Antibody fragments such as single domain antibodies (dAbs), antigen binding fragment (Fabs), and single chain variable fragments (scFv) have unique properties that can make them better suited to certain therapeutic conditions than full size antibodies. Whereas larger molecules can fail to penetrate or persist in tissue, smaller fragments can effectively reach their targets. They can also be produced in microbial, rather than mammalian cells, thanks to their non-glycosylated structure. Antibody fragment work is an area of interest and potential for the biopharmaceutical industry, but it presents some purification challenges.

Increased interest in antibody fragments

How are antibody fragments made

Various methods are used for this process, including genetic engineering and proteolytic cleavage. Fragments can be covalently or recombinantly enhanced to add extra functional properties. In previous work, they have been coupled with various elements known as antibody-drug conjugates (ADC), including enzymes, toxins, and radionuclides for cancer treatment.

What types of fragments are being used in bioprocessing?

There are three types of antibody fragments being used in bioprocessing:

Fabs are considered the first generation of antibody fragments. They were originally generated by cleavage of an intact antibody using an enzyme, such as papain, but are now produced using modern genetic engineering approaches. Papain cleavage yields two monovalent Fab fragments, each composed of one variable heavy chain (VH) and one variable light chain (VL), linked by disulfide bonds and displaying a single antigen-binding site.

scFvs are monovalent structures, with affinity for a single antigen. With an approximate size of Mr 25 000, an scFv contains the variable regions of an antibody’s heavy and light chains fused into a single polypeptide chain via a short flexible linker. An scFv comprises the complete antigen-binding site of its parental antibody molecule.

dAbs are some of the smallest functional antibody fragments that retain full antigen-binding specificity, as they consist of the VH or VL domains. The dAb is approximately one-tenth of the molecular weight of a normal antibody. Although dAbs contain only three of the six complementary determining regions from the parent antibody, they exhibit antigen binding specificity and affinity. A dAb can be remarkably stable under harsh conditions of temperature, pressure, and denaturing chemicals.

 

Fig1-antibody-and-antibody-fragments_v2 

Production and purification of antibody fragments

Antibody fragment expression and purification

Because antibody fragments are small and have a non-glycosylated nature, it’s possible to use simpler and less costly prokaryotic and yeast cell expression systems. But to deal with the demands on culture clarification, step cross-flow filtration is advised for viscous and high-solid feeds to allow for microbial fermentation. Hollow fiber filters are well suited to facilitate the capture.

Several antibody fragments have been purified using cation exchange chromatography (CIEX) as the capture step. For complex purification conditions, resins providing more than one type of interaction between ligand and sample components (multimodal resins) can be used.

For intermediate purification and final polishing, separation based on different selectivity than the primary technique (orthogonal) can involve anion exchange (AIEX) or hydrophobic interaction chromatography (HIC).

Due to the diversity of antibody fragments and the lack of an FC region, development of a specific purification protocol is required for each fragment. The platform approach that has worked so well in monoclonal antibody purification with Protein A has been more difficult to achieve with antibody fragments because of this diversity.

A purification platform approach to fragments

Typically, complete protein molecules can be purified using a platform approach, where standard unit operations and operating conditions are held constant. The Fc region common to mAbs means that a near-generic approach is possible. However, fragments are far more diverse molecules than whole proteins, which means a broader approach must be taken to purification.

Novel affinity chromatography resins offer new possibilities for a platform approach compatible with antibody fragments. Our BioProcess affinity chromatography resins have high selectivity and excellent pressure flow capabilities to capture the majority of protein fragments, including a range of Fab, scFv, and dAb lambda and kappa light chains.

Capturing antibody fragments containing kappa light chains

Because of its particular binding specificities, protein L offers interesting options for the purification of antibody fragments. Native protein L interacts with Ig kappa light chains and will bind to representatives of most antibody classes, including IgG, IgM, IgA, IgE, and IgD. It has no immunoglobulin class restrictions. Approximately 60% of mammalian IgG light chains are kappa chains, with the remaining 40% being lambda chains that lack binding sites for protein L. This means that protein L offers the potential for being a broadly useful affinity ligand–though perhaps not as generally useful as protein A.

New resins such as Capto L have made it possible to purify antibody fragments at industrial scale. With its rigid base matrix, allowing for high flow rates and high productivity as well as low ligand leakage, it is suitable for large-scale manufacturing. Its broad affinity for a range of antibody fragments of different sizes that contain the variable region of the kappa light chains is also potentially useful for a wide range of purification applications for conventional Fabs, scFv, and dAbs. KappaSelect is an affinity resin that binds to the constant region of the kappa light chain and can be used to capture Fabs containing kappa light chain in conditions where Capto L has been found to be less suitable.

Capturing antibody fragments containing lambda light chains

Alternatively, if you want to target the capture of Fabs containing the lambda light chain, choose LambdaFabSelect–an affinity resin that binds to the constant region of the lambda light chain.

Between them, Capto L and LambdaFabSelect cover nearly all Fabs, as well as a majority of the smaller antibody fragments.

Capturing antibody fragments containing heavy chains

If you want to capture heavy chain dAbs that contain the VH3 domain subtype, you might want to consider MabSelect. It is an affinity resin which binds to the VH3 domain subtype of human IgG Fabs in addition to binding in the Fc region.

Antibody fragment purification in action

See how you can make efficiency gains in purification by reading our white paper A platform approach to purification of antibody fragments.

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