materials for a base matrixEssentially any material that could be formed into porous beads with good mechanical properties
O SiO Si O
O Si
O Si
OSiO
SiO Si
OSi
Si OO Si O
SiOSi
OH
OH OH
OH
OH
OHOH
OH
Silica, Glass,Ceramic, Carbon etc
Agarose, Cellulose,Dextran, etc
Polystyrene, Acrylamide,Methacrylate, etc
Mechanical properties and chemical stability are key issues to be considered
Inorganic materials Bio-polymers Synthetic polymers
Agarose an ideal material?
Hydrophilic and neutral
“Protein friendly”
Chemically and physically stable
CIP using NaOH
Low material content
Potentially high capacity
Open pore structure
Fast mass transport
..for chromatography media
The new High Flow Agarose platform
Bead size
Pore sizeRigidity
Sepharose 4/6B - 1966
Sepharose CL- 1977
Sepharose™ FF - 1985
High Flow Agarose - 2001
Capto™ and MabSelect™ media for high productivity processes
Chemical modification of agarose
O
OO
OO O
O
O
C H2 O H
OH
R
R
O
OO H
O HO O
O
O
C H2 O H
OH
Increasing the window of operation
The increased rigidity of the media allows forhigher flow rates at large scale and smaller foot print
Introduction
Performance
Screening and optimization
Screening strategy
Conclusions
Outline
Process needs
Designing a base matrix
Novel Affinity and Multimodal media
Improved productivity in IgG process
Summary
Ligands for plasma processing
The ligandThe key component for selectivity and capacity in combination with the nature of the base matrix!
Affinity ligands Conventional IEX Multimodal
O O N+
O H
O H O H
O O N+
O H
O H O HSO3
O -
O NOH
+
+O
N+
Established Affinity ligands
Heparin
Affinity chromatography is already a well established technique in plasma processing
Dyes
A collaboration between BAC and GE Healthcare to develop industrial affinity media
- IgG binder- FVIII binder- AAV binder- kappa fab binder- custom ligands
New Affinity LigandsFrom Single Domain Antibodies
BAC IgG004:10_UV1_280nm BAC IgG004:10_Inject BAC IgG004:10_Logbook
0
500
1000
1500
2000
2500
3000
mAU
0.0 5.0 10.0 15.0 20.0 ml
Column: Tricorn™ 5/100, 10 cm bed height
Flow rate: 250 cm/h, corresponding to 2.4 min residence time
Loading material: Human serum
Equilibration: 20 mM phosphate, 150 mM NaCl, pH 7.4, 10 column volumes
Wash: Same as equilibration buffer, 7 column volumes
Elution: 0.1 M glycine, pH 3.0, 5 column volumes
Yield: 90-94 %
LMW
Serum
Wash
Elution diluted1:5
Elution diluted1:10
LMW
Com
mercialIVIG
Phosforylas B 97KdAlbumin 66 Kd
Ovalbumin 45 Kd
Carbanhydrase 30 Kd
Trypsin inhibitor 20.1 Kd
A-Lactalbumin 14.4 Kd
Human serum albumin
Heavy chain IgG
Light chain IgG
LMW
Serum
Wash
Elution diluted1:5
Elution diluted1:10
LMW
Com
mercialIVIG
Phosforylas B 97KdAlbumin 66 Kd
Ovalbumin 45 Kd
Carbanhydrase 30 Kd
Trypsin inhibitor 20.1 Kd
A-Lactalbumin 14.4 Kd
Human serum albumin
Heavy chain IgG
Light chain IgG
IgG Subclass Distribution, Load vs. Eluate
Serum feedIgSelect
eluate
MabSelect SuRe™
eluate
IgG1 62.6% 62.3% 62.9%
IgG2 28.4% 28.2% 34.2%
IgG3 5.9% 5.9% 0.5%
IgG4 3.2% 3.7% 2.5%
IgSelectA new affinity resin for IgG purification
Yield: 92%
IgSelect - CIP stability
Ligand screened for high chemical stabilityExcellent low pH stability
For long lifetime phosphoric acid is suggestedCombination with 0.1 M NaOH can be consideredLeakage assay available from BAC (www.bac.nl)
0
20
40
60
80
100
0 10 20 30 40 50 60 70 80 90 100
Number of CIP cycles
Capa
city
(% o
f ref
eren
ce)
0.1 M NaOH + 1 M NaCl
0.5 M phosphoric acid
Multimodal chromatography
HIC/RPC(Hydrophobicity)
Ion exchange(Charge)
Affinity(Biorecognition)
Different types of multimodal media
S N
O OO
Capto™ MMC (high salt tolerant)
N+
OH
Capto adhere
(polishing of MAbs, non binding mode)
Multimodal chromatography will give multiple type of interactions, provide new selectivities, and are in some cases designed for specific applications.
