title: Optimal conditions for the papain digestion of polyclonal ovine IgG for the production of bio-therapeutic Fab fragments.
abstract: 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 bio-therapeutic 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(R) (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 bio-therapeutic 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 degrees 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.
authors.affiliation: Process Development Group, Protherics U.K. Ltd, Blaenwaun, Ffostrasol, Llandysul, Wales SA44 5JT, U.K.
authors.forename: Chrissie
authors.initials: C
authors.lastname: Cresswell
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Newcombe
authors.forename: Susannah
authors.initials: S
authors.lastname: Davies
authors.forename: Ian
authors.initials: I
authors.lastname: Macpherson
authors.forename: Paul
authors.initials: P
authors.lastname: Nelson
authors.forename: Kieran
authors.initials: K
authors.lastname: O'Donovan
authors.forename: Richard
authors.initials: R
authors.lastname: Francis
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000378:metabolism; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D010206:Papain / Q000737:chemistry* / Q000494:pharmacology; D012756:Sheep; D013696:Temperature; D013997:Time Factors
chemical_list: D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010206:Papain