Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells.
Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.
Zhao
Yonghong
Y
Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA 19087, USA. yzhao23@its.jnj.com
Gutshall
Lester
L
Jiang
Haiyan
H
Baker
Audrey
A
Beil
Eric
E
Obmolova
Galina
G
Carton
Jill
J
Taudte
Susann
S
Amegadzie
Bernard
B
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D006639:Histidine / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008027:Light; D009842:Oligopeptides / Q000235:genetics; D010206:Papain / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012542:Scattering, Radiation; D014961:X-Ray Diffraction
D000911:Antibodies, Monoclonal; C471213:His-His-His-His-His-His; D007140:Immunoglobulin Fab Fragments; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010206:Papain