Planung, Aufbau und Evaluation einer Membranadsorber-basierten Periodic-Counter-Current-Chromatographie (PCCC)-Anlage für die kontinuierliche Aufreinigung von Proteinen

Obwohl Batch-Chromatographie viele Nachteile wie die ineffiziente Nutzung der Kapazität und hohe Leerlaufzeiten aufweist, überwiegt bisher der Einsatz im Vergleich zur kontinuierlichen Chromatographie. Vor allem unter dem Gesichtspunkt der hohen Prozesskosten im Bereich des Downstream Processings, würde der Einsatz von kontinuierlichen Methoden zu Produktivitätssteigerungen und damit zur Senkung der Prozesskosten führen. In dieser Arbeit wurde ein kontinuierliches Chromatographie-System entwickelt und nach dem Periodic-Counter-Current-Chromatographie (PCCC)-Prinzip in Betrieb genommen. Das System wurde für (Einweg-)Membranadsorber als Alternative zu Säulen ausgelegt und soll damit aufgrund des erhöhten konvektiven Massentransports zu weiteren Produktivitätssteigerungen führen. Zunächst wurde das System mit drei Membranadsorbern und schließlich mit vier Membranadsorbern zur Optimierung des Systems und Steigerung der Flexibilität betrieben. Durch das Beladungsprinzip der PCCC können die Membranadsorber über die dynamische Bindungskapazität hinaus beladen werden. Das führt z. B. zur besseren Kapazitätsnutzung, verringertem Pufferverbrauch und zur Steigerung der Raum-Zeit-Ausbeute. Im Rahmen dieser Arbeit wurden drei Applikationsbeispiele zur kontinuierlichen Aufreinigung mit dem PCCC-System untersucht: die Aufreinigung einer Candida antarctica Lipase B, einer Patchoulolsynthase und eines monoklonalen Antikörpers. Das System eignet sich vor allem für die Aufreinigung mittels Affinitätschromatographie, da hier das Prinzip der PCCC bestmöglich ausgenutzt wird. Für die drei Applikationsbeispiele wurden Produktivitätssteigerungen zwischen 20 und 50 % sowie eine Steigerung der Kapazitätsnutzung von 20 % erreicht. Generell kann jedoch keine pauschale Aussage über die zu erzielenden Steigerungen getroffen werden, sodass jeder Prozess individuell bewertet werden sollte

Development and Testing of a 4-Columns Periodic Counter-Current Chromatography System Based on Membrane Adsorbers

Continuous chromatography can surmount the disadvantages of batch chromatography like low productivities and extensive usage of consumables. In this work, a 4-column continuous chromatographic system based on the principle of periodic counter-current chromatography (PCCC) was developed and tested with a model protein mixture of BSA and lysozyme. The PCCC system was specially designed for membrane adsorbers as an alternative to conventional columns to facilitate the use of disposable process units and to further increase the productivity due to higher convective mass transport in the membrane adsorber. Membrane adsorber Sartobind® Q was used to continuously purify BSA from the protein mixture. The usage of PCCC led to an increased capacity utilization (here 20%) and higher space–time-yields, and thus to a remarkable productivity increase and cost savings

Membrane Adsorber for the Fast Purification of a Monoclonal Antibody Using Protein A Chromatography

Monoclonal antibodies are conquering the biopharmaceutical market because they can be used to treat a variety of diseases. Therefore, it is very important to establish robust and optimized processes for their production. In this article, the first step of chromatography (Protein A chromatography) in monoclonal antibody purification was optimized with a focus on the critical elution step. Therefore, different buffers (citrate, glycine, acetate) were tested for chromatographic performance and product quality. Membrane chromatography was evaluated because it promises high throughputs and short cycle times. The membrane adsorber Sartobind® Protein A 2 mL was used to accelerate the purification procedure and was further used to perform a continuous chromatographic run with a four-membrane adsorber-periodic counter-current chromatography (4MA-PCCC) system. It was found that citrate buffer at pH 3.5 and 0.15 M NaCl enabled the highest recovery of >95% and lowest total aggregate content of 0.26%. In the continuous process, the capacity utilization of the membrane adsorber was increased by 20%

Medienoptimierung für die pDNA-Produktion im Mikrotiterplattenformat

Das Ziel der Arbeit ist die Steigerung der pDNA-Ausbeute von verschiedenen Stamm Plasmid-Kombinationen. Dabei wird die statistische Versuchsplanung eingesetzt, um Trends für diese Optimierung zu liefern

Membrane Adsorber for the Fast Purification of a Monoclonal Antibody Using Protein A Chromatography

Monoclonal antibodies are conquering the biopharmaceutical market because they can be used to treat a variety of diseases. Therefore, it is very important to establish robust and optimized processes for their production. In this article, the first step of chromatography (Protein A chromatography) in monoclonal antibody purification was optimized with a focus on the critical elution step. Therefore, different buffers (citrate, glycine, acetate) were tested for chromatographic performance and product quality. Membrane chromatography was evaluated because it promises high throughputs and short cycle times. The membrane adsorber Sartobind® Protein A 2 mL was used to accelerate the purification procedure and was further used to perform a continuous chromatographic run with a four-membrane adsorber-periodic counter-current chromatography (4MA-PCCC) system. It was found that citrate buffer at pH 3.5 and 0.15 M NaCl enabled the highest recovery of >95% and lowest total aggregate content of 0.26%. In the continuous process, the capacity utilization of the membrane adsorber was increased by 20%

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism