High efficiency reduction capability for the formation of Fab׳ antibody fragments from F(ab)2 units (1)
Immunoglobulins, or antibodies, are large proteins produced by the immune system that have found numerous applications in many different fields. Some examples include the use of these proteins in therapeutics, immunoaffinity capillary electrophoresis, immunoaffinity chromatography, protein microarray technology, and immunosensors. Certain applications require the use of only specific parts of the antibody, such as the F(ab)2, Fab, or Fc fragments. One of the most commonly encountered classes of antibodies is the immunoglobulin G (IgG). The structure of a general IgG antibody contains four protein chains – two pairs of two chains, with each pair consisting of a heavy and light chain. The portion of the antibody that binds to specific antigens is called the fragment–antigen binding (Fab) unit. Each antibody contains two Fab fragments and one tail Fc fragment. A common approach for the fragmentation of antibodies is through the use of proteolytic enzymes. Papain, pepsin, bromelain, ficin, lysyl endopeptidase are frequently used enzymes. Papain cleaves the antibody above the two key hinge disulfide bridges. Alternatively, pepsin cleaves the antibody below the two disulfide bridges, resulting in an F(ab)2 fragment and an Fc fragment. The F(ab)2 fragment may then be reduced to yield two Fab׳ fragments with C-terminal nucleophilic sulfides. mouse IgG1 cannot be cleaved using pepsin whereas mouse IgG2a and IgG2b are very reactive towards pepsin. Although pepsin has an optimum cleavage efficiency in very acidic solutions (pH=2.0), antibody denaturation and diminished antigen-binding ability are also associated with such harsh conditions.
Commonly used reducing agents are β-mercaptoethanol (β-ME), tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and mercaptoethylamine (MEA). DTT offers the benefit of performing an intramolecular attack of the disulfide once the first thiol has attached to the molecule being reduced. The resulting oxidized DTT product is a cyclic disulfide that is thermodynamically favorable due to its steric specifications. Dithiobutylamine (DTBA) has desirable characteristics since its hydrochloride salt is nearly odorless and is highly soluble in water. Having lower pKa values allows for DTBA to have more nucleophilic deprotonated sulfides present at any given pH compared to DTT and MEA.
The reduction process was nearly complete in 3 h time using DTBA, whereas both DTT and MEA were far from completion. The SDS-PAGE results showed a decrease in the intensity of the F(ab)2 band around 110 kDa and an increase in the intensity of the Fab׳ fragment band around 55 kDa. The changes in the two bands are most noticeable in the 90, 180, and 300 min time points. There is an increase in the band intensity of the band located near the 26 kDa marker on the SDS-PAGE for the DTT reduction. This increase in intensity can be attributed to the cleavage of the interchain disulfides between the light and heavy chains of the antibody. This undesirable cleavage was not observed with the DTBA reduction of the polyclonal antibodies. DTBA is still the more efficient reducing agent at physiological temperature, however, not by as large of a difference as is observed at room temperature.
All of the tested concentrations of DTBA proved to cleave F(ab)2 fragments faster than the 2.0 mM DTT or 2.0 mM MEA concentrations indicating the kinetic superiority of DTBA with polyclonal anti-goat IgG antibodies.
The reduction kinetics for the monoclonal anti-human IgG1 antibodies showed that Cleavage of the F(ab)2 with MEA was significantly slower than with the other reducing agents and appeared to have slowed down drastically after the first five minutes. These results show that DTBA and DTT are able to cleave through the tougher hinge region of the mouse antibodies, whereas MEA has a much more difficult time. The concentration of the monoclonal Fab׳ fragments increased significantly during the DTT and DTBA reductions. Most likely, DTT and DTBA were participating in interchain disulfide cleavages, resulting in an increase in the concentration of the light chains in solution.
With the cleavage of polyclonal rabbit anti-goat IgG F(ab)2, DTBA was ~213 times faster than DTT and ~71 times faster than MEA. This trend was observed with the cleavage of the monoclonal mouse anti-human IgG1 F(ab)2 as DTBA was ~2 times faster than DTT and ~10 times faster than MEA. However, DTT and DTBA were both observed to cleave the interchain disulfide bonds of the antibody fragments in the monoclonal antibody reductions. It is very important that antibody cleavage is optimized in order to maximize the yield of the desirable Fab׳ fragments.