Antibody Purification Techniques

Antibody Purification Methods Overview

Antibody purification methods are used to isolate polyclonal antibodies from serum, or monoclonal antibodies from ascites fluid or the culture supernatant. There are various ways to purify antibodies. These purification methods can purify antibodies from very crude to highly specific.

Animals immunized with prepared antigens will produce specific antibodies against the antigen. When purified from serum or hybridoma cell lines that are prepared from tissue of the immunized animal, the antibody can be used directly (or after labeling with enzyme or fluorescent tags) to probe the specific antigen in Western blotting, ELISA or a variety of other applications. Monoclonal antibody purification processes typically involve a two or three step purification strategy of capture, intermediate and polish purification.

Three Phase Strategy: considerations when developing the purification scheme
Fig 1. Three Phase Strategy: considerations when developing the purification scheme

Antibody Capture Method

Because antibodies have predictable structure, including relatively invariant domains, it has been possible to identify certain protein ligands that are capable of binding generally to antibodies, regardless of the antibody's specificity to antigen. Protein A, Protein G and Protein L are three bacterial proteins whose antibody-binding properties have been well characterized. These proteins have been produced recombinantly and used routinely for affinity purification of key antibody types from a variety of species. These antibody-binding proteins are available immobilized to beaded agarose resin.

Binding sites of antibody-binding proteins
Fig 2. Binding sites of antibody-binding proteins

Proteins A, G and L have unique properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). It is important to realize that use of Protein A, G or L results in purification of general immunoglobulin from a crude sample. Depending on the sample source, antigen-specific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2-5% of total IgG in mouse serum is specific for the antigen used to immunize the animal.

The interaction between the various proteins and IgG is not equivalent for all species or all antibody subclasses. Figure 3. will help you decide which affinity protein is best for your application.

Proteins used to immobilize antibodies to beaded support show specificity to different antibody domains. Protein A and G bind to the heavy chains of the antibody Fc region, while Protein L specifically binds the light chains of the two Fab regions of the F(ab')2 antibody fragment. Protein G can also bind Fab fragments in certain conditions.

Table 1. Characteristics of immunoglobulin-binding proteins

  Recombinant Protein A Recombinant Protein G Recombinant Protein L
Native Source Staphylococcus aureus Streptococcus Peptostreptococcus magnus
Production Source E. coli E. coli E. coli
Molecular Weight 44,600 21,600 35,800
Apparent Mass by SDS-PAGE 45kDa 32kDa 36kDa
Binding Sites for Ig 5 2 4
Albumin Binding Site No No No
Optimal Binding pH 8.2 5 7.5
Ig Binding Target Fc Fc VL-kappa

The three proteins bind almost exclusively with the IgG class of antibodies, but their binding properties differ among species and subclasses of IgG. Protein A is generally preferred for rabbit, pig, dog and cat IgG. Protein G has better binding capacity for a broader range of mouse and human IgG subclasses (IgG1, IgG2, etc.). Protein L binds to certain immunoglobulin kappa light chains. Because kappa light chains occur in members of all classes of immunoglobulin (i.e., IgG, IgM, IgA, IgE and IgD), Protein L can purify these different classes of antibody. However, only those antibodies within each class that possess the appropriate kappa light chains will bind. Generally, empirical testing is required to determine if Protein L is effective for purifying a particular antibody.

Table 2. Binding characteristics of immunoglobulin-binding proteins

Species Antibody Class Protein A Protein G Protein L
Human Total IgG S S S
IgG1 S S S
IgG2 S S S
IgG3 W S S
IgG4 S S S
IgM W NB S
IgD NB NB S
IgA W NB S
Fab W W S
scFv W NB S
Mouse Total IgG S S S
IgM NB NB S
IgG1 W W S
IgG2a S S S
IgG2b S S S
IgG3 S S S
Rat Total IgG W M S
IgG1 W M S
IgG2a NB S S
IgG2b NB W S
IgG2c S S S
Total IgG W S NB
IgG1 W S NB
IgG2 S S NB
Goat Total IgG W S NB
IgG1 W S NB
IgG2 S S NB
Sheep Total IgG W S NB
IgG1 W S NB
IgG2 S S NB
Horse Total IgG W S ?
IgG(ab) W NB ?
IgG(c) W NB ?
IgG(T) NB S ?
Rabbit Total IgG S S W
Guinea Pig Total IgG S W ?
Pig Total IgG S W S
Dog Total IgG S W ?
Cat Total IgG S W ?
Chicken Total IgY NB NB NB

W=weak binding; M=medium binding; S=strong binding; NB=no binding; ?=information not available

Binding to Protein L will occur only if the immunoglobulin has the appropriate kappa light chains. The stated binding affinity refers only to species and subtypes with appropriate kappa light chains. Lambda light chains and some kappa light chains will not bind.

