Antibody purification

Antibody Purification

Sulov Saha

30 Nov, 2015

Contents

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Sources of antibodies

• Different methods of purifying antibodies

• Why “purified antibodies” are not all the same

• How to purify antibodies easily without specialist knowledge orequipment

• Considerations for conjugating antibodies to enzymes, dyes andnanoparticles

•

Introduction

Introduction

• The production and use of specific antibodies as detection probes and purification ligands – often called immunodetection or immunotechnology – has revolutionized bioresearch and diagnostic technologies. Animals immunized with prepared antigens will produce specific antibodies against the antigen.

• Once they are purified (and possibly after labeling them with an enzyme or fluorescent tag), these antibodies can be used directly to probe the specific antigen in Western blotting, ELISA and other applications.

Sources of antibodies

• Animal serum – polyclonal antibodies

• Ascites – monoclonal antibodies

• Cell culture supernatant – monoclonal antibodies

• Eggs – avian antibodies

• Bacterial expression systems – recombinant antibodies

Each of these sources has contains the specific antibody of interest amongstother proteins, lipids and other components.

Different methods of purifying antibodies

• Fractionation by globulin precipitation

• Ion exchange chromatography

• Size exclusion chromatography

• Protein A affinity chromatography

• Protein G affinity chromatography

• Protein L affinity chromatography

• Antigen affinity chromatography

Physicochemical fractionation

Class-specific affinity

Globulin precipitation

• First published in 1899 by James Atkinson in J. Exp. Med. – separatingalbumin from the anti-toxic components of a horse antiserum usingmagnesium sulphate.

• Ammonium sulphate and sodium sulphate are more commonly used,taking advantage of the principle of “salting-out”. At the appropriateconcentration these precipitate globulins, whilst many other proteins,including albumin, remain in solution.

• Whilst this is a simple and gentle procedure, it only provides a partiallypure preparation if starting from a complex mixture such as serum.

• This step is often combined with further purification using one of theother methods discussed in this presentation.

Ion exchange chromatography

• Ion exchange chromatography (IEC) uses positively or negatively chargedresins to bind proteins based on their net charges in a given buffer system(pH).

• Typically, a complex mixture is added to the column in a certain set of bufferconditions (e.g. low salt), and the buffer conditions are then changed eitherstep-wise or on a gradient basis.column in differing conditions.

Different proteins are released from the

• IEC is perhaps more often used to purify polyclonal antibodies than formonoclonals – note that each monoclonal is unique in terms of charge, andwill therefore be released under a specific set of conditions – something thatis useful for repeated purifications of the same antibody, but also means thatoptimisation is required for each antibody.

• There are various IEC resins available, with perhaps the most widely usedbeing DEAE-Sepharose.

Ion exchange chromatography

Starting buffer counter-ions

Substances to be separated

Gradient ions

Size exclusion chromatography

• Separation of complex mixtures on the basis of size or molecular shapecan be achieved by size exclusion (SEC) or gel filtration chromatography.

• The molecular sieving process takes place as a solute passes through apacked bed stationary phase. Separation depends on the differentabilities of the various molecules to enter the pores of the bead-basedstationary phase.

• Large molecules, which cannot enter the pores, are excluded and passthrough the column quickly. Smaller molecules that can enter the poresare retarded and move through the column more slowly. Very smallmolecules, such as salt, are able to fully permeate and elute last.


• SEC tends not to be used for primary purification of antibodies otherthan IgMs. However, it may be a valuable “polishing step” followingpurification by other methods.

• Ranges of chromatography resins are available that separate proteinswithin different ranges of size (e.g. AcA22, AcA34, AcA44 from Ultrogel®,and S200, S300, S400 in the Sephadex range)

DE-SALTING COLUMNS

• So called de-salting columns are used to undertake a bufferexchange, or to remove small size contaminants. This is simplya special case of SEC, with the most commonly used gel beingSephadex G-25, often in pre-prepared PD10 columns.


Different sizedproteins

Mixture ofdifferent sizedproteins

Gel f

iltra

tion

colu

mn

Protein A/G/L chromatography

• In their native form these proteins are expressed bybacteria as part of their defense mechanisms against themammalian immune response.

• All bind to mammalian immunoglobulins via constantregions – Protein A and G to the Fc region, and Protein L tothe kappa light chain.

• Recombinant versions are used immobilised to variousmatrices to purify IgG by a form of affinitychromatography.



• Samples are added to the Protein A/G/L matrix in asuitable binding buffer – pH and ionic strength should beconsidered

• Column is washed, which effectively leaves only IgG bound

• Low pH elution is carried out, which may be with differentbuffer formulations and may differ depending upon theisotype of the antibody being purified. It may beimportant to know the basic formulation of the elutionbuffer for downstream processing

• Low pH elution requires neutralisation to maintainantibody integrity. Often carried out with Tris buffer, but isthis sufficient for your downstream needs?


