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The development, optimization, and analysis of downstream processes are challenged by a high number of potentially critical process parameters that need to be investigated using lab-scale experiments. These process parameters are spread across multiple unit operations and potentially show interactions across unit operations. In this contribution, we present a novel strategy for bioprocess development that considers the risk of parameter interactions across unit operations for efficient experimental design. A novel risk assessment tool (interaction matrix) is introduced to the Quality by Design (QbD) workflow. Using this tool, the risk of interaction across unit operations is rated. Subsequently, a design of experiments (DoE) across unit operations is conducted that has the power to reveal multivariate interdependencies. The power of the presented strategy is demonstrated for protein isolation steps of an inclusion body process, focusing on the quality attribute inclusion body purity. The concentration of Triton X-100 in the course of inclusion body (IB) purification was shown to interact with the g-number of the subsequent centrifugation step. The presented strategy targets a holistic view on the process and allows handling of a high number of experimental parameters across unit operations using minimal experimental effort. It is generically applicable for process development along QbD principles.

Recombinant mixed feed bioprocesses are characterized by the controlled feeding of multiple defined carbon sources aiming at increased productivities. However, mixed feed process design is challenging due to physiological constraints such as adaptation times and catabolite repression. A novel soft-sensor assisted dynamic method that allows the science-based process design with respect to co-utilization of primary and secondary substrate was developed. The method is based on the control of the specific uptake rates of primary and secondary substrate via a combination of a rate-based soft sensor and in-line infrared spectroscopy. Maximum secondary substrate specific uptake rates and adaptation times are determined by a combination of dynamic pulse and ramp experimentation. The power of the presented method was demonstrated on a recombinant Escherichia coli pBAD mixed feed process with d -glucose as primary and l -arabinose as secondary carbon source. Onset of catabolite repression was observed once a total specific substrate uptake rate of 1.0 g/gh was exceeded. Adaptation times to l -arabinose were determined as ∼10 min. The presented method can be considered generically applicable for the physiological investigation of mixed feed systems. Furthermore, metabolic capabilities of the promising but yet unexplored E. coli pBAD mixed feed system were explored for the first time.

The exploitation of dynamic process conditions increasingly finds attention for strain screening and bioprocess development of biotechnological products. Prerequisite for successful dynamic experimentation is the controlled deflection of process parameters, e.g. temperature, pH or specific substrate uptake rates, while the latter can be considered the most challenging. Here, a soft-sensor based control strategy capable of dynamic control of specific uptake rates is presented, opening new perspectives for efficient process development. A soft-sensor on the basis of a redundant equation system involving the Degree of Reduction (DoR) and Carbon (C) balance was used for the estimation of the real-time biomass concentration and used for closed-loop control of specific uptake rates using a mass balance based control approach. The power of the presented soft sensing approach for bioprocess development was demonstrated on microbial bioprocess development tasks: (a) the control of the specific substrate uptake rates in a non-induced E. coli decellerostat culture aiming at the determination of the specific acetate production as a function of the specific substrate uptake, (b) the static control of the specific substrate uptake rate in an induced P. Pastoris AOX expression system, (c) the dynamic control of specific substrate uptake rates under induced conditions in a E. coli pET expression system and (d) the simultaneous dynamic control of the specific glucose and arabinose uptake rates in an E.coli pBAD expression system. In this contribution the dynamic controllability of multiple specific substrate uptake rates using a first-principle soft sensor independent of known or dynamically changing yield coefficients in recombinant bioprocesses is demonstrated for the first time. The presented control strategy holds potential to become a key process analytical technology (PAT) tool for bioprocess development.

Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h−1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of −0.8 ± 0.3 h−1. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products. Electronic supplementary material The online version of this article (doi:10.1007/s00253-017-8281-x) contains supplementary material, which is available to authorized users.

The Bacterial Ghost (BG) platform technology is an innovative system for vaccine, drug or active substance delivery and for technical applications in white biotechnology. BGs are cell envelopes derived from Gram-negative bacteria. BGs are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structures. Using BGs as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of BGs are targeting the carrier itself to primary antigen-presenting cells. Furthermore, BGs exhibit intrinsic adjuvant properties and trigger an enhanced humoral and cellular immune response to the target antigen. Multiple antigens of the native BG envelope and recombinant protein or DNA antigens can be combined in a single type of BG. Antigens can be presented on the inner or outer membrane of the BG as well as in the periplasm that is sealed during BG formation. Drugs or supplements can also be loaded to the internal lumen or periplasmic space of the carrier. BGs are produced by batch fermentation with subsequent product recovery and purification via tangential flow filtration. For safety reasons all residual bacterial DNA is inactivated during the BG production process by the use of staphylococcal nuclease A and/or the treatment with β-propiolactone. After purification BGs can be stored long-term at ambient room temperature as lyophilized product. The production cycle from the inoculation of the pre-culture to the purified BG concentrate ready for lyophilization does not take longer than a day and thus meets modern criteria of rapid vaccine production rather than keeping large stocks of vaccines. The broad spectrum of possible applications in combination with the comparably low production costs make the BG platform technology a safe and sophisticated product for the targeted delivery of vaccines and active agents as well as carrier of immobilized enzymes for applications in white biotechnology.

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.

