Premium
Optimal conditions for the papain digestion of polyclonal ovine IgG for the production of biotherapeutic Fab fragments
Author(s) -
Cresswell Chrissie,
Newcombe Anthony R.,
Davies Susannah,
Macpherson Ian,
Nelson Paul,
O'Donovan Kieran,
Francis Richard
Publication year - 2005
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20050020
Subject(s) - papain , polyclonal antibodies , digestion (alchemy) , chemistry , immunoglobulin fab fragments , antibody , chromatography , monoclonal antibody , biochemistry , enzyme , biology , immunology , complementarity determining region
In the present paper, we describe a rapid method for the determination of optimum conditions for papain digestion of polyclonal ovine IgG (purified by Na 2 SO 4 precipitation) for the production of biotherapeutic Fabs (antigen‐binding fragments). To determine the optimum conditions for digestion, a factorial approach to the design of experiments was undertaken. The resulting experimental data were used to construct the mathematical models using Design Expert 6.06® (Stat‐Ease, Minneapolis, MN, U.S.A.) to predict the optimum conditions for a robust IgG digestion step. Optimum conditions were evaluated experimentally, and the applicability of the conditions for large‐scale manufacture of biotherapeutic Fab fragments was assessed. The results and methods described in the present paper suggest that, provided the time and temperature are maintained at the high settings evaluated (24 h, 40 °C), the modelled data predict IgG digestion close to 100% for all the papain concentrations used. Provided papain is used at >2.5% (w/w), either time and/or temperature may be reduced. The results and methods described in the present paper may also be applicable to the generation of therapeutic Fab fragments from other immunoglobulins, including monoclonal antibodies purified from mammalian cell culture.