Recombinant Protein A Recombinant Protein G Recombinant Protein A/G Recombinant Protein L Recombinant Protein A/G/L

Native SourceStaphylococcus aureus

Streptococcus N/A Peptostrepto- coccus magnus

N/A

Binding Sites for Ig 5 2 6 4 13

Optimal Binding pH 8 - 9 5 5 to 8.2 7.5 7.5

Typical elution pH 3.0 – 7.0(isotype dependent)

2.5 – 3.0 2.5 – 3.0 2.0 – 3.0 2.5 – 3.0

Ig Binding Target Fc Fc Fc VL-kappa Fc + VL-kappa


Species Immunoglobulin Binding to Protein A Binding to Protein G Binding to Protein L

Human IgG1 Strong Strong Strong

IgG2 Strong Strong Strong

IgG3 Negligible Strong Strong

IgG4 Strong Strong Strong

Mouse lgG1 Weak Strong Strong

lgG2a Strong Strong Strong

lgG2b Medium Medium Strong

lgG3 Weak/medium Medium Strong

Rat lgG1 Negligible Weak Strong

lgG2a Negligible Strong Strong

lgG2b Negligible Medium Strong

lgG2c Negligible Medium Strong

Goat lgG Weak Medium Negligible

Rabbit lgG Strong Medium Weak

Sheep lgG Weak Medium Negligible

Antigen affinity chromatography

• In this case a specific chromatography resin is prepared to which the actualantigen that the antibodies bind to is immobilised.

• The sample containing the specific antibodies is then passed over the column,allowing only specific antibodies to bind, with all other contaminants,including other antibodies, being washed away.

• Buffer conditions are then changed to elute the specific antibodies from thecolumn.

• Typical examples of the uses of affinity chromatography are in preparation ofspecies specific secondary antibodies, in purifying antibodies to peptideantigens and for purifying proteins with epitope tags (e.g. Myc, HIS etc).

Antigen affinity chromatography

Preparation of gel matrix

Matrix Ligand ImmobilizedLigand

Application of sample

Sample Complex Impurities

Elution of purified antibody

Why “purified antibodies” are not all the same

Many antibodies are sold by companies as “purified” – but what does that mean?

• Concentration

• Preservatives / stabilisers – have any been added back in to thepreparation after purification?

• Buffer components – including contaminants from elution etc.

• Level of purity - e.g. Ig fraction vs. total IgG vs. specific IgG

• Unexpected contaminants – e.g. bovine IgG

• Endotoxin etc.


Your final buffer……..

• The final buffer that your antibody is in may have a significanteffect on what you can use your antibody for. For instance, thepresence of glycine in a buffer can inhibit conjugation reactions, orsodium azide may kill live cells. Some buffers may be suitable forconjugating to proteins, but not to nanoparticles.

• If you undertake a dialysis step post-purification to transfer anantibody into a particular buffer, you need to be sure that thedialysis has been effective.

• e.g. To remove (i.e. to 1nM level) 100mM glycine from 10ml ofantibody you need to dialyse against 1 million litres of PBS.Alternatively, changing the dialysis 4 times will achieve the sameeffect with only 4 litres of PBS.

• However, a single dialysis into 1 litre leaves glycine present at 1mM.


Total IgG vs. specific IgG

• The serum produced when any polyclonal antibody is raised will containonly approximately 10% of antibodies that are specific to the antigen ofinterest

• Therefore if you purify all of the IgG present, only 10% of that IgG will bespecific for the antigen.

• Only antigen affinity chromatography effectively provides IgG that is100% specific for antigen – Protein A/G/L chromatography provides totalIgG, albeit apparently 100% pure when analysed by SDS-PAGE.

• These differences lead to significant differences in activity – a workingdilution of an antigen affinity purified antibody is likely to be 10-foldhigher than that of a Protein A/G purified antibody.


Unexpected contaminants – e.g. bovine IgG

• By definition, your purified monoclonal antibody must be 100%pure. Is that right?

• Be aware that if your cell culture media contains foetal/newbornbovine serum there may be significant levels of bovine IgGpresent – which may co-purify with your monoclonal antibodywhen using media such as Protein A or G.

• Avoid these either by using a mouse IgG specific purificationsystem or converting all of your hybridoma culture to serum freemedia.

• If you use ascites you avoid bovine IgG, but you will have normalmouse IgG present which also co-purifies.

Conjugation considerations

You need to know some things about your reagent.Conjugations are really simplebut you need protein in the right format to work effectively.

Concentration – 1mg/ml or higher is preferred

Purity – ensure other proteins have been removed,and also make sure they haven’t been put back again afterwards!

Buffer formulation – most common formulations are suitable,but ensure that amines such as glycine are truly absent,as well as thiols such as DTT or mercaptoethanol. Tris is OK up to 20mM

Preferred buffer formulations for the antibodies to be labelled with thevarious kits differ (especially for nanoparticles), and purification methodsare important to understand

Future of Antibody Purification

Strategies include :• decreasing the number of steps• expanded and simulated moving beds• disposable and process analytical technology• membrane chromatography • non-chromatographic methods such as flocculation, precipitation, crystallization and aqueous two-phase systems.

1

More potential 2

Less expensive 3

Large scale productions

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Antibody purification