(9h) High-Throughput Bioprocess Development for Polymer-Polymer Aqueous Two-Phase Systems

The market for therapeutic proteins is currently increasing at a remarkable pace. In many cases, therapeutic proteins, such as monoclonal antibodies, must be administered in relatively large dosages. This large dosage requirement (> 100 mg dose^-1) (Shire et al., 2004), coupled with the high demand for these drugs, has resulted in an increased pressure on pharmaceutical companies to deliver high quality drugs at sufficient quantities. As a result, there has been an increasing amount of research & development (R&D) aimed at improving upstream efficiency, which has resulted in increased cell titres during the cell culture/fermentation stage of protein production. To date, improvements in upstream technology have, however, not been matched by improvements in downstream technology. Consequently expensive chromatography columns must be scaled accordingly to cope with the increasing amounts of material generated, which is proving to be a process bottleneck due to their capacity and cost, and has highlighted the need to re-evaluate current downstream separation methodologies.

Aqueous two-phase extraction (ATPE) has been shown to be a promising alternative downstream separation technology (Rito-Palomares, 2004). Systems can be formed from combining two incompatible polymers or a polymer and salt above a critical composition. Although having many advantages such as low surface-tension, easy scalability and a gentle environment for biopharmaceuticals, there is still a lack of widespread use of such systems. This can be attributed in part to the difficulty involved in characterising the large experimental space required for optimisation and effective process development. Factors such as charge, hydrophobicity, solute affinity, polymer molecular weight and concentration all play an important role on partitioning in ATPE (Asenjo and Andrews, 2011) and characterising all of the above factors manually is very resource intensive.

The high-throughput characterisation of polymer-salt aqueous two-phase systems has been explored (Oelmeier et al., 2011, Bensch et al., 2007) however, polymer-polymer aqueous two-phase systems behave somewhat differently and have so far not been considered although they have a clear potential for high separation efficiency. To help address this issue, we present an approach that utilises automated liquid handling systems for the high-throughput determination of polymer-polymer phase diagrams and protein partition behaviour. Such an approach allows for rapid exploration of the large experimental space provided by such systems, which in turn will allow for easier and more rapid development of optimised separation systems. Conventional techniques used to determine the binodial curve can be cumbersome and time consuming. An automated approach for polymer-polymer aqueous two-phase systems based on the determination of cloud point by light obscuration measurements is presented, and demonstrated for a system of PEG and Dextran. In addition, phase diagram tie-lines are generated by integration of automated phase volume determination with the lever arm rule. Measurement of protein partitioning is determined by measurements of UV absorbance at 280 nm with an appropriate control system as a blank.

The developed methodology will allow for screening of the wide variety of factors known to affect aqueous two-phase systems, such as polymer molecular weight, salt type, salt concentration, pH etc., thereby allowing the potential benefits of ATPE to be more readily explored. Future work would include the integration of more sophisticated assays into the high-throughput methodology to characterise components in complicated feed material such as impurities and products of interest. In addition, the integration of the experimental methodology with mechanistic models via parameter estimation is of particular interest as this would further speed up process development.

Acknowledgement:

We gratefully acknowledge the part-funding and support from BioMarin.

References:

Asenjo, J. A. & Andrews, B. A. 2011. Aqueous two-phase systems for protein separation: A perspective. Journal of Chromatography A, 1218, 8826-8835.

Bensch, M., Selbach, B. & Hubbuch, J. 2007. High throughput screening techniques in downstream processing: Preparation, characterization and optimization of aqueous two-phase systems. Chemical Engineering Science, 62, 2011-2021.

Oelmeier, S. A., Dismer, F. & Hubbuch, J. 2011. Application of an Aqueous Two-Phase Systems High-Throughput Screening Method to Evaluate mAb HCP Separation. Biotechnology and Bioengineering, 108, 69-81.

Rito-Palomares, M. 2004. Practical application of aqueous two-phase partition to process development for the recovery of biological products. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 807, 3-11.

Shire, S. J., Shahrokh, Z. & Liu, J. 2004. Challenges in the development of high protein concentration formulations. Journal of Pharmaceutical Sciences, 93, 1390-1402.