A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris

<p>Abstract</p> <p>Background</p> <p><it>Pichia pastoris </it>is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. To date, strain specific parameters, which are needed to set up feeding profiles for fed batch cultivations, are determined by time-consuming continuous cultures or consecutive fed batch cultivations, operated at different parameter sets.</p> <p>Results</p> <p>Here, we developed a novel approach based on fast and easy to do batch cultivations with methanol pulses enabling a more rapid determination of the strain specific parameters specific substrate uptake rate q_s, specific productivity q_pand the adaption time (Δtime_adapt) of the culture to methanol. Based on q_s, an innovative feeding strategy to increase the productivity of a recombinant <it>Pichia pastoris </it>strain was developed. Higher specific substrate uptake rates resulted in increased specific productivity, which also showed a time dependent trajectory. A dynamic feeding strategy, where the setpoints for q_swere increased stepwise until a q_s_maxof 2.0 mmol·g^-1·h^-1resulted in the highest specific productivity of 11 U·g^-1·h^-1.</p> <p>Conclusions</p> <p>Our strategy describes a novel and fast approach to determine strain specific parameters of a recombinant <it>Pichia pastoris </it>strain to set up feeding profiles solely based on the specific substrate uptake rate. This approach is generic and will allow application to other products and other hosts.</p

Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

10.1186/1472-6750-11-81BMC Biotechnology118

Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

<p>Abstract</p> <p>Background</p> <p>Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1) gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described.</p> <p>Results</p> <p>Five commercially available animal-component free, serum-free media (SFM) were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log₁₀TCID₅₀/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log₁₀TCID₅₀/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log₁₀TCID₅₀/ml were achieved.</p> <p>Conclusions</p> <p>We describe for the first time the production of Influenza viruses using Vero cells in commercially available animal-component free, serum-free medium. This work can be used as a basis for efficient production of attenuated as well as wild type Influenza virus for research and vaccine production.</p

Heavy Quarks and Heavy Quarkonia as Tests of Thermalization

We present here a brief summary of new results on heavy quarks and heavy quarkonia from the PHENIX experiment as presented at the "Quark Gluon Plasma Thermalization" Workshop in Vienna, Austria in August 2005, directly following the International Quark Matter Conference in Hungary.Comment: 8 pages, 5 figures, Quark Gluon Plasma Thermalization Workshop (Vienna August 2005) Proceeding

How to Realise Corporate Value from Enterprise Architecture

The design and ongoing management of an Enterprise Architecture is widely accepted as an established way of managing an organisation’s complexities in relating business and Information Technology capabilities. While the focus of related research is predominantly on frameworks, tools and methodologies, only limited attention has been given to understand and improve the value realisation process of Enterprise Architecture. We adopted the DeLone and McLean model of IS success for the design of a value realisation model specific to Enterprise Architectures. Thereby we identified service quality and actual use as two major catalysts fostering an Enterprise Architecture’s overall success. The amendments made were based on findings from two major case studies involving an Australian utility company and a Swiss insurance company

Protein expression in Pichia pastoris - a novel strategy for fast bioprocess development

