Cresswell Chrissie, Newcombe Anthony R, Davies Susannah, Macpherson Ian, Nelson Paul, O'Donovan Kieran, Francis Richard
Process Development Group, Protherics U.K. Ltd, Blaenwaun, Ffostrasol, Llandysul, Wales SA44 5JT, U.K.
Biotechnol Appl Biochem. 2005 Oct;42(Pt 2):163-8. doi: 10.1042/BA20050020.
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.
在本论文中，我们描述了一种快速方法，用于确定木瓜蛋白酶消化多克隆绵羊IgG（通过Na₂SO₄沉淀纯化）以生产生物治疗性Fab（抗原结合片段）的最佳条件。为了确定消化的最佳条件，采用了析因实验设计方法。所得实验数据用于使用Design Expert 6.06®（Stat-Ease，美国明尼阿波利斯，MN）构建数学模型，以预测稳健的IgG消化步骤的最佳条件。通过实验评估最佳条件，并评估这些条件在大规模生产生物治疗性Fab片段中的适用性。本论文中描述的结果和方法表明，只要时间和温度保持在所评估的高设置（24小时，40℃），对于所有使用的木瓜蛋白酶浓度，模型数据预测IgG消化接近100%。如果木瓜蛋白酶的使用量>2.5%（w/w），时间和/或温度可以降低。本论文中描述的结果和方法也可能适用于从其他免疫球蛋白生成治疗性Fab片段，包括从哺乳动物细胞培养物中纯化的单克隆抗体。
