Process, development for industrial scale bacterial ghost production

Diese Doktorarbeit ’Prozessentwicklung zur Herstellung von Bacterial Ghosts im Industriemaßstab’ beschreibt die Entwicklung eines Produktionsprozesses für BGs von einem Prozess mit niedriger Zellausbeute in komplexem Medium bis hin zu einem Fed-batch Prozess in einem Minimalmedium. Gezielte Veränderung in den Fermentations- und Ernte- Prozeduren ermöglichten es, höhere Ausbeuten zu erzielen und gleichzeitig die Produktqualität zu steigern. Zudem wurden Parameter zur Echtzeit-Bewertung der Prozessleistung eingeführt, die die Produktion effektiver machen. In einem ersten Schritt wurde ein Prozess mit niedriger Zellausbeute für Shigella flexneri 2a vorgestellt. Dieser Prozess kombiniert die E-Lyse mit der Verwendung von Staphylokokken Nuclease A (SNUC). Letztere ermöglichte eine erweiterte Herabsetzung der Viabilität und zudem den Abbau von DNA im Produkt. In einer Konsistenzstudie aus fünf unter identischen Bedingungen durchgeführten Fermentationen wurde eine mittlere E-Lyseeffizienz von 99.94% erreicht. Nach 90 min E-Lyse und weiteren 180 min SNUC-Aktivität war die Viabilität um beinahe sechs Zehnerpotenzen abgesunken. Darüber hinaus wurde gezeigt, dass der DNA Gehalt des Produkt bis zum Detektionslimit der real-time PCR reduziert werden konnte. Das bedeutet, dass das Produkt im wesentlichen als DNA-frei einzuordnen ist. Chemische Inaktivierung mit 0.75‰ (v/vf) beta-Propiolacton vor der Produkternte durch einen Separator lieferte ein keimfreies Produkt, was durch Sterilitäts-Tests vor und nach der Gefriertrocknung belegt wurde. In der Ernteprozedur für BG-Produkte wurde in der Folge die mechanischen Separation durch Querstromfiltration (TFF) ersetzt. Die Kulturbrühe von Sf2a BG Fermentationen mit niedriger Zellausbeute (≤ 1.0E+09 Zellen pro ml) wurden vor dem Inaktivierungsschritt zehnfach aufkonzentriert, dabei wurde die absolute Menge an freier DNA in der Suspension um 90% reduziert. Rasterexperimente mit Sf2a zeigten, dass die Menge an benötigtem BPL durch diese Maßnahme ebenfalls um den Faktor zehn auf nurmehr 0.075‰ (v/vf) reduziert werden konnte. Diese Ergebnisse wurden für BGs auf Fermentationen mit Escherichia coli bestätigt. Die Anwendungstemperatur entsprach jeweils der Temeratur zur Induktion der E-Lyse: 44°C für Sf2a und 42°C für E. coli. Die Einbindung eines Diafiltrationsschrittes in die E-Lyse Phase bei Fermentationen von E. coli Nissle (EcN) BGs mit mittleren Zelldichten (≤ 1.0E+10 Zellen pro ml) führte zu einer DNA-Abreicherung von mindestens 30% noch vor der Aufkonzentrierung der Kulturbrühe. Dies spiegelte sich direkt im BPL-Bedarf wider, der nur 0.50‰ (v/vf) betrug. So konnte gezeigt werden, dass die benötigte Menge an BPL aus der Zelldichte berechnet werden kann, solange die verbleibende Menge an freier DNA zuverlässig abgeschätz werden kann. Eine Methode zur Beurteilung von E-Lyse Prozessen mittels Durchflusszytometrie (FCM) wurde vorgestellt. Ausgehend von einer existierenden Methode wurde ein verbessertes Assay etabliert. Die größere Durchsichtigkeit der Zellhüllen nach der E-Lyse verändert die Lichtstreuung im vergleich zu vollen Zellen, kombiniert man diese Eigenschaft mit einem geeigneten lebend/tot-Fluoreszenzfarbstoff wie DiBAC4(3) zur Erfassung der Zellviabilität, so lassen sich lebende Zellen, volle aber tote Zellen und leere BGs gut unterscheiden. Die zusätzliche Verwendung des Fluoreszenzfarbstoffs RH414 zur Markierung von Zellmembranen verbesserte die Qualität der FCM Daten wesentlich, da nicht-zelluläres Rauschen effektiv unterdrückt werden konnte. Die Methode liefert quantitativ hochwertige Daten und eignet sie sich daher als Parameter zur Beurteilung von BG Produktionsprozessen in Echtzeit. Durch die Verwendung eines definierte Mediums zur Herstellung von E. coli Nissle 1917 BGs konnte die Produktivität des Batch-Prozesses im Vergleich zu den Fermentationen mit niedrigen Zellausbeuten um den Faktor 10 gesteigert werden (mittlere Zelldichten), ohne dass die E-Lyseeffizienz abnahm. Eine gute Übereinstimmung zwischen Zellzahlbestimmungen durch FCM und herkömmlich ermittelten Zellzahlen mit der Methode der Kolonie-bildenden Einheiten (KBE) wurde gezeigt. Bei der Fermentation mit mittleren Zelldichten war die Anwendung von Querstromfiltration von großem Wert. Da durch das Ausstoßen des Zytoplasma bei höheren Zelldichten die Viskosität des Medium ansteigt, kam es zu verstärkter Schaumbildung. Das Einbinden eines ersten Diaflitrationsschrittes in die E-Lyse Phase half, diesem Effekt entgegen zu wirken und bewirkte als Nebeneffekt auch die bereits erwähnte Abreicherung freier DNA aus der Kulturbrühe. Die gute Konsistenz des Batch-Prozesses wurde in insgesamt neun Läufen gezeigt. Die durchschnittliche Zelldichte vor der E-Lyse war 7.35E+09 Zellen pro ml, die mittlere E-Lyseeffizienz lag bei 99.78%. Die Produktinaktivierung mit 0.50‰ (v/vf) BPL war in allen Fällen ausreichend und lieferte ein keimfreies Produkt nach der Gefriertrocknung. Zusätzlich wurde ein Hochdichte Fed-batch Prozess in einem Minimalmedium vorgestellt. Erste Versuche zeigten, dass es möglich ist, die Zelldichten im Vergleich zum beschriebenen Batch-Prozess mindestens nochmals um den Faktor 10 zu erhöhen (durchschnittliche Zellausbeute vor E-Lyse: 7.06E+10 Zellen pro ml). Es wurde nachgewiesen, dass nach der E-Lyse Induktion eine weiterlaufende, wenn auch exponentiell abnehmende Zufütterungs- Strategie notwendig ist. Bei geeigneter Zufütterung lassen sich E-Lyseeffizienzen > 99.8% erreichen.This thesis ’Process Development for Industrial Scale Bacterial Ghost Production’ describes the development of a