Gesine Cornelissen

Soft sensors in bioprocessing: A status report and recommendations

The following report with recommendations is the result of an expert panel meeting on soft sensor applications in bioprocess engineering that was organized by the Measurement, Monitoring, Modelling and Control (M3C) Working Group of the European Federation of Biotechnology - Section of Biochemical Engineering Science (ESBES). The aim of the panel was to provide an update on the present status of the subject and to identify critical needs and issues for the furthering of the successful development of soft sensor methods in bioprocess engineering research and for industrial applications, in particular with focus on biopharmaceutical applications. It concludes with a set of recommendations, which highlight current prospects for the extended use of soft sensors and those areas requiring development.

Fully automated production of potential Malaria vaccines with Pichia pastoris in integrated processing

Here, we have studied the setup of an integrated bioprocess for the production of artificial Malaria vaccine candidates with Pichia pastoris. Production of pharmaceutically relevant proteins such as vaccines has high demands regarding protein processing in the bioreactor and for subsequent purification. To implement this challenging protein expression process, a highly instrumented bioreactor was configured for repeated fed batch cultivations and supplemented with an at‐line monitoring of the target protein production via HPLC. The integration of a fast in situ purification of the sensitive products using an expanded bed adsorption for a sequential integrated bioprocess allows cyclic product separation. Thus, a fully automated production of artificial malaria vaccines was achieved.

Sequential/parallel production of potential Malaria vaccines – A direct way from single batch to quasi-continuous integrated production

An intensification of pharmaceutical protein production processes can be achieved by the integration of unit operations and application of recurring sequences of all biochemical process steps. Within optimization procedures each individual step as well as the overall process has to be in the focus of scientific interest. This paper includes a description of the development of a fully automated production plant, starting with a two step upstream followed by a four step downstream line, including cell clarification, broth cleaning with microfiltration, product concentration with ultrafiltration and purification with column chromatography. Recursive production strategies are developed where a cell breeding, the protein production and the whole downstream is operated in series but also in parallel, each main operation shifted by one day. The quality and reproducibility of the recursive protein expression is monitored on-line by Golden Batch and this is controlled by Model Predictive Multivariate Control (MPMC). As a demonstration process the production of potential Malaria vaccines with Pichia pastoris is under investigation.

Sequential injection analyzer for glycerol monitoring in yeast cultivation medium

A smart and versatile flow system for the at-line monitoring of glycerol based on sequential injection analysis is proposed. Formaldehyde, generated by oxidation of glycerol with sodium periodate, is transformed into 2,4-diacetyl-1,4-dihydrolutidine applying the Hantzsch condensation reaction with acetylacetone and ammonium. Dual-wavelength detection was carried out to minimize the contribution of the schlieren effect using a single blue LED. In-line sample dilution is accomplished applying the concept of zone-penetration and a new concept of sample splitting. Under optimized physical and chemical variables, regression curves over two dynamic working ranges of 0.1-4 and 1-40g l(-1) were attained. The injection throughputs were 14 and 12h(-1), respectively. Applying on-line data evaluation and conditional inquiries, the smart and independent selection of the adequate analytical procedure for the required working range was accomplished. The system was successfully applied to the at-line monitoring of glycerol in a continuous, cell-free medium flow from a yeast cultivation process during batch and fed-batch phase with glycerol as the only carbon source.

Development of an Integrated Bioprocess for production of potential Malaria vaccines with Pichia pastoris

Abstract Pichia pastoris was used for the expression of an artificial fusion protein D1M1 – a potential Malaria vaccine. A fully automated integrated bioprocess was set up which combined upstream and downstream operations and has been performed in alternating fed batch cycles with parallel protein capture by Expanded Bed Adsorption Chromatography. Design of Experiments was used to study the influence of procedural factors on protein recovery and purity. As a result it was achieved to purify the protein to a purity of up to 87 % and a recovery of 51 % was reached in a single downstream operation.

A Cybernetic Approach for Modelling of Complex Dynamic Expression in Recombinant Protein Production with Pichia pastoris

Abstract This paper demonstrates the dynamic behaviour of recombinant pharmaceutical protein production with Pichia pastoris. Fully automated experiments with PAT applications deliver reproducible and reliable data. Based on this, complex expression and secretion courses are modelled with simple kinetic and cybernetic elements, including a small number of parameters. The model is useful for process analysis and optimization as well as for development of integrated production processes.

Monitoring and Control of Pharmaceutical Protein Production with Sequential Integrated Down Stream Processes

Abstract The methylotrophic yeast Pichia pastoris is used for expression of pharmaceutical proteins like 1-8del MCP-1 and vMIP-II. Here the proteins are secreted and these extra cellular products open new strategies in production processes. The combining of complex cultivations and primary down stream steps with cross flow filtration units leads to integrated bioprocessing. The development of optimised sequential production strategies in research and industrial scale needs on-line measurement of substrates and products up to the recombinant protein itself. Full automated processes are designed to analyse integrated filtration steps for modeling of cross flow units and combined bioreaction. The transfer of research work into industrial pilot plant scale is also demonstrated.

Integrated Bioprocess Development for Production of Recombinant Proteins in High Cell Density Cultivation with Pichia Pastoris

Abstract An integrated bioprocess is built up for production of an antagonist of recombinant monocyte chemoattractant protein-1 with Pichia pastoris. The process line based on full automated cultivation procedures includes micro-and ultra-filtration membrane modules for in-situ interactions as well as liquid chromatography methods for post cultivation opcrations.