Tobias Käppler

Characterization of magnetic ion-exchange composites for protein separation from biosuspensions

Downstream processing is a major issue in biotechnological production. A multitude of unit operations with nonsatisfying yield are often used to reach the desired product purity. Direct recovery technologies such as high-gradient magnetic fishing (HGMF) are advantageous because of their ability to separate the desired product in early stages from crude cultivation broths. However, the use of magnetic particles to capture valuable biotechnological products is often linked to the drawback that support particles are expensive and not available in greater quantities. This current work presents new composite magnetic particles that can be used in biotechnology. They are manufactured by a spray drying process. During this process, the nanosized magnetite particles as well as functional ion-exchange nanoparticles are integrated into one particle in which they are linked by a matrix polymer. The production procedure is flexible, scalable, and therefore economical. These particles have good adsorption capacities of up to 85 mg/g adsorbed protein and good binding kinetics. They are resistant to harsh conditions such as short ultrasonic treatment or extreme pHs. In order to test their usefulness in biosuspensions, model proteins were separated using these particles. The anion and cation exchanger particles separated lysozyme (LZ) or BSA from cultivation suspensions. The selectivity of recovery was dependent on other proteins present as is usual for ion-exchange binding mechanisms.

In situ magnetic separation for extracellular protein production

A new approach for in situ product removal from bioreactors is presented in which high‐gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non‐porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production. Biotechnol. Bioeng. 2009;102: 535–545.

Semi-continuous in situ magnetic separation for enhanced extracellular protease production—modeling and experimental validation†

In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations. Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98% because proteolytic degradation and potential inhibition of the protease in the medium could be minimized. Furthermore, integration of semi‐continuous ISMS increased the overall process efficiency due to reduction of the medium consumption. Process simulation revealed a deeper insight into protease production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172.

Elektrofiltration - Einsatzgebiet Enzymproduktion

Untersucht wird der Einsatz der Elektrofiltration im Dead-End-Verfahren fur Anwendungen in der Enzymproduktion, vor allem die Applikationen Biomasseabtrennung und Enzymkonzentrierung. Fur den Prozess ist die Filtrationsgeschwindigkeit von entscheidender Bedeutung. Auch die Ausbeuten an aktivem Wertprodukt werden ermittelt, wobei insbesondere die Einwirkung des elektrischen Feldes auf die Aktivitat der Biopolymere uberpruft wird. In einem Ausblick wird die Weiterentwicklung der Elektrofiltration durch ein Kerzenfiltersystem aufgezeigt, das die Effizienz hinsichtlich des Energieverbrauchs steigern soll.

Electrofiltration: Mature enzyme production [Elektrofiltration - Einsatzgebiet enzymproduktion]

Untersucht wird der Einsatz der Elektrofiltration im Dead-End-Verfahren fur Anwendungen in der Enzymproduktion, vor allem die Applikationen Biomasseabtrennung und Enzymkonzentrierung. Fur den Prozess ist die Filtrationsgeschwindigkeit von entscheidender Bedeutung. Auch die Ausbeuten an aktivem Wertprodukt werden ermittelt, wobei insbesondere die Einwirkung des elektrischen Feldes auf die Aktivitat der Biopolymere uberpruft wird. In einem Ausblick wird die Weiterentwicklung der Elektrofiltration durch ein Kerzenfiltersystem aufgezeigt, das die Effizienz hinsichtlich des Energieverbrauchs steigern soll.