Antibody Polish Method

A single, rapid capture step using affinity chromatography is often sufficient to achieve the level of purity and quantity of product required for research purposes. Antibodies or their fragments can be adequately purified for further use, and a polishing step is sufficient to remove unwanted impurity. If affinity chromatography cannot be used, or if a higher degree of purity is required, alternative techniques need to be combined effectively into a polish strategy. A significant advantage when working with native or recombinant antibodies or fragments is that there is often considerable information available about the product and contaminants, as shown in Table 2.

Table 3. Characteristics of IgG

Molecular weight (Da) 150,000–160,000
Isoelectric point (pI) 4–9, most > 6.0, often more basic than other serum proteins
Hydrophobicity IgG is more hydrophobic than many other proteins
Solubility IgG very soluble in aqueous buffers. Lowest solubility (specific to each antibody) near pI or in very low salt concentration.
Temperature stability Relatively stable at room temperature (but specific to each antibody)
pH stability Relatively stable at room temperature (but specific to each antibody)
Carbohydrate Content 2–3%, most carbohydrate is associated with Fc region of the heavy chains

Antibodies are purified using purification techniques that separate according to differences in specific properties, as shown in Table 3.

Ion-exchange (IEX) chromatography

Ion-exchange (IEX) chromatography refers to the separation of proteins based on charge. Columns can either be prepared for anion exchange or cation exchange. Anion exchange columns contain a stationary phase with a positive charge that attracts negatively charged proteins. Cation exchange columns are the reverse, negatively charged beads which attract positively charged proteins. Elution of the target antibody is done by changing the pH in the column, which results in a change or neutralization of the charged functional groups of each protein.

Gel filtration (GF) chromatography

Gel filtration (GF) chromatography, also known as size-exclusion chromatography, separates larger proteins from small ones since the larger molecules travel faster through the cross-linked polymer in the chromatography column. The large antibodies do not fit into the pores of the polymer whereas smaller antibodies do, and take longer to travel through the chromatography column, via their less direct route. Eluate is collected in a series of tubes separating antibodies based on elution time. Gel filtration is a useful tool for concentrating an antibody sample since the target antibody is collected in a smaller elution volume than was initially added to the column. Similar filtration techniques might be used during large-scale antibody production because of their cost-effectiveness.

Hydrophobic interaction (HIC) chromatography

Hydrophobic interaction (HIC) chromatography is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC is based on interactions between hydrophobic (aliphatic or aromatic) ligands on the stationary phase with hydrophobic patches on the surface of proteins. This interaction is enhanced by high-ionic-strength buffer, which makes HIC an excellent purification step after precipitation with ammonium sulfate or elution in high salt during IEX.

Table 4. Protein properties used during purification

Protein property Technique
Charge Ion exchange (IEX)
Size Gel filtration (GF)
Hydrophobicity Hydrophobic interaction (HIC)

Every chromatographic technique offers a balance between resolution, capacity, speed and recovery. as shown in Figure 3.

Considerations in different chromatographic techniques
Fig 3. Considerations in different chromatographic techniques

Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample that can be loaded will be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein.

Speed is most important at the beginning of purification where contaminants, such as proteases, must be removed as quickly as possible.

Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and by unfavorable conditions on the column.

Resolution is achieved by the selectivity of the technique and the efficiency of the chromatography matrix in producing narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties.

Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step.

A guide to the suitability of each purification technique for polish is shown in Table 5. Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in conditions suitable for the start conditions of the next column (see Table 5).

Table 5. Suitability of purification techniques

Technique Main features Capture Intermediate Polishing Sample start condition Sample end condition
IEX high resolution
high capacity
high speed
★★★ ★★★ ★★★ low ionic strength
sample volume not limiting
high ionic strength or pH change
concentrated sample
HIC good resolution
good capacity
high speed
★★ ★★★ high ionic strength
sample volume not limiting
low ionic strength
concentrated sample
AC high resolution
high capacity
high speed
★★★ ★★★ ★★ specific binding conditions
sample volume not limiting
specific elution conditions
concentrated sample
GF High resolution   ★★★ limited sample volume (<5% total column volume) and flow rate range buffer exchanged (if required)
diluted sample
RPC high resolution   ★★★ sample volume usually not limiting
additives maybe required
in organic solvent, risk loss of biological activity

IEX: Ion-exchange chromatography;HIC: Hydrophobic interaction chromatography;AC:Affinity chromatography;GF: Gel filtration chromatography;RPC:Reverse Phased Chromatography.

References

1. Low, D., O'Leary, R., & Pujar, N. S. (2007). Future of antibody purification. Journal of Chromatography B, 848(1), 48-63.
2. Huse, K., Böhme, H. J., & Scholz, G. H. (2002). Purification of antibodies by affinity chromatography. Journal of biochemical and biophysical methods, 51(3), 217-231.
3. Ayyar, B. V., Arora, S., Murphy, C., & O'Kennedy, R. (2012). Affinity chromatography as a tool for antibody purification. Methods, 56(2), 116-129.
4. Ghose, S., Tao, Y., Conley, L., & Cecchini, D. (2013, September). Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions. In MAbs (Vol. 5, No. 5, pp. 795-800). Taylor & Francis.

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