Bacterial Ghost (BG) production process from a low density batch process in complex medium towards a high density fed-batch approach in minimal medium. Specific changes in the fermentation and harvesting procedures allowed to reach higher yields while at the same time product quality was improved. Real-time parameters for the evaluation of process performance were introduced to make the production more efficient. In a first step a stable low density production process for Shigella flexneri 2a (Sf2a) BGs was presented. This process combined the features of E-lysis and staphylococcal nuclease A (SNUC). The latter effected enhanced reduction of viability and moreover significant degradation of DNA in the product. In a consistency study of five production runs performed under identical conditions a mean E-lysis efficiency of 99.94% was obtained. After 90 min of E-lysis and another 180 min of SNUC activity the viability of the cultures was overall reduced by an average of almost six decimal powers. Furthermore, it could be shown that the DNA contents in the product was reduced to the detection limit of real-time PCR by the nuclease indicating the product was essentially DNA-free. Chemical inactivation with 0.75‰ (v/vf) beta-propiolactone (BPL) before harvesting by mechanical separation yielded a bioburden-free product which was verified by sterility tests before and after lyophilization. The harvesting procedure for BG production was changed from mechanical separation to tangential flow filtration (TFF). The culture broth of low density Sf2a BG fermentations (cell densities ≤ 1.0E+09 cells per ml) was concentrated by a factor 10 before inactivation thereby reducing the total amount of free DNA in the suspension by 90%. Screening experiments with Sf2a indicated that by this measure the required amount of BPL could also be reduced by 90% to now 0.075‰ (v/vf). Results were confirmed for low density fermentations of E. coli BGs. The application temperature is determined by the respective temperature of E-lysis induction: 44°C for Sf2a and 42°C for E. coli. For medium density fermentations of E. coli Nissle (EcN) BGs with ten-fold higher cell densities (≤ 1.0E+10 cells per ml) where dia-filtration was incorporated into the E-lysis phase the concentration of free DNA could be reduced by at least 30% even before concentration of the broth. This reflected in the amount of required BPL which was only 0.50‰ (v/vf). This showed that in case the amount of residual free DNA in the concentrate can be reliably estimated the required amount of BPL for total inactivation of BG products can be calculated as a function of cell density. A flow cytometry-based (FCM) method for the evaluation of E-lysis in E. coli was presented. Based on a previously suggested assay an improved method was established. Utilizing the change in translucency caused by E-lysis which impacts the cells forward scatter signal and combining this with viability information obtained from the fluorescence signal of membrane potential-sensitive dye DiBac4(3) gave good differentiation between live, whole dead and E-lysed bacteria. Using red-fluorescent dye RH414 as a cell membrane marker enhanced the quality of FCM data as non-celular noise could be effectively excluded. The described method allowed to use FCM as an eligible quality criterion for BG production in quasi realtime. Using a minimal medium for batch production of Escherichia coli Nissle 1917 BGs the productivity could be increased by the factor 10 (medium cell density) as compared to the standard low density fermentation without losing E-lysis efficiency. Good conformance between FCM and conventional cfu data was shown. For this medium density approach using tangential flow filtration was of great value. Due to expulsion of the cytoplasm at elevated cell densities the culture broth viscosity changed significantly and foaming issues occurred. Incorporating a first dia-filtration step into E-lysis phase resolved those problems and also had the beneficial side-effect of removing free DNA from the broth mentioned above. Over a total of nine production cycles a high degree of reproducibility was shown for medium density production of EcN BGs. Average cell densities were 7.35E+09 live EcN per ml while the average E-lysis efficiency was 99.78%. BPL treatment with 0.50‰ (v/vf) was sufficient in all cases yielding a bioburden-free product after lyophilization. In addition, a high-density fed-batch process in minimal medium was presented. Initial data generated showed that it is possible to further increase cell densities by at least a factor 10 (average cell density: 7.06E+10) with respect to the medium density process. It was evident that feeding has to be maintained into E-lysis phase, the feed profile should be decelerated exponentially. With an adequate feeding strategy after E-lysis induction lysis efficiencies > 99.8% were reached

An Integrated Downstream Process Development Strategy along QbD Principles

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

Fed-Batch Production of Bacterial Ghosts Using Dielectric Spectroscopy for Dynamic Process Control

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

A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E

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.Morphoplant GmbH, Bochum (RCPE)560356141

MOESM2 of A novel one-step expression and immobilization method for the production of biocatalytic preparations

Additional file 1: Figure S2. Comparison of β-galactosidase activities without detergent treatment. The activity of equal amounts of β-galactosidase before and after detergent treatment are compared