Affinity Separation and Crystallization of F_c Fragments

Immunoglobulins are used by the immune system present in serum and tissue fluids to identify and neutralize foreign targets. Immunoglobulins are “Y”-shaped molecules comprised of two identical light chains (L) and two identical heavy chains (H) linked via disulfide covalent forces and via non-covalent interactions. Each chain has constant (C_L and C_H) and variable (V_L and V_H) domains (Holt et al. 2003). These form two antigen-binding fragments (F_ab) that specifically recognize and bind to antigen molecule and a constant fragment (F_c) which is responsible for the effector function and biodistribution of the antibody (Beale 1987). Proteolytic digestion provides important information to determine an antibody molecule’s structure and function. Antibody molecules can be cleaved into fragments and each of these fragments has a distinct activity. A number of enzymes like papain, pepsin, ficin, bromelain, and elastase can be used for this purpose, papain being the most frequently used (Luo et al. 2002). Because of their smaller size as functional components of the whole molecule, antibody fragments offer several advantages over intact antibodies for use in certain immunochemical techniques and experimental applications. For this purpose, researchers have attempted to use smaller, more stable counterparts of antibodies and have also been searching for alternative ways to obtain antibody-like receptors (Haupt et al. 1998). Thus, bioengineered antibody fragments that are smaller than antibodies and have the ability to bind antigen bivalently have been promoted by the researchers (Cortese 1995). IgG molecule can be digested with papain for gaining F_ab and F_c fragments.

For the separation of F_ab and F_c fragments, HiTrap_r Protein A column can be used and collected the fragments by the fraction unit of the fast protein liquid chromatography (FPLC) system (Ertürk et al. 2011; Bereli et al. 2013; Aslıyüce et al. 2013). As shown in Fig. 1, a successful separation was observed and F_ab and F_c fragments were separately collected. The retention times of the fragments were achieved at 3.06 and 11.64 min, respectively. The calculated chromatographic parameters such as t_R, N, k′, α, and Rs values are summarized and given in Table 1. R_s value was calculated as 4.70 for F_c fragment. Because the R_s value should be higher than 1.0 for a good resolution of two peaks in such a chromatography system, the results for the resolution of F_ab fragment/F_c fragment can be accepted as good resolution values.

Affinity Separation and Crystallization of Fc Fragments, Fig. 1

FPLC chromatogram of IgG after digestion with papain (Ertürk et al. 2011)

Affinity Separation and Crystallization of Fc Fragments, Table 1 Chromatographic separation data