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  • 1
    Language: English
    In: Journal of Biotechnology, 20 February 2013, Vol.163(4), pp.362-370
    Description: ► A novel on-line monitoring device is described. ► We show the high frequent precise determination of multiple components in cultivations. ► The data quality was demonstrated by extraction of information. ► Physiological responses of microbial cultures upon media limitations were discussed efficiently. On-line monitoring devices for the precise determination of a multitude of components are a prerequisite for fast bioprocess quantification. On-line measured values have to be checked for quality and consistency, in order to extract quantitative information from these data. In the present study we characterized a novel on-line sampling and analysis device comprising an automatic photometric robot. We connected this on-line device to a bioreactor and concomitantly measured six components ( glucose, glycerol, ethanol, acetate, phosphate and ammonium) during different batch cultivations of . The on-line measured data did not show significant deviations from off-line taken samples and were consequently used for incremental rate and yield calculations. In this respect we highlighted the importance of data quality and discussed the phenomenon of error propagation. On-line calculated rates and yields depicted the physiological responses of the cells in unlimited and limited cultures. A more detailed analysis of the physiological state was possible by considering the off-line determined biomass dry weight and the calculation of specific rates. Here we present a novel device for on-line monitoring of bioprocesses, which ensures high data quality in real-time and therefore refers to a valuable tool for Process Analytical Technology (PAT).
    Keywords: On-Line Monitoring ; Process Analytical Technology ; Bioprocess Quantification ; Bioprocess Development ; Media Limitations ; Engineering
    ISSN: 0168-1656
    E-ISSN: 1873-4863
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  • 2
    Language: English
    In: Microbial Cell Factories, Oct 27, 2011, Vol.10, p.85
    Description: 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.
    Keywords: Molecular Biology -- Research ; Methanol -- Physiological Aspects ; Methanol -- Research ; Recombinant Proteins -- Physiological Aspects ; Recombinant Proteins -- Research
    ISSN: 1475-2859
    Source: Cengage Learning, Inc.
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  • 3
    Language: English
    In: Microbial Cell Factories, Oct 27, 2011, Vol.10, p.85
    Description: 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.
    Keywords: Molecular Biology -- Research ; Methanol -- Physiological Aspects ; Methanol -- Research ; Recombinant Proteins -- Physiological Aspects ; Recombinant Proteins -- Research
    ISSN: 1475-2859
    Source: Cengage Learning, Inc.
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  • 4
    Language: English
    In: Microbial Cell Factories, March 3, 2011, Vol.10, p.14
    Description: Background Pichia pastoris 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. Results 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.sub.s , specific productivity q.sub.p and the adaption time ([DELTA]time.sub.adapt ) of the culture to methanol. Based on q.sub.s , an innovative feeding strategy to increase the productivity of a recombinant Pichia pastoris 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.sub.s were increased stepwise until a q.sub.s .sub.max of 2.0 mmol*g.sup.-1.sup.*h.sup.-1 .sup.resulted in the highest specific productivity of 11 U*g.sup.-1.sup.*h.sup.-1.sup.. Conclusions Our strategy describes a novel and fast approach to determine strain specific parameters of a recombinant Pichia pastoris 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.
    Keywords: Methanol -- Analysis ; Methanol -- Methods ; Proteins -- Analysis ; Proteins -- Methods
    ISSN: 1475-2859
    Source: Cengage Learning, Inc.
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  • 5
    Language: English
    In: Applied Microbiology and Biotechnology, 2010, Vol.88(2), pp.461-475
    Description: Over the last decade, adherent MDCK (Madin Darby canine kidney) and Vero cells have attracted considerable attention for production of cell culture-derived influenza vaccines. While numerous publications deal with the design and the optimization of corresponding upstream processes, one-to-one comparisons of these cell lines under comparable cultivation conditions have largely been neglected. Therefore, a direct comparison of influenza virus production with adherent MDCK and Vero cells in T-flasks, roller bottles, and lab-scale bioreactors was performed in this study. First, virus seeds had to be adapted to Vero cells by multiple passages. Glycan analysis of the hemagglutinin (HA) protein showed that for influenza A/PR/8/34 H1N1, three passages were sufficient to achieve a stable new N- glycan fingerprint, higher yields, and a faster increase to maximum HA titers. Compared to MDCK cells, virus production in serum-free medium with Vero cells was highly sensitive to trypsin concentration. Virus stability at 37 °C for different virus strains showed differences depending on medium, virus strain, and cell line. After careful adjustment of corresponding parameters, comparable productivity was obtained with both host cell lines in small-scale cultivation systems. However, using these cultivation conditions in lab-scale bioreactors (stirred tank, wave bioreactor) resulted in lower productivities for Vero cells.
