A new enzymatic method to obtain high-yield F(ab)2 suitable for clinical use from mouse IgGl.
Four different proteases were screened for their capability of selectively digesting murine monoclonal IgGl to obtain active F(ab)2. For the screening, a series of five different mouse monoclonal antibodies (IgGl, k) was used, recognizing different tumor-associated antigens and currently used for radioimmunoimaging studies. The enzymes (pepsin, bromelain, ficin and elastase) showed different fragmentation capability and the fragments obtained showed different stability and immunoreactivity. No digestion was noticed using elastase. Pepsin gave discontinuous results, in that its activity ranged from reduction of IgG to small inactive fragments to an inability to digest the immunoglobulin. Pepsin activity was strongly pH-dependent and immunoreactivity of the obtained fragments was not always conserved. Bromelain and, in particular, ficin gave excellent results. Digestion was always rapid and stable, all five MAbs were reduced to F(ab)2 in a comparable time range and with high yields. Moreover, ficin-obtained F(ab)2 showed a highly conserved immunoreactivity. Therefore, ficin was selected as the murine monoclonal IgGl digestion enzyme to obtain active bivalent antibody fragments. The digestion procedure gave a uniform result for all five different MAbs and was easily scaled up to produce hundreds of milligrams of F(ab)2.
Mariani
M
M
Biochemical Oncology Laboratory, SORIN Biomedica, Saluggia (VC), Italy.
Camagna
M
M
Tarditi
L
L
Seccamani
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001963:Bromelains / Q000378:metabolism; D005361:Ficain / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry*; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D010196:Pancreatic Elastase / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D001963:Bromelains; D010196:Pancreatic Elastase; D005361:Ficain; D010434:Pepsin A