Die metyhlotrophe Hefe P. pastoris ist ein weit verbreiteter Wirtsorganismus für die Herstellung von rekombinanten Proteinen.Derzeitige Strategien für die Prozessentwicklung hinsichtlich Erhöhung des Prozessverständnisses und Prozessoptimierung basieren meist auf empirischen Erfahrungen. Derartige Strategien sind oft stammspezifisch und können nicht generell auf andere Wirtssysteme transferiert und angewendet werden. Die hier vorgestellte Studie beschreibt eine neue Prozessentwicklungsmethode, die auf physiologischen und skalierbaren Prozessparametern basiert und zur Herleitung einer Fütterungsmethode für Fed-Batch Prozesse entwickelt wurde. Die Methode kann zur schnellen Quantifizierung möglicher Kandidaten für die Herstellung von rekombinanten Proteinen verwendet werden. Exemplarisch ist die Methode erfolgreich für unterschiedliche P. pastoris Systeme (Phänotyp, Produktion von verschiedenen Proteinen) verwendet worden.Stammspezifische Parameter konnten mit Hilfe von Batch-Verfahren zuverlässig bestimmt und in Fed-Batch-Verfahren erfolgreich transferiert werden. Die spezifische Substrataufnahmerate qs stellt die Basis für die beschrieben Methode dar und kam in Einzel- sowie Gemischtsubstratsystemen für die Prozesskontrolle und Optimierung zur Anwendung. Dadurch konnte das Prozessverständnis hinsichtlich der Korrelation von qs und der Produktbildung vertieft werden. Die beschriebene Quantifizierungsmethode diente erfolgreich zur Detektierung von produktspezifischen Interaktionen des Methanolstoffwechsels in P.pastoris. Zusätzlich wurde ein neues on-line Messsystem für die Bestimmung von Substraten und Stoffwechselprodukten etabliert, welches auch zur Quantifizierung und Qualifizierung des Produktes verwendet werden konnte und unterstützte somit die Prozessentwicklung hinsichtlich einer Echtzeitdatengenerierung.Die beschriebene Strategie ist durch die Verwendung von transferierbaren und skalierbaren Parametern leicht auf andere bioprozesstechnische Systeme im wissenschaftlichen sowie industriellen Umfeld anwendbar.The methylotrophic yeast has evolved to a widely used expression host for the production of recombinant proteins in the past few years.However, most strategies in process development for increased process understanding and optimization are based on empiricism. Such strategies are, caused by product or host specific interactions, generally not transferable to other production systems. In the present study, a novel physiologically based process development strategy for the fast and scalable determination of key parameter sets for fed batch production processes with P. pastoris strains was developed. The strategy was applied to various phenotypes expressing different recombinant products. Certain strain specific parameters were reliably quantified in batch pulse experiments and successfully transferred into fed batch feeding regimes. The method based on the specific substrate uptake rate qs as a key parameter for process control and optimization, was applied in single as well as in mixed feed substrate systems. Increased process understanding regarding the correlation of qs to product generation was achieved and physiological changes caused by genetic modifications of the methanol utilization pathway were quantified reliably. Additionally, a novel on-line device for fast process monitoring of physiological relevant components as well as product quantity and quality was introduced and supported the process development strategy to increase process understanding by real time data extraction. With the present strategy, enhanced bioprocess information for recombinant protein expression in P. pastoris was achieved within a short time. The strategy represents a valuable and transferable tool for fast early process development in academic as well as in industrial environments, where several strains have to be quantitatively screened for their potential use in later production processes.16

A fast approach to determine a fed batch feeding profile for recombinant Pichia pastoris strains

Abstract Background The microorganism Pichia pastoris is a commonly used microbial host for the expression of recombinant proteins in biotechnology and biopharmaceutical industry. To speed up process development, a fast methodology to determine strain characteristic parameters, which are needed to subsequently set up fed batch feeding profiles, is required. Results Here, we show the general applicability of a novel approach to quantify a certain minimal set of bioprocess-relevant parameters, i.e. the adaptation time of the culture to methanol, the specific substrate uptake rate during the adaptation phase and the maximum specific substrate uptake rate, based on fast and easy-to-do batch cultivations with repeated methanol pulses in a batch culture. A detailed analysis of the adaptation of different P. pastoris strains to methanol was conducted and revealed that each strain showed very different characteristics during adaptation, illustrating the need of individual screenings for an optimal parameter definition during this phase. Based on the results obtained in batch cultivations, dynamic feeding profiles based on the specific substrate uptake rate were employed for different P. pastoris strains. In these experiments the maximum specific substrate uptake rate, which had been defined in batch experiments, also represented the upper limit of methanol uptake, underlining the validity of the determined process-relevant parameters and the overall experimental strategy. Conclusion In this study, we show that a fast approach to determine a minimal set of strain characteristic parameters based on easy-to-do batch cultivations with methanol pulses is generally applicable for different P. pastoris strains and that dynamic fed batch strategies can be designed on the specific substrate uptake rate without running the risk of methanol accumulation.</p