    Keywords: Influenza virus ; Vaccine production ; TCID stability ; Trypsin ; Glycosylation ; Bioreactor
    ISSN: 0175-7598
    E-ISSN: 1432-0614
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  • 6
    Language: English
    In: Microbial Cell Factories, 01 March 2011, Vol.10(1), p.14
    Description: Abstract Background Pichia pastoris 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. Results 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 qs, specific productivity qp and the adaption time (Δtimeadapt) of the culture to methanol. Based on qs, an innovative feeding strategy to increase the productivity of a recombinant Pichia pastoris 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 qs were increased stepwise until a qs max of 2.0 mmol·g-1·h-1 resulted in the highest specific productivity of 11 U·g-1·h-1. Conclusions Our strategy describes a novel and fast approach to determine strain specific parameters of a recombinant Pichia pastoris 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.
    Keywords: Engineering
    ISSN: 1475-2859
    E-ISSN: 1475-2859
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  • 7
    Language: English
    In: Microbial Cell Factories, 01 October 2011, Vol.10(1), p.85
    Description: 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.
    Keywords: Pichia Pastoris ; Strain Characterization ; Specific Substrate Uptake Rate ; Batch Cultivation ; Methanol Pulse ; Dynamic Feeding Profile ; Engineering
    ISSN: 1475-2859
    E-ISSN: 1475-2859
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  • 8
    Language: English
    In: Protein Expression and Purification, December 2012, Vol.86(2), pp.89-97
    Description: ► Glycosylated HRP was efficiently purified from a fermentation broth. ► The successful purification strategy comprised only two steps. ► Strategies were switched from standard to flowthrough mode. The enzyme horseradish peroxidase (HRP), which is frequently applied in industry and medicine, is primarily isolated from plant. This purification procedure is costly and the obtainable amount of HRP from the horseradish root is low. However, recombinant HRP (rHRP) produced in yeast is hyperglycosylated rendering the subsequent purification cumbersome and the recombinant production of HRP in yeast not competitive. In this study, we screened different common techniques to develop a fast and efficient purification strategy for hyperglycosylated rHRP expressed in . We demonstrated that the extensive glycosylation pattern on the surface of rHRP masked its physico-chemical properties, which is why standard purification strategies were rather unsuccessful. Only switching the strategies to a flowthrough mode gave satisfactory results. After determining the optimal operation conditions in a multivariate Design of Experiments approach, we present a simple 2-step strategy for the purification of hyperglycosylated rHRP. Combining a hydrophobic charge induction chromatography operated in flowthrough mode and a size-exclusion chromatography, we were able to purify rHRP more than 12-fold from a specific activity of 80 U/mg to more than 1000 U/mg.
    Keywords: Horseradish Peroxidase ; Pichia Pastoris ; Glycosylation Pattern ; Protein Purification ; Flowthrough Mode ; Specific Activity ; Chemistry ; Anatomy & Physiology
    ISSN: 1046-5928
    E-ISSN: 1096-0279
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  • 9
    Language: English
    In: BMC Biotechnology, August 11, 2011, Vol.11, p.81
    Description: Background 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. Results 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 x 10^6 cells/ml, whereas other SFM achieved about 1.2 x 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 Log.sub.10 TCID.sub.50 /ml was achieved using trypsin concentration of 10 [mu]g/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 x 10^6 cells/ml and virus titre of 8.3 Log.sub.10 TCID.sub.50 /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 Log.sub.10 TCID.sub.50 /ml were achieved. Conclusions 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.
    Keywords: Influenza Viruses -- Health Aspects ; Influenza Vaccines -- Production Processes ; Influenza Vaccines -- Health Aspects ; Virus Replication -- Health Aspects ; Swine Influenza -- Health Aspects ; Trypsin -- Health Aspects
    ISSN: 1472-6750
    Source: Cengage Learning, Inc.
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  • 10
    Language: English
    In: BMC Biotechnology, 01 August 2011, Vol.11(1), p.81
    Description: Abstract Background 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. Results 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 Log10 TCID50/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 Log10 TCID50/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 Log10 TCID50/ml were achieved. Conclusions 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.
    Keywords: Influenza ; Vero ; Microcarrier ; Ns1 ; Bioreactor ; Engineering
    ISSN: 1472-6750
    E-ISSN: 1472-6750
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