Scale down model in industrial cell culture processes – A powerful tool to ensure reliable production

Reliability and reproducibility of therapeutic protein production is one of the key objectives of GMP manufacturing with respect to product quality and supply to patients. But even in the age of chemical defined media, QbD and well characterized cell culture processes during manufacturing deviations can occur. While those deviations caused by human errors, equipment failures or process control (e.g. pH) can be assessed in a straightforward manner coming to satisfying results in an appropriate period of time, deviation caused by raw material lot variations are usually hard to detect. Development of risk mitigation strategies is complex since sophisticated analytical methods need to be applied and most often, additionally, vendors have to be involved in the root cause analysis. For risk mitigation change control assessments and QC testing covering a broad spectrum of parameters and quality attributes are applied before those materials are released for use in manufacturing. Even with this tight mesh of control quite a number of incidences can be reported, demonstrating that deviations based on raw material issues have a huge impact on our business. Here we present several case studies, where neither QC testing nor vendor and customer change control could predict the deviating process behavior observed when these materials had been used in the cell culture process at production scale. As a consequence cell culture use tests by validated scale down models have been introduced for several raw materials in the meantime. These test procedures will be presented

Elimination of Fetal Calf Serum (FCS) in industrial cell culture media for manufacturing of diagnostic Anti-bodies

Here we demonstrate the suitability of different substitutes for the use of fetal calf serum (FCS) in cell culture media. Due to decreasing availability of FCS on the global market whilst growing demand of cell culture media to support Anti-body production for more than 200 different invitro-diagnostic kits media development efforts had to be taken. A benchmark of different substitutes, such as NBS (new born serum), human thrombocytes lysate, and chemically defined media (CDM) will be presented. Case studies from lab scale (10Ltr) up to full production scale (2,000Ltr) will be shown

Improving the batch-to-batch reproducibility in microbial cultures during recombinant protein production by guiding the process along a predefined total biomass profile

In industry Escherichia coli is the preferred host system for the heterologous biosynthesis of therapeutic proteins that do not need posttranslational modifications. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of a therapeutic hormone is described. The strategy is to guide the process along a predefined profile of the total biomass that was derived from a given specific growth rate profile. This profile might have been built upon experience or derived from numerical process optimization. A surprisingly simple adaptive procedure correcting for deviations from the desired path was developed. In this way the batch-to-batch reproducibility can be drastically improved as compared to the process control strategies typically applied in industry. This applies not only to the biomass but, as the results clearly show, to the product titer also

Advanced monitoring & control in microbial cultivation processes for recombinant protein production

Verglichen mit dem rasanten Fortschreiten in den molekularbiologischen Wissenschaften während der letzten Jahrzehnte, konnten die Entwicklungen, der in der industriellen Praxis verwendeten Fermentations- und Downstream-Techniken, bei weitem nicht mithalten. Daraus ergibt sich ein Ungleichgewicht zwischen neuen biologischen Systemen und den industriellen Produktionstechnologien, was zu einer fortwährenden Abnahme der jährlichen Neuanmeldungen von Produktionsprozessen für Biologics führt. In ihrer PAT-Initiative (PAT = Process Analytical Technology) benennt die für die Zulassung von Pharmaprodukten zuständige US-amerikanische Aufsichtsbehörde FDA die Problempunkte, welche verbessert werden müssen, und fordert die Hersteller gleichzeitig auf in wissenschaftlich basierte Ansätze zur Problemlösung zu investieren. Diese Doktorarbeit zeigt am Beispiel der rekombinanten Proteinproduktion mit Escherichia coli - Bakterien verschiedene bioverfahrenstechnische Ansätze, wie diesen Forderungen nachgekommen werden kann. Konkret werden robuste Prozessführungsstrategien vorgestellt, die, mit entsprechenden Überwachungs- und Regelungsalgorithmen ausgestattet, reproduzierbare Prozessverläufe garantieren und damit die Gesamtqualität des Produktionsverfahrens signifikant steigern.Compared to the immense achievements in fundamental molecular biological sciences, the improvements in the fermentation and downstream processing technologies used in industry have been less spectacular over the last decade. Hence, there is a misbalance between new cellular systems and production technologies, resulting in a decreasing annual rate of approvals for protein manufacturing processes. In its process analytical technology (PAT) initiative, the U.S. Food and Drug Administration identified the issues that must be improved to compensate for this development and forces manufactures towards a more scientific approach of solving the problems. In this doctoral dissertation, methods of bioprocess engineering science have been used to meet the demands. Recombinant protein production processes, where Escherichia coli were used as host cells, are taken as a concrete example. Concretely, a design strategy for improved robust process operational procedures was developed that can be tightly supervised and automatically controlled.von Marco Jenzsc