Antibody Purification - Tips and Tricks

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-Tips & Tricks

Antibody Purification

Content

IntroductionAffinity ChromatographyAntibody purification techniques

Tips & HintsSample related challenges

Immunoglobulin typeSample source

Method related challengesYieldPurityHomogeneityAcid sensitive antibodiesThroughputColumn life-time

Summary

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Summary

What is affinity chromatography?

Affinity chromatography is a liquid chromatography technique which separates biomolecules based on highly specific biological interactions

Why use affinity chromatography?

• Simple to do − one step• Concentrates• High purity, often > 90 %

• Easy to achieve otherwise difficult separations

– Separate native from denatured and functionally different forms

• Fast separations

Tools for affinity capture

Magnetic beads

Multiwell plates

Pre-packed columns

Gravity columns

µg mg

Spin columns

Mag Sepharose™ SpinTrap™ MultiTrap™ GraviTrap™

HiTrap™

HiScreen™

etc.

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Cipp principle in short

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Antibody purification techniques

Target immunoglobuli

n

Affinity ligand

Any immunoglobulin

Immobilized antigen

IgG Protein A, protein G (protein L)

IgG fragment (with kappa chains)

Protein L

IgM ThiolphilicIgY Thiophilic

CaptureAffinity chromatography

Intermediate purification & polishingSize exclusion chromatographyCation exchange chromatographyMultimodal anion exchange

IgG

IgMIgG fragments

Antibody purification techniques

Protein A ligandProtein G ligand

Protein L ligand

Kappa and Lambda ligands

Two heavy chains Two light chainsKappa or Lambda

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Summary

Immunoglobulin type related challengesTarget molecule

Challenge

IgG Identifying the optimal affinity capture resin with respect to species and subclass selectivity.

Acid sensitive antibodies Polyclonal IgG Enrichment of IgG that is directed towards the antigen of

interest – removal of the irrelevant IgG.IgG fragments Identifying the optimal affinity capture resin IgM Often poor stability.

Technology issues since most separation techniques were developed for proteins << 700 kD.

IgY (Removal of lipids in egg yolks)

Sample source related challenges

Start material

Challenge Remedy

Serum Clarification 0.45µm filtrationCell culture medium

Phenol red removal Desalting column

Ascites Lipid removal; clarification

Centrifugation; 0.45µm filtration

E. coli lysate Clarification Centrifugation; 0.45µm filtrationEgg yolk Lipid removal;

clarificationExtraction, centrifugation; 0.45µm filtration

Species Protein A Protein G

binding bindingMonkey (rhesus) ++++ ++++MouseIgG1 + ++++IgG2a ++++ ++++IgG2b +++ +++IgG3 ++ +++IgM* variable -Pig +++ +++Rabbit ++++ +++RatIgG1 - +IgG2a - ++++IgG2b - ++IgG3 + ++Sheep +/- ++

IgG binding specificities of immobilized protein G and protein A differ

Species Protein A Protein Gbinding binding

HumanIgA variable -IgD - -IgEIgG1 ++++ ++++IgG2 ++++ ++++IgG3 - ++++IgG4 ++++ ++++IgM* variable -Avian egg yolkIgY† - -Cow ++ ++++Dog ++ +Goat - ++Guinea PigIgG1 ++++ ++IgG2 ++++ ++Hamster + ++Horse ++ ++++Koala - +Llama - +

+ relative binding strength– weak or no bindingProtein A and protein G binds to the Fc region of IgG, and also has a weak affinity for the Fab region.

Purification of mouse IgG1

Sample: Cell culture supernatant containing mouse IgG1

Column: HiTrap™ Protein G HP 1 mlBinding buffer: 20 mM sodium phosphate, pH 7.0Elution buffer: 0.1 M glycine-HCl, pH 2.7System: ÄKTAprime™

Mouse IgG1

Inject

Mouse IgG1 binds strongly to protein G

Polyclonal antibodies – getting the desired specificity

Polyclonal antibody preparation.Crude serum or purified on protein A or

protein G

Affinity enrichmenton immobilized target antigen

Affinity depletionon immobilized irrelevant antigen

030213 S345:1_UV1_280nm 030213 S345:1_Cond

0

200

400

600

800

mAU

0 100 200 300 400 500 600 700 ml

Flow-through(collected)

Eluted fraction(discarded)

Flow-through(discarded)

Eluted fraction(collected)

Challenge:Polyclonal sera contains both irrelevant and antigen-specific IgG. Protein A and protein G can not discriminate between the two.

Preparation of the affinity column for affinity enrichment or affinity depletion

• Dissolve the antigen in (amine free) coupling buffer• Inject antigen solution• Block excess active groups

HiTrapTM NHS-activated HP

+H2N

HN +

Image from RCSB PDB (www.pdb.org)

Purification of antibody fragments (Fab)Column: HiTrapTM Protein L 1 mlSample: 2 mg Polyclonal mouse FabFlow rate: 0.25 ml/min (residence time 4 min)Gradient: pH 7.4 – 2.3 (linear gradient, 10 column

volumes)System: ÄKTAavantTM 25

Lambda light chain

Kappa light chain

A 280

nm (m

AU)

Sample: Monoclonal IgM in cell culture supernatant, 0.5 M potassium sulphate Column: HiTrap™ IgM Purification HP 1 ml Binding buffer: 20mM sodium phosphate,

0.5 M potassium sulphate, pH 7.5Elution buffer: 20 mM sodium phosphate, pH 7.5Cleaning buffer: 20 mM sodium phosphate, 30% isopropanol, pH 7.5

1.5

1.0

2.0

0.5

0

00

20

60

80

50

40

100

100

ml

AU280nm mS/cm

Elutionbuffer

Cleaningbuffer

N

SH

IgM

Ligand:2-mercaptopyridine

IgM

1. LMW standard2. Starting material3. Human IgM, standard4. Human IgG, standard5. Flow-through 6. Eluted IgM

Purification of IgMThiophilic adsorption chromatography

Purification of IgYThiophilic adsorption chromatography N

SHLigand:

2-mercaptopyridine

Sample: 45 ml egg yolk extract (corresponding to ¼ egg

yolk) containing a-Hb IgY, in 0.5 M potassium

sulphate,filtered through 0.45µm

Column: HiTrap™ IgY Purification HP 1ml Binding buffer: 20 mM sodium phosphate,

0.5 M potassium sulphate, pH 7.5Elution buffer: 20 mM sodium phosphate, pH 7.5Cleaning buffer: 20 mM sodium phosphate,

30% isopropanol, pH 7.5

IgY

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Summary

Low yield in affinity capture (1)

Diagnosis: Antibody appears in the flow-through fractionCause RemedySelectivity issues 1. Try another affinity ligand. Protein G, protein A and

protein L are all suitable for IgG purification but they differ in species and subclass selectivity.

2. Change binding conditions

Column is overloaded 1. Increase bed volume2. Increase residence time

Low yield in affinity capture (2)

Diagnosis: Poor mass recovery, but not due to antibody appearing in flow-throughCause RemedyInsufficient elution conditions

Increase mass yields from protein A, protein G and protein L by lowering the elution pH. The collected fractions should be neutralized immediately to preserve activity.

Precipitation on column Change one or several of: binding, wash and elution conditions.

Insufficient purity

Diagnosis: There are remaining irrelevant proteins as assayed by e.g., SDS-PAGE or ELISA.

