ISBN 978-3-8439-3298-1

978-3-8439-3298-1, Reihe Technische Chemie

Julia Sieberz
A Contribution to Protein Purification with Polyelectrolytes

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

Zusammenfassung / Abstract

Precipitation with polyelectrolytes is a promising alternative to conventional methods for the purification of monoclonal antibodies due to its advantages like low additive (polyelectrolyte) consumption and the simple technical implementation. The technique is based on the complex formation between oppositely charged polyelectrolytes and proteins. Under specific conditions these complexes form insoluble aggregates. The purification of mAbs with polyelectrolytes can be performed with two possible modes, capture and clarification. Capture is the direct precipitation of mAb with anionic polyelectrolytes. Clarification is the precipitation of impurity proteins with cationic polyelectrolytes, leaving the antibody in supernatant. Numerous parameters like the pH value, the polyelectrolyte concentration and molecular weight, the impurity and salt concentration affect the complex formation. Aim of this thesis is to provide a contribution to the understanding of the influence of these parameters on the complex formation. For this purpose, different anionic polyelectrolytes for capture and cationic polyelectrolytes for clarification were screened with a model system, consisting of a monoclonal antibody, the model impurity protein BSA and the model salt NaCl, to identify suitable candidates for both methods. The systematic investigation of influencing parameters was performed on basis of Design of Experiments (DoE) which provided a fast identification of influencing parameters and parameter interactions with a minimal number of experiments. Additionally, the influence of different model impurity proteins (BSA, Myoglobin, Ovalbumin, α-Lactalbumin und β-Lactoglobulin) with different properties (molecular weight, isoelectric point and surface charge distribution) on the precipitation of the mAb precipitation was investigated. To demonstrate that precipitation with anionic polyelectrolytes can be integrated as first purification step after fermentation in a typical mAb production process, precipitation of a monoclonal antibody out of a complex protein mixture and a first approach for the further chromatographic purification after re-dissolving the precipitate was investigated.