ISBN 9783843931717

978-3-8439-3171-7, Reihe Thermodynamik

Christian Kress
Hybrid approach to estimate protein phase behavior in aqueous two-phase extraction (Band 25)

144 Seiten, Dissertation Technische Universität Dortmund (2017), Softcover, A5

Zusammenfassung / Abstract

The market of therapeutic proteins like monoclonal antibodies (mAbs) has experienced enormous growth within the last decades. This is especially driven by the increased demand for medical treatment of cancer, immunological disorders, and other fatal diseases. The production of mAbs, and in particular their purification is very expensive and needs optimization mainly due to the use of cost-intensive chromatography. In this context the extraction of mAbs using aqueous two-phase systems (ATPS) under mild and biocompatible conditions is a promising alternative to state of the art downstream processing. Within aqueous two-phase extraction (ATPE) the protein partitioning can be influenced by a displacement agent (DA) leading to a selective purification of the target protein from impurities.

In this work a new estimation approach was developed in order to access the protein phase behavior for an optimized extraction of pharmaceutical proteins by ATPE. For this purpose the second osmotic virial coefficient B22 and cross virial coefficient B23 determined by composition gradient multi-angle light scattering were used to account for the protein-protein and protein-solute (phase-former, DA) interactions. B22 was used as predictive key parameter on protein precipitation affinity induced by solutes of the ATPS. B23 could be successfully applied in order to identify applicable DAs. The approach of this work provided suitable salt (phosphate, citrate)-PEG ATPS and DAs allowing for an efficient purification of the target protein immunoglobulin G (IgG) from the impurity protein human serum albumin (HSA).

Additionally a hybrid shortcut model based on B23 (protein-solute interaction) and the concentration difference between the low molecular-weight components of the ATPS was developed. Based on this shortcut model the partition coefficients of IgG and HSA within different salt (phosphate, citrate)-PEG ATPS including DAs (NaCl, LiBr) could be successfully determined independently on the protein concentration within the ATPS.

Finally the influence of NaCl on the IgG solubility within the ATPS was investigated. A combination of ATPS phase composition data with the IgG ATPS solubility allowed for the selection of an optimal IgG feed concentration.

The results of this thesis contribute to an improved implementation of aqueous two-phase extraction within the downstream processing of pharmaceutical proteins.