designing next generation chromatography media for modern...

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

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 strategy

Conclusions

Outline

Process needs




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 strategy

Conclusions

Outline

Process needs




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 strategy

Conclusions

Outline

Process needs




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 strategy

Conclusions

Outline

Process needs




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 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.

GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.

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