Multiple types of interactionsionic interactionhydrophobic interactionhydrogen bondingthiophilic
Multimodal media in plasma process
• Replacing Q Sepharose™ Fast Flow in IgG process. Non-binding mode
• Initial study: sample loading increased by 100%
• Doubled capacity for binding impurities
• Yield: 4.3 g IgG/L starting plasma (vs. 3.4 IgG/L in reference process)
• Purity of IgG: 97% (gel filtration)
DEAE Sepharose FF
Adjustments
Q Sepharose FF
pH adjustment
UF/DF
S/D treatment
CM Sepharose FF
Euglobulin prec.
Desalting
Plasma
IgG
DEAE Sepharose FF
Adjustments
Capto adhere
pH adjustment
Euglobulin prec.
Desalting
Plasma
Reference process Alternative process
UF/DF
S/D treatment
CM Sepharose FF
IgG
DEAE Sepharose FF
Adjustments
Q Sepharose FF
pH adjustment
UF/DF
S/D treatment
CM Sepharose FF
Euglobulin prec.
Desalting
Plasma
IgG
DEAE Sepharose FF
Adjustments
Capto adhere
pH adjustment
Euglobulin prec.
Desalting
Plasma
Reference process Alternative process
UF/DF
S/D treatment
CM Sepharose FF
IgG
Virus clearance Capto™ adhere
3.6 ± 0.430MuLV
4.5 ± 0.410MuLV
≥ 5.9 ± 0.330MVM
≥ 5.8 ± 0.310MVM
LRF 95% confidence limit
Conductivity (mS/cm)
Virus
3.6 ± 0.430MuLV
4.5 ± 0.410MuLV
≥ 5.9 ± 0.330MVM
≥ 5.8 ± 0.310MVM
LRF 95% confidence limit
Conductivity (mS/cm)
Virus
Very good log10 reduction factors even for conditions where traditional ion exchangers
do not work!
IgG1 pool from MabSelect SuRe™ spikedwith stock solutions of:
MVM (Minute Virus of Mice)Single stranded DNA virusNon-enveloped, 20-26 nm
MuLV (Murine leukemia Virus)Singel stranded RNAEnveloped, 80-110 nm
Applied to Capto adhere in flow throughmode, pH 6.75, 10 and 30 mS/cm*
* Performed by NewLab BioQuality AG
Introduction
Performance
Screening and optimization
Screening strategy
Conclusions
Outline
Process needs
Designing a base matrix
Novel Affinity and Multimodal media
Improved productivity in IgG process
Summary
Plasma
Cryosupernatant
Supernatant I
Delipidated SNI
Delipidated & Diafiltered SNI
Delipidated & Euglobulin depleted SNI
Sepharose DEAE-FF chromatography
pH adjusted Crude IgGCM Sepharose FF chromatography
pH adjusted & concentrated Albumin
Sephacryl S200HR chromatography
Albumin Bulk
Low pH caprylate incubation
MacroPrep HQ chromatography
Concentrated & Diafiltered Pure IgG
Pasteurisation
Pasteurised Bulk IgG
Formulation (Albumin) Formulation (Immunoglobulin)
Current Albumin & IgG process at CSL
0
500
1000
1500
2000
2500
3000
3500
400 500 600 700 800 900 1000 1100 1200
IgG in flow through
Elution volume (ml)
Abs
orba
nce
280
nm (m
AU
)
albumin eluted at
pH 4.5
0
500
1000
1500
2000
2500
3000
3500
400 500 600 700 800 900 1000 1100 1200
IgG in flow through
Elution volume (ml)
Abs
orba
nce
280
nm (m
AU
)
albumin eluted at
pH 4.5
Plasma
Albumin IgG
Objective of the study
• Evaluate Capto™ DEAE as replacement of DEAE Sepharose™ Fast Flow in IgG / Albumin process
• Investigate effects of increasedprotein loadflow ratesbed height
• Scale-up studies
• Life time study
• Initial discussion with regulatory bodies
DEAE Sepharose™ Fast Flow
Capto™ DEAE
Effect of increased protein load
020406080
100120
65 80
020406080
100120
65 80 100 120 137Protein load (g/L)
Flow through
EluateProtein load (g/L)
% o
f tot
al lo
aded
020406080
100120
65 80
0
50
100
150
65 80 100 120 137Protein load (g/L) Protein load (g/L)
IgG IgA IgM Transferrin Albumin
Protein load could be increased to 100 g/L using Capto DEAE
% o
f tot
al lo
aded
% o
f tot
al lo
aded
% o
f tot
al lo
aded
XK16/40 column h=17.5 cm. Flow rate of 100 cm/h. , Equilibration pH 5.2Albumin eluted at pH 4.5 , Washed with 1 M NaCl.
