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p. 66-70 / PROCESS DEVELOPMENT/OPTIMIZATION
Implementation of aqueous two-phase extraction combined with precipitation in a monoclonal antibody manufacturing process
PETRA GRONEMEYER, REINHARD DITZ, JOCHEN STRUBE*
*Corresponding author
Institute for Separation and Process Technology, Clausthal University of Technology,
Leibnizstraße 15, D-38678 Clausthal-Zellerfeld, Germany

KEYWORDS: Process development, downstream processing, process integration, host cell proteins, ATPE, precipitation.

ABSTRACT: 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.

 

INTRODUCTION

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 chromatography steps) and processing costs (6). This problem is intensified by a change in type and concentration of impurities which emerge from process changes (7-10). They become more similar to the product regarding characteristic properties like isoelectric point, molecular weight or hydrophobicity (6).

New approaches of optimisation strategies, technologies and methods are required in order to solve this “downstream bottleneck”, like e.g. the integration of modeling and simulation and the use of mini-plant facilities (1, 11, 12). Innovative technologies for antibody purification include aqueous two-phase extraction (ATPE/ATPS) (13-19), precipitation (20-22) and membrane-based adsorption (23-24), among others.

ATPE has the potential to separate cells and undissolved components, impurities and monoclonal antibodies (15-25). This non-chromatographic unit operation is considered as simple and low-cost technology compared to Protein A chromatography (6, 25). It can integrate clarification, concentration and partial purification in one step. ATPE´s is considered as a technology of high selectivity, capacity and biocompatibility. It allows easy scale-up and continuous operation mode (5, 6, 14, 19) and presents an alternative towards a...In order to continue reading this article please register to our website – registration is for free and no fees will be applied afterwards to download contents.

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