Flow cytometry (FCM) is a tool for the analysis of single-cell properties in a cell suspension. In this contribution, we present an improved FCM method for the assessment of E-lysis in Enterobacteriaceae. The result of the E-lysis process is empty bacterial envelopes-called bacterial ghosts (BGs)-that constitute potential products in the pharmaceutical field. BGs have reduced light scattering properties when compared with intact cells. In combination with viability information obtained from staining samples with the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylarbituric acid) trimethine oxonol (DiBAC4(3)), the presented method allows to differentiate between populations of viable cells, dead cells, and BGs. Using a second fluorescent dye RH414 as a membrane marker, non-cellular background was excluded from the data which greatly improved the quality of the results. Using true volumetric absolute counting, the FCM data correlated well with cell count data obtained from colony-forming units (CFU) for viable populations. Applicability of the method to several Enterobacteriaceae (different Escherichia coli strains, Salmonella typhimurium, Shigella flexneri 2a) could be shown. The method was validated as a resilient process analytical technology (PAT) tool for the assessment of E-lysis and for particle counting during 20-l batch processes for the production of Escherichia coli Nissle 1917 BGs.

In diesem Beitrag wird das Modell der Fertigung (MFERT) als konzeptionelle Basis für markt- und prozeßorientierte Controlling-Informationssysteme in Logistik und Produktion vorgestellt. Anhand eines Beispiels werden der Modellierungsansatz und ein Vorgehensmodell für seine Anwendung verdeutlicht. Das Beispiel ist fiktiv, beruht aber auf Erfahrungen aus mehreren Industrieprojekten.

Background Science-based recombinant bioprocess designs as well as the design of statistical experimental plans for process optimization (Design of Experiments, DoE) demand information on physiological bioprocess boundaries, such as the onset of acetate production, adaptation times, mixed feed metabolic capabilities or induced state maximum metabolic rates as at the desired cultivation temperature. Dynamic methods provide experimental alternatives to determine this information in a fast and efficient way. Information on maximum metabolic capabilities as a function of temperature is needed in case a reduced cultivation temperature is desirable (e.g. to avoid inclusion body formation) and an appropriate feeding profile is to be designed. Results Here, we present a novel dynamic method for the determination of the specific growth rate as a function of temperature for induced recombinant bacterial bioprocesses. The method is based on the control of the residual substrate concentration at non-limiting conditions with dynamic changes in cultivation temperature. The presented method was automated in respect to information extraction and closed loop control by means of in-line Fourier Transformation Infrared Spectroscopy (FTIR) residual substrate measurements and on-line first principle rate-based soft-sensors. Maximum induced state metabolic capabilities as a function of temperature were successfully extracted for a recombinant E. coli C41 fed-batch bioprocess without the need for sampling in a time frame of 20 hours. Conclusions The presented method was concluded to allow the fast and automated extraction of maximum metabolic capabilities (specific growth rate) as a function of temperature. This complements the dynamic toolset necessary for science-based recombinant bacterial bioprocess design and DoE design.

The Bacterial Ghost (BG) platform technology evolved from a microbiological expression system incorporating the ϕX174 lysis gene E. E-lysis generates empty but structurally intact cell envelopes (BGs) from Gram-negative bacteria which have been suggested as candidate vaccines, immunotherapeutic agents or drug delivery vehicles. E-lysis is a highly dynamic and complex biological process that puts exceptional demands towards process understanding and control. The development of a both economic and robust fed-batch production process for BGs required a toolset capable of dealing with rapidly changing concentrations of viable biomass during the E-lysis phase. This challenge was addressed using a transfer function combining dielectric spectroscopy and soft-sensor based biomass estimation for monitoring the rapid decline of viable biomass during the E-lysis phase. The transfer function was implemented to a feed-controller, which followed the permittivity signal closely and was capable of maintaining a constant specific substrate uptake rate during lysis phase. With the described toolset, we were able to increase the yield of BG production processes by a factor of 8–10 when compared to currently used batch procedures reaching lysis efficiencies >98%. This provides elevated potentials for commercial application of the Bacterial Ghost platform technology.

Die rasante Entwicklung der Informationstechnologie ermoglicht es Unternehmen ihre Kunden- und Lieferantenbeziehungen heute deutlich besser zu managen. Im Bereich des Kundenmanagements sind seit wenigen Jahren unter dem Begriff des Customer Relationship Managements (CRM) eine Vielzahl an unterschiedlichen DV-Losungen am Markt erhaltlich (Schwetz 2000). Innerhalb der Wertschopfungskette ringen traditionelle Anbieter von ERP-Systemen und Spezialanbieter von Supply Chain Management Systemen (SCM-Systemen) um die Vormachtstellung im SCM-Markt.

Stetig abnehmende Produktlebenszyklen, zunehmende Anforderungen an die Lieferflexibilitat und gleichzeitig ein diagnostizierbarer Trend hin zu „virtuellen“ Unternehmensnetzwerken stellen erhohte Anforderungen an die Planungs- und Koordinationsfahigkeiten von Unternehmen. Unternehmen, die nachhaltig erfolgreich in derartigen Netzwerken agieren wollen, mussen hinsichtlich der Supply Chain Planungsfahigkeiten einerseits ihre eigene, zunehmend im Werksverbund stattfindende interne Planung beherrschen, gleichzeitig aber auch sicherstellen, dass eigene Planungsprozesse mit denen von Kunden und Zulieferern „synchronisiert“ sind (Hillek 2001).

Dieser Beitrag beschreibt Zielsetzung, Vorgehen und Ergebnisse eines Kooperationsprojektes zwischen dem HEINZ NIXDORF INSTITUT und der ITT AUTO-MOTIVE Europe GmbH1. Im Rahmen dieses Projektes ist ein Ansatz und ein Werkzeug im Bereich des Produktions- und Supply-Chain-Management (PSCM) konzipiert und realisiert worden. Ein wichtiger Fortschritt ist hierbei vor allem die Vermeidung fixer Ubergangszeiten und eine dynamische Planung, die die Ent-wicklung eines Logistik- und Produktionsnetzwerkes abhangig von der Belastung unternehmensubergreifend uber mehrere Wertschopfungsstufen berechnet.