Antibody Purification Sulov Saha 30 Nov, 2015 Contents

Antibody Purification

Sulov Saha

30 Nov, 2015

Contents

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Sources of antibodies

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Different methods of purifying antibodies

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Why “purified antibodies” are not all the same

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How to purify antibodies easily without specialist knowledge or equipment

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Considerations for conjugating antibodies to enzymes, dyes and nanoparticles

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Introduction

Introduction

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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.

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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

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Animal serum – polyclonal antibodies

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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 amongst other proteins, lipids and other components.

Different methods of purifying antibodies

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Fractionation by globulin precipitation

• Ion exchange chromatography
• Size exclusion chromatography

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Protein A affinity chromatography

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Protein G affinity chromatography

• Protein L affinity chromatography
• Antigen affinity chromatography

Physicochemical fractionation

Class-specific affinity

Globulin precipitation

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First published in 1899 by James Atkinson in J. Exp. Med. – separating albumin from the anti-toxic components of a horse antiserum using magnesium sulphate.

• Ammonium sulphate and sodium sulphate are more commonly used, taking advantage of the principle of “salting-out”. At the appropriate concentration these precipitate globulins, whilst many other proteins, including albumin, remain in solution.
• Whilst this is a simple and gentle procedure, it only provides a partially pure preparation if starting from a complex mixture such as serum.

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This step is often combined with further purification using one of the

other methods discussed in this presentation.

Ion exchange chromatography

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

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Typically, a complex mixture is added to the column in a certain set of buffer conditions (e.g. low salt), and the buffer conditions are then changed either step-wise or on a gradient basis. Different proteins are released from the column in differing conditions.

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

• There are various IEC resins available, with perhaps the most widely used being 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 shape

can be achieved by size exclusion (SEC) or gel filtration chromatography.

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The molecular sieving process takes place as a solute passes through a packed bed stationary phase. Separation depends on the different abilities of the various molecules to enter the pores of the bead-based stationary phase.

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

Size exclusion chromatography

• SEC tends not to be used for primary purification of antibodies other than IgMs. However, it may be a valuable “polishing step” following purification by other methods.
• Ranges of chromatography resins are available that separate proteins within 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 buffer exchange, or to remove small size contaminants. This is simply a special case of SEC, with the most commonly used gel being Sephadex G-25, often in pre-prepared PD10 columns.

Size exclusion chromatography

Different sized proteins

Mixture of different sized proteins

Gel filtration column

Protein A/G/L chromatography

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In their native form these proteins are expressed by bacteria as part of their defense mechanisms against the mammalian immune response.

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

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Recombinant versions are used immobilised to various matrices to purify IgG by a form of affinity chromatography.

Protein A/G/L chromatography

Protein A/G/L chromatography

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Samples are added to the Protein A/G/L matrix in a suitable binding buffer – pH and ionic strength should be considered

• Column is washed, which effectively leaves only IgG bound

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Low pH elution is carried out, which may be with different buffer formulations and may differ depending upon the isotype of the antibody being purified. It may be important to know the basic formulation of the elution buffer for downstream processing

• Low pH elution requires neutralisation to maintain antibody integrity. Often carried out with Tris buffer, but is this sufficient for your downstream needs?

Protein A/G/L chromatography

	Recombinant Protein A	Recombinant Protein G	Recombinant Protein A/G	Recombinant Protein L	Recombinant Protein A/G/L
Native Source	Staphylococcus 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

Protein A/G/L chromatography

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

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In this case a specific chromatography resin is prepared to which the actual antigen 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.

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Buffer conditions are then changed to elute the specific antibodies from the

column.

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

Antigen affinity chromatography

Preparation of gel matrix

Matrix

Ligand

Immobilized

Ligand

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?

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Concentration

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Preservatives / stabilisers – have any been added back in to the preparation after purification?

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Buffer components – including contaminants from elution etc.

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

• Unexpected contaminants – e.g. bovine IgG
• Endotoxin etc.

Your final buffer……..

Why “purified antibodies” are not all the same

• The final buffer that your antibody is in may have a significant effect on what you can use your antibody for. For instance, the presence of glycine in a buffer can inhibit conjugation reactions, or sodium azide may kill live cells. Some buffers may be suitable for conjugating to proteins, but not to nanoparticles.

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If you undertake a dialysis step post-purification to transfer an antibody into a particular buffer, you need to be sure that the dialysis has been effective.

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

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

Why “purified antibodies” are not all the same

Total IgG vs. specific IgG

• The serum produced when any polyclonal antibody is raised will contain only approximately 10% of antibodies that are specific to the antigen of interest
• Therefore if you purify all of the IgG present, only 10% of that IgG will be specific for the antigen.

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

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

Why “purified antibodies” are not all the same

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/newborn bovine serum there may be significant levels of bovine IgG present – which may co-purify with your monoclonal antibody when using media such as Protein A or G.
• Avoid these either by using a mouse IgG specific purification system or converting all of your hybridoma culture to serum free media.
• If you use ascites you avoid bovine IgG, but you will have normal mouse IgG present which also co-purifies.

Conjugation considerations

You need to know some things about your reagent.

Conjugations are really simple

but 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 the various kits differ (especially for nanoparticles), and purification methods are 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.

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More potential

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Less expensive

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Large scale productions