Implementation of aqueous two-phase extraction combined with precipitation in a monoclonal antibody manufacturing process


*Corresponding author
Institute for Separation and Process Technology, Clausthal University of Technology,  Leibnizstraße 15, D-38678 Clausthal-Zellerfeld, Germany


Increasing antibody concentrations result in a manufacturing bottleneck and, therefore, in rising costs in the downstream processing. New technologies, like aqueous two-phase extraction and precipitation, present an option to solve this problem. In this work, a combination of both unit operations is investigated in order to present an alternative path to centrifugation and Protein A chromatography. At first, a screening was carried out to identify possible precipitation reagents which can selectively precipitate IgG or HCP. At the end of a DoE-based optimisation process, the purification process consists of an ATPE, followed by a precipitation at the isoelectric point and another precipitation using cold ethanol.


In upstream processing, challenges of manufacturing new and innovative economically priced monoclonal antibodies (mAb) have been addressed by increasing process efficiency and product concentrations (1-5). Antibody concentrations of 3-5 g/L can be achieved regularly (2-4) and newest cell retention devices result in even higher titers of up to 25 g/L (5). Traditionally, antibodies are purified by centrifugation and Protein A chromatography as capture step, followed by several membrane filtrations and at least two additional chromatography separations. A pH shift is carried out for virus inactivation and an additional filtration to remove viruses.

This benchmark process can be applied for almost all monoclonal antibodies produced by mammalian fermentation (6).

The resulting volumes of the fermentation contain 15-100 kg mAb/batch at titers of 5 g/L in 20–25 kL bioreactors (3, 4) leading to a physical and, therefore, a capacity limitation of the DSP equipment which was designed for much lower amounts of antibody. This results in an increase of processing time, material consumption (e.g. membranes or buffers and resins for different chro ...