Remedy:• Optimize affinity capture

– focus on wash step • Add purification steps

IgG heavy chain

IgG light chain

Optimized wash step for improved purity

Sample application

Wash 1: PBS

Wash 2: 2M urea, 10% isopropanol,0.5M NaCl, pH 5.5Elution, pH 3.4

0

1

2

3

0 50 100 150 ml

4

AU

Sample: 27 mg mAb/ml resinColumn: HiScreenTM MabSelect SuReTM

Wash 1 only

Wash 1 + Wash 2

IgG recovery (%)

93 94

Aggregates (%)

1.5 1.7

HCP (ppm) 3137 419

Insufficient homogeneity

Diagnosis: Presence of aggregates monitored by e.g., size exclusion.

Remedy: Add a purification step- size exclusion- Cation exchange - Multimodal anion exchange

size exclusion requires little or no optimization. Cation exchange and multimodal ion exchange provide higher throughput but will require optimization.

Mouse IgG1 purification with protein G followed by size exclusionAffinity chromatography

HiTrap™ Protein G HP 1 mlSize exclusion

HiLoad™ 16/60 Superdex™ 200 pg

• Removal of dimers and aggregates• Buffer exchangeH chain

L chain

3.0

Low pH

IgG aggregate removal

Cation exchange Multimodal anion exchange

IgG monomer

Dimers,aggregate

s

CaptoTM SP ImpRes

Alternatives to size exclusion as a second purification step after affinity capture with protein A or protein G

Capto adhere

IgG monomer

peak

Aggregates

Two step process Yield Dimers/aggregates Host cell protein (%) (%) (ppm)Start material 100 130000MabSelect SuRe 95 0.7 55Capto adhere 95 <0.1 7.5

O O N+

OH

OH OH

O O N+

OH

OH OH

Particularly acid sensitive antibodies

Diagnosis: Acceptable mass recovery but poor activity recovery (precipitation at acidic pH).

Remedies: 1. Collect the antibody, when eluted at acidic pH, into tubes pre-

filled with a small volume of e.g., 1M Tris, pH 8.0. 2. Use non-affinity technique for capture (e.g., ion exchange).3. Use alternatives to acidic elution from protein A or protein G.

Exploring alternative elution strategies Protein A and protein G

Start

materia

l pH

2.94M

MgCl 2

Start

materia

l pH

2.910

mM

NaOH

Protein G Mag Sepharose

Xtra

Protein A Mag Sepharose™ Xtra

IgG

bin

ding

act

ivit

y

4M MgCl2 and 10 mM NaOH can be used for elution from protein A and protein G resins,

respectively

Throughput

Parallel LC system

- 4 Parallel

purifications per unit

- Automated multi-step

purifications.

ÄKTAxpress™ MAb

Multiwell filter plates- 96 parallel

- Manual (multi-pipette)- Automated (robot)

Gravity columns- Simple, manual

affinity capture- Up to ~10 parallel

Magnetic beads- Small scale

purifications- Manual (MagRack)- Automated (robot)

AC1 AC2 AC3 AC4

DS

Unattended two-step mAb purification

Sample: 20 ml human mAb in cell culture supernatant, 0.5 mg/mlColumns: Affinity chromatography (AC): HiTrap™ MabSelect SuRe™ 1 ml

Desalting (DS): HiPrep™ 26/10 DesaltingFlow: 1 ml/minSystem: ÄKTAxpress™

AC1

AC2

AC3

AC4

DS DS DS DS

MabSelectTM MabSelect

0.1M NaOH 15 min

MabSelectTM

MabSelect SuReTM

CIP protocol: 0.1M NaOH, 15 minProtein A

E D A B C

Improved column life time

Alkali-stabilized protein A

Summary

• Protein A, protein G and protein L provide excellent selectivities for affinity capture of IgG and IgG fragments.

- Neutralize the collected material quickly after elution

- Consider additional wash step to increase purity- Consider alternatives to pH elution for particularly

acid sensitive IgG’s

• Thiophilic adsorption chromatography for purification of IgY and IgM.

• Affinity enrichment or affinity depletion with immobilized antigen yields monospecific, polyclonal antibodies

• size exclusion, cation exchange or multimodal chromatography are efficient techniques for removal of IgG aggregates

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