Antibody Purification: Tips and Tricks

For all species, classes or size (FL vs. fragments)

Whatever the type of antibody production (hybridoma or recombinant protein production in bacteria or in mammalian cells), you need to isolate your antibody of interest from other proteins present in the sample (sera, ascites fluid, medium, cell lysate…).

In order to isolate the antibody of interest, you need to select the right strategy depending on the nature of the antibody (species, class, tagged or not), the purity required and the intrinsic antibody physico-chemistry.

Since 2000, tebu-bio provides antibody services from production to purification, we can help you further determine the most appropriate strategy by considering all parameters, including intrinsic antibody characteristics and following experimental requirements.

This article is your first guide to prepare the best angle of attack to put into action!

1.Antibody isotype differences

Antibodies, known as immunoglobulins, are generally Y-shaped. Their main function is to control and stop pathogens and to assist in an immune response. The antibodies are subdivided into different isotypes and the constant region varies according to the type of isotype (see our post on recombinant antibody and figure 2).

2.Purification affinity of antibodies

Purification affinity of monoclonal antibodies has been largely confined to the use of Protein A, Protein G and Protein L chromatography. Nevertheless, the binding capacity depends on the species and on the antibody classes. Indeed, beads complexed with Protein A and G are the most commonly used for the capture step of human IgG, but they are not efficient enough and/or adapted to isolate human IgA, IgD, IgE and IgM. Therefore, you may need to choose other strategies. Notably, the quality of the resin is very important, and the binding capacity can be very different of a resin from one to another supplier.  That is why we recommend some resin suppliers below to ensure the quality of your experiments.

Protein A beads

Protein A is a cell wall component produced by several strains of Staphylococcus aureus. It’s a single polypeptide chain of 42 kDa that contains few or no carbohydrates and is covalently coupled to beaded agarose gel. This peptide can bind specifically to the Fc region of immunoglobulin molecules, especially with IgG of several species of mammals, with high affinity.
The difference in binding efficiency of Protein A to different sub-classes of IgG allows to separate one IgG type from another. Moreover, antibodies that do not bind to immobilized Protein A may be recovered by collecting the Flow-Through fractions during the binding step.

Protein G beads

Protein G is a bacterial cell wall protein isolated from group G streptococci. It’s a highly charged protein weighing 22kDa. The immunoglobulin-binding sites of protein A and protein G are very different. Although the tertiary structures of these proteins are similar, their amino acid compositions are significantly different. Protein G can bind Fc and Fab regions of several Ig from different species.

Generally, the Protein G resins that I used were Protein G Sepharose FF resins from GE with a binding capacity up to 25 mg human IgG/ml medium.

Protein L beads

Protein L is an immunoglobulin-binding protein of 35.8 kDa from bacteria Peptostreptococcus magnus. It binds k light chain of the VL domain of Ig from different classes (IgG, IgM, IgA, IgE, IgD and IgY with different binding affinities). Consequently, the prerequisite is that the Ig of interest presents a k light chain… Please note that this efficiency depends on the species of the antibody (see Fig. 3).

Protein L beads, such as Capto L resins from GE, can capture h&m IgG, mIgM and antibody fragments (Fab, ScFv, Dab…) with a high efficiency.

In most of cases, these classical resins are highly effective to capture antibodies. Nevertheless, for certain antibody species, classes or fragments, protein of interest will be difficult to isolate. For this reason, we have developed internal innovative solutions to optimize the isolation.

3.Innovative strategies to purify antibodies

Conventional strategies, such as the use of protein A, G and L resins for the purification of antibodies, constitute the first approach to follow. Indeed, they are easier and cheaper than other strategies. However, in some cases, a new approach can help you better achieve your purification goals.
Antibody fragments and some classes or species of antibodies are very difficult to capture. The only strategy usually used in such case is to perform an ion exchange purification at first. But separating an antibody from other contaminants only by using the charge of the protein is not very effective. Many purification steps must be performed, for example, hydrophobic chromatography, another Ion exchange chromatography and finally a Gel filtration step to separate contaminants by selecting their size, these steps can be very time consuming and costly.
Below are solutions that we have developed to help you to better control time and budget spent on experiments. In addition, these innovative strategies are particularly effective when an optimization step has been well designed, performed and interpreted.

NHS activated beads

NHS ou N-hydroxysuccinimide activated beads (from Purolite or GE) allow the ligand to be coupled to a preactivated medium via its primary amine group. It offers a lot of possibilities, for example, capturing an antibody based on its affinity for the target… Do not hesitate to test a lot of binding conditions to optimize this step which is the most important step here.

Of course, tebu-bio’s lab is always ready to provide you with the most appropriate advice and services.

Multimodal beads

Multimodal beads or mixed-mode chromatography resins combine usually ion exchange and hydrophobic or affinity properties. Such type of purification requires an extended optimization step to find the best conditions for the binding and the elution steps. It’s not so easy but it can really be helpful in certain complicated situations, e.g. when other strategies are not efficient to purify the Antibody (Full Length or fragment versions), when the protein stability poses a problem, when we want to reduce the number of purification steps… Such a strategy can be a very helpful and an efficient way both in the first and second purification steps to eliminate contaminants and/or as a polishing purification step.
GE and BIO-RAD propose different multimodal resins that have different efficiency depending on the protein specific characteristics.
We can help you to screen different types of multimodal resins at the same time to find the most efficient purification strategy for your protein of interest by using specific Design of Experiments (DoE).
I recommend resin candidates such as: Capto adhere and Capto MMC +/- ImpRess (GE), CHT™ ceramic hydroxyapatite and CFT™ ceramic fluoroapatite resin (BIO-RAD).

Lambda/Kappa Select beads

KappaSelect and LambdaFabSelect are affinity chromatography media for purifying kappa and lambda Fab fragments respectively. They enable efficient capture with high purity and yield. Both are used to capture Fab with binding affinities up to 5mg/mL medium for KappaSelect and up to 7 mg/mL medium for LambdaFabSelect. What you should be aware of here is that the elution buffers are not the same for the 2 resins (glycine vs acetate buffer) but both have low pH (2.5 and 3.5).

We have prepared a summary table to show the binding efficiency of all above strategies which cover 17 antibody species, 13 antibody classes, 6 type of fragments.

4. Conclusion

The antibody purification science may be challenging for you. However, guided by the information in this article, you can design a first approach to purify your antibody or fragments of interest. Don’t hesitate to go off the beaten track and explore innovative strategies to find new solutions!

Of course, we will be pleased to assist you in designing effective and innovative strategies and running experiments to get high purity antibodies.

Feel free to contact our lab experts for further information and welcome to leave your comments below.