0
500
1000
1500
2000
2500
3000
3500
400 500 600 700 800 900 1000 1100 1200
Effect of increased flow rate and bed height
XK16/40 column h=25cm. Flow rates of 100, 130 and 150 cm/h. Equilibration pH 5.2 Albumin eluted at pH 4.5 Washed with 1 M NaCl.
Elution volume (ml)
Abs
orba
nce
280
nm (m
AU
)
17.5, 25 and 30 cm100, 130 and 150 cm/h
Level of IgA depending on residence time – could be maintained at same level as today’s processPossibility to increase the column size by increasing the bed heightPossible to reduce the number of cycles from 10 to 5 or 4
0
500
1000
1500
2000
2500
3000
3500
4000
0 5000 10000 15000 20000 25000 30000 35000
Volume (mL)
Abso
rban
ce @
280
nm
Scale-up experiments
Flow through Average (3 batches)
% recovery % of total
IgG
IgA
IgM
Albumin
Transferrin
α2-macroglobulin
106 73
26 4
60 1
0.3 1
85 17
22 4
Eluate Average (3 batches)
% recovery % of total
IgG
IgA
IgM
Albumin
Transferrin
α2-macroglobulin
2 <1
21 1
57 1
105 98
5 <1
8 <1
Life time study
0
500
1000
1500
2000
2500
3000
3500
50 100 150 200 250Time (min)
Abs
orba
nce
at 2
80 n
m
Cycle 1
Cycle 364
No change in performance after >360 cycles including CIP
Product equivalence of formulated IgG
115118= 60% EPBRPFc function
PassPassClear or slightly opalescent and colourless or pale yellow
Appearance
15.715.3≥ 1 IU/mLTetanus Antitoxin
16.718.7≥ 3 IU/mLHepatitis A Antibody
2.72.45≥ 0.03 IU/mLHepatitis B Antibody
< 1< 1Anti-D Titre ≤ 1:1
5.36.7Anti-B Titre ≤ 1:64
88Anti-A Titre ≤ 1:64ABD Titres
< 1< 1≤ 28.6 IU/mLPKA
3.72.3≤ 10 CH50/mg/hourFreedom from ACA
0.50.2Fragments – For information
99.599.7Monomer + Dimer ≥ 90%
00.1Aggregates ≤ 3% Protein Composition B (SEHPLC)
Production-scale batches (n = 3)
Capto DEAE Laboratory-scale
batches (n = 3)
Limits / Expected valuesTest type
115118= 60% EPBRPFc function
PassPassClear or slightly opalescent and colourless or pale yellow
Appearance
15.715.3≥ 1 IU/mLTetanus Antitoxin
16.718.7≥ 3 IU/mLHepatitis A Antibody
2.72.45≥ 0.03 IU/mLHepatitis B Antibody
< 1< 1Anti-D Titre ≤ 1:1
5.36.7Anti-B Titre ≤ 1:64
88Anti-A Titre ≤ 1:64ABD Titres
< 1< 1≤ 28.6 IU/mLPKA
3.72.3≤ 10 CH50/mg/hourFreedom from ACA
0.50.2Fragments – For information
99.599.7Monomer + Dimer ≥ 90%
00.1Aggregates ≤ 3% Protein Composition B (SEHPLC)
Production-scale batches (n = 3)
Capto DEAE Laboratory-scale
batches (n = 3)
Limits / Expected valuesTest type
Introduction
Performance
Screening and optimization
Screening strategy
Conclusions
Outline
Process needs
Designing a base matrix
Novel Affinity and Multimodal media
Improved productivity in IgG process
Summary
Summary
Constant improvements of process chromatography media
Give possibility for plasma process improvements
Some examples:New base matrix, e.g. High Flow AgaroseEstablished ligands on new base matrixNew affinity ligand constructsMultimodal chromatography
Acknowledgement
GE Healthcare
Inger Andersson
Anders Ljunglöf
Anna Grönberg
Lise Lundh
Klas Allmér
CSL
Karl McCann
Joe Bertolini
Introduction
Performance
Screening and optimization
Screening strategy
Conclusions
Thank you
Capto, MabSelect, MabSelect SuRe, MabSelect Xtra, Sepharose, Sephacryl, SOURCE and Tricornare trademarks of GE Healthcare Ltd , a General Electric Company. GE, Imagination at work and GE Monogram are trademarks of General Electric Company.
All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. General Electric Company reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation.
© 2006 General Electric Company – All rights reserved.
GE Healthcare Bio-Sciences AB, a General Electric Company.
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