Datenbestand vom 09. Dezember 2024
Verlag Dr. Hut GmbH Sternstr. 18 80538 München Tel: 0175 / 9263392 Mo - Fr, 9 - 12 Uhr
aktualisiert am 09. Dezember 2024
978-3-8439-3042-0, Reihe Ingenieurwissenschaften
Ulrike Weichsel Investigation of Seeded Protein Crystallization Using Foreign Particles
170 Seiten, Dissertation Universität Erlangen-Nürnberg (2016), Softcover, A5
The crystallization of proteins in industrial scale is mainly used as an effective separation technique which combines product recovery and purification in one single step. Here it is the aim to obtain large, compact and well-defined crystals to facilitate further downstream processing. Protein crystallization processes on a technical scale are often difficult to control as high supersaturations are needed which as well might lead to the formation of undefined, denatured and highly aggregated solid material. For this reason the controlled addition of seed material acting as hetergeneous nuclei is proposed.
In the present work the impact of different inorganic seed particles on the crystallization of lysozyme (LSZ) is investigated. In the first part the importance of electrostatic interactions between LSZ and seed particles and of the adsorption behavior of LSZ on these particles is discussed. Then seeded crystallization experiments both in μl- and in ml-scale and thus close to a technically relevant scale were performed. A clearly extended crystallization window upon the addition of seed particles was found which underlines the influence of the protein-particle interactions on protein crystal formation. Moreover, induction times of crystal formation and crystallization times were considerably reduced. In general, with the addition of seed particles a shift of the final crystal size distribution (CSD) to larger structures is observed which is beneficial in terms of subsequent down-stream processing steps.
The third part of the present work deals with oriented attachment as it presumably plays a major role in seeded growth of protein crystals. It was shown that the agglomeration of the seed particles/bioconjugates is advantageous for the product quality in terms of larger and more defined LSZ crystals and in terms of accelerated reaction kinetics. Population balance equations (PBE) were used to model the observed attachment of growth and agglomeration units. Finally, the applied model allows reproducing particle/crystal sizes distributions ranging from only a few nanometers up to the micrometer-scale. Thus, by the combination of PB modeling and experimentally determined crystallization parameters, insights into the crystal formation mechanism were obtained.