Membrane bioreactor separator system for integrated IgG fragmentation and Fab purification

Abstract

This paper discusses a membrane based bioreactor system for producing pure Fab from human IgG. The bioreactor consisted of a stack of microporous anion-exchange membrane discs housed in a temperature controlled module. IgG was adsorbed on the membrane followed by its fragmentation with papain under optimized conditions. Fab was recovered in the reaction flow through while other fragments remained membrane bound and were subsequently eluted using high salt concentration buffer. By using the membrane bioreactor–separator system the overall process for producing Fab was simplified and high product purity and recovery were achieved. The pH of the feed solution had a significant effect on Fab recovery. The rate of IgG fragmentation by papain observed with the membrane bioreactor was about three times higher than that in an equivalent liquid phase reaction.

Introduction

Fab fragments obtained from immunoglobulin G (IgG) by papain digestion are widely used as immunochemical reagents and biopharmaceuticals (Yamaguchi et al., 1995, Cohen et al., 2000, Ljunglöf et al., 2007). The enzymatic fragmentation process is generally carried out as a liquid phase reaction in buffered medium at 30–40 °C, typical reaction time ranging from 2–24 h (Rousseaux et al., 1983). IgG fragmentation is followed by Fab purification, commonly using ultrafiltration and column chromatography (Roque et al., 2004, Ljunglöf et al., 2007). Such multi-step approach is cumbersome, increases manufacturing cost and reduces product recovery. One way to address these issues is by developing integrated reaction-separation techniques based on membrane bioreactors.

We recently developed a reactant adsorptive membrane bioreactor separator (or RAMBS) system based on microporous hydrophobic interaction membranes for integrated enzymatic fragmentation of IgG and purification of Fab (Yu and Ghosh, 2009). The main limitation of using hydrophobic interaction for IgG immobilization is the requirement of high salt (typically ammonium sulfate) concentration in the binding buffer which limits the concentration of IgG in the feed solution. In this paper we describe the use of a RAMBS system based on microporous anion-exchange membranes for integrated production of pure Fab from IgG. The membrane stack housed within a thermostatic module was loaded with IgG by anion exchange and then pulsed with papain solution. The differences in isoelectric points of the different IgG fragments were then utilized to sequentially release these into the effluent stream, Fab being obtained in the reaction flow-through. The reaction and separation conditions were optimized to achieve efficient IgG fragmentation, and high purity and recovery of Fab. The results obtained are discussed.