Reiner Luttmann

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.

A multi‐bioreactor system for optimal production of malaria vaccines with Pichia pastoris

The successful development of optimal multistage production processes for recombinant products with Pichia pastoris needs to meet three pre‐conditions. These pre‐conditions are (i) strategies for performing fully automated and observable processes, (ii) characterization of the host cell‐specific reaction parameters in order to make an adapted process layout for feeding and aeration strategies, and (iii) knowledge of optimal operation parameter conditions for maximizing the expression productivity of target protein amount and/or quality. In this report, an approach of a fully automated multi‐bioreactor plant is described that meets all these requirements. The expression and secretion of a potential malaria vaccine with Pichia pastoris was chosen as an example to demonstrate the quality of the bioreactor system. Methods for the simultaneous identification of reaction kinetics were developed for strain characterization. Process optimization was carried out by applying a sequential/parallel Design of Experiments. In the view of Process Analytical Technology (PAT)‐applications and in order to develop fully automated and globally observable production processes, methods for quasi on‐line monitoring of recombinant protein secretion titers and the immunological quality of the products are also discussed in detail.

High-level production of bacillus subtilis glycine oxidase by fed-batch cultivation of recombinant Escherichia coli Rosetta (DE3).

A fed‐batch process for the high cell density cultivation of Escherichia coli Rosetta (DE3) and the production of the recombinant protein glycine oxidase (GOX) from Bacillus subtilis was developed. GOX is a deaminating enzyme that shares substrate specificity with d‐amino acid oxidase and sarcosine oxidase and has great biotechnological potential. The B. subtilis gene coding for GOX was expressed in E. coli Rosetta under the strong inducible T7 promotor of the pET28a vector. Exponential feeding based on the specific growth rate and a starvation period for acetate utilization was used to control cell growth, acetate production, and reconsumption and glucose consumption during fed‐batch cultivation. Expression of GOX was induced at three different cell densities (20, 40, and 60 g·L−1). When cells were induced at intermediate cell density, the amount of GOX produced was 20 U·g−1 cell dry weight and 1154 U·L−1 with a final intracellular protein concentration corresponding to approximately 37% of the total cell protein concentration. These values were higher than those previously published for GOX expression and also represent a drastic decrease of 26‐fold in the cost of the culture medium.

Designing a fully automated multi-bioreactor plant for fast DoE optimization of pharmaceutical protein production.

The identification of optimal expression conditions for state‐of‐the‐art production of pharmaceutical proteins is a very time‐consuming and expensive process. In this report a method for rapid and reproducible optimization of protein expression in an in‐house designed small‐scale BIOSTAT® multi‐bioreactor plant is described. A newly developed BioPAT® MFCS/win Design of Experiments (DoE) module (Sartorius Stedim Systems, Germany) connects the process control system MFCS/win and the DoE software MODDE® (Umetrics AB, Sweden) and enables therefore the implementation of fully automated optimization procedures. As a proof of concept, a commercial Pichia pastoris strain KM71H has been transformed for the expression of potential malaria vaccines. This approach has allowed a doubling of intact protein secretion productivity due to the DoE optimization procedure compared to initial cultivation results. In a next step, robustness regarding the sensitivity to process parameter variability has been proven around the determined optimum. Thereby, a pharmaceutical production process that is significantly improved within seven 24‐hour cultivation cycles was established. Specifically, regarding the regulatory demands pointed out in the process analytical technology (PAT) initiative of the United States Food and Drug Administration (FDA), the combination of a highly instrumented, fully automated multi‐bioreactor platform with proper cultivation strategies and extended DoE software solutions opens up promising benefits and opportunities for pharmaceutical protein production.

An integrated scale-down plant for optimal recombinant enzyme production by Pichia pastoris

An integrated bioprocess was created in a scale‐down production plant by developing a two‐stage enzyme production process with Pichia pastoris, containing a cell‐breeding reactor and a production reactor in combination with a three‐stage downstream process. To harvest the secreted enzymes, a disc separator and a cross‐flow microfiltration clear the broth from the cells. Purification with hydrophobic interaction chromatography removes other proteins, concentrates the product, and prepares the enzyme solution for lyophilization. Fully automated and broad observable multi‐stage parallel process courses have been developed using industrial process control systems and at‐line measurements for enzyme concentration and enzyme activity. Optimal process conditions were found by application of Design of Experiments (DoE) for the production process.

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.

On-Line Simulation Techniques for Bioreactor Control Development

During the last decade, bioprocess development automation tasks have become more significant for overall process performance. Quality requirements on the one hand and growing process knowledge on the other have resulted in applied control strategies of increasing complexity. At the same time modern information technology facilitates higher flexibility of the technical systems used for practical realization of control tasks [1, 2]. Nevertheless, one of the most important goals in bioprocess automation is the manipulation of the process to meet desired performance criteria. Examples are the control of p02 to a prescribed limit or the realization of an exponential substrate feeding strategy. In practice, the process and the controller form a closed loop, often realized in a feedback fashion (Fig.6.1a). For design and stability investigations of these control-loops both process and controller require treatment using a theoretical mathematical model (Fig.6.1b). Obviously the time-dependent changes of the relevant process variables are of major interest for these applications. Therefore the underlying model is highly dynamic in nature and typically a set of differential equations based on mass and energy balances is used for simulation of the process behavior.

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.