Process adapted calibration improves fluorometric pH sensor precision in sophisticated fermentation processes.

Miniaturization and automation have become increasingly popular in bioprocess development in recent years, enabling rapid high-throughput screening and optimization of process conditions. In addition, advances in the bioprocessing industry have led to increasingly complex process designs, such as pH and temperature shifts, in microbial fed-batch fermentations for optimal soluble protein expression in a range of hosts. However, in order to develop an accurate scale-down model for bioprocess screening and optimization, small-scale bioreactors must be able to accurately reproduce these complex process designs. Monitoring methods, such as fluorometric-based pH sensors, provide elegant solutions for the miniaturization of bioreactors, however, previous research suggests that the intrinsic fluorescence of biomass alters the sigmoidal calibration curve of fluorometric pH sensors, leading to inaccurate pH control. In this article, we present results investigating the impact of biomass on the accuracy of a commercially available fluorometric pH sensor. Subsequently, we present our calibration methodology for more precise online measurement and provide recommendations for improved pH control in sophisticated fermentation processes.

Implementation of a Fully Automated Microbial Cultivation Platform for Strain and Process Screening.

Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of potential cultivations is multiplied. Exploiting this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols. Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed-batch processes, including at-line sampling, deep freezer off-line sample storage, and complete data handling. To enable scalable high-throughput early-stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot are combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom-built hardware and software, fully automated milliliter-scale microbial fed-batch cultivation, sample handling, and data storage are realized. The initial results of cultivations with two different expression systems and three different process conditions are compared using 5 L scale benchmark cultivations, which provide identical rankings of expression systems and process conditions. Thus, fully automated high-throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early-stage bioprocess development.

Integrated Process Modeling-A Process Validation Life Cycle Companion.

During the regulatory requested process validation of pharmaceutical manufacturing processes, companies aim to identify, control, and continuously monitor process variation and its impact on critical quality attributes (CQAs) of the final product. It is difficult to directly connect the impact of single process parameters (PPs) to final product CQAs, especially in biopharmaceutical process development and production, where multiple unit operations are stacked together and interact with each other. Therefore, we want to present the application of Monte Carlo (MC) simulation using an integrated process model (IPM) that enables estimation of process capability even in early stages of process validation. Once the IPM is established, its capability in risk and criticality assessment is furthermore demonstrated. IPMs can be used to enable holistic production control strategies that take interactions of process parameters of multiple unit operations into account. Moreover, IPMs can be trained with development data, refined with qualification runs, and maintained with routine manufacturing data which underlines the lifecycle concept. These applications will be shown by means of a process characterization study recently conducted at a world-leading contract manufacturing organization (CMO). The new IPM methodology therefore allows anticipation of out of specification (OOS) events, identify critical process parameters, and take risk-based decisions on counteractions that increase process robustness and decrease the likelihood of OOS events.

Workflow for Criticality Assessment Applied in Biopharmaceutical Process Validation Stage 1.

Identification of critical process parameters that impact product quality is a central task during regulatory requested process validation. Commonly, this is done via design of experiments and identification of parameters significantly impacting product quality (rejection of the null hypothesis that the effect equals 0). However, parameters which show a large uncertainty and might result in an undesirable product quality limit critical to the product, may be missed. This might occur during the evaluation of experiments since residual/un-modelled variance in the experiments is larger than expected a priori. Estimation of such a risk is the task of the presented novel retrospective power analysis permutation test. This is evaluated using a data set for two unit operations established during characterization of a biopharmaceutical process in industry. The results show that, for one unit operation, the observed variance in the experiments is much larger than expected a priori, resulting in low power levels for all non-significant parameters. Moreover, we present a workflow of how to mitigate the risk associated with overlooked parameter effects. This enables a statistically sound identification of critical process parameters. The developed workflow will substantially support industry in delivering constant product quality, reduce process variance and increase patient safety.

Expression of recombinant human flavin monooxygenase and moclobemide-N-oxide synthesis on multi-mg scale.

A panel of human flavin monooxygenases were heterologously expressed in E. coli to obtain ready-to-use biocatalysts for the in vitro preparation of human drug metabolites. Moclobemide-N-oxide (65 mg) was the first high-priced metabolite prepared with recombinant hFMO3 on the multi-milligram scale.

Manejo farmacológico coadyuvante al tratamiento endodóntico: [revisión] / Use of pharmacological agent in endodontic treatment: [review]

Durante la terapia endodóntica, el dolor, inflamación e infección son unos de los principales motivos de consulta que afronta el endodoncista. El control de estos signos y síntomas pueden ser tratados mediante la acción de procedimientos locales, medicación local o tópica, así como de coadyudantes farmacológicos.

During endodontic therapy, pain, inflammation and infection are among the main chief complaints. The control of these signs and symptoms can be managed by the action of local procedures, local medication topical as well as pharmacological.

High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris.

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

Reliability and validity of the Spanish version of the TAPQOL: a health-related quality of life (HRQOL) instrument for 1- to 5-year-old children.

OBJECTIVES: To evaluate the reliability, and construct validity of the Spanish version of the TNO-AZL preschool children quality of life (TAPQOL). METHODS: A consecutive sample of children (3 months to 5 years old) was recruited from primary care centers and two teaching hospitals in Spain. The TAPQOL and a set of questions related to their child's health status were administered to parents. Clinical diagnoses were collected from clinical records. Principal component analysis (PCA) with varimax rotation was used to analyze the instrument's structure. Effect size (ES) and analysis of variance (ANOVA) were used to analyze differences between subgroups known to be in poor health compared to the healthy subgroup. RESULTS: A total of 228 children participated in the study (response rate=95%). Ten of the 12 scales showed more than 30% ceiling effect. All dimensions except one had Cronbach's alpha coefficients greater than 0.7. PCA explained 75% of the variance. Healthy children in general had better scores than the other subgroups. Children at risk of poor health outcomes and those with respiratory problems scored lower in several scales than the healthy subgroup. CONCLUSIONS: Although the Spanish TAPQOL shows a non-negligible ceiling effect, it seems to be a reliable and valid instrument for Spanish infants and toddlers, and with similar psychometric characteristics to the original version. Future studies should try to improve questionnaire's structure and assess its sensitivity to change.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst.

BACKGROUND: Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. RESULTS: As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. CONCLUSIONS: Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Real-time PCR-based determination of gene copy numbers in Pichia pastoris.

Pichia pastoris is a preferred host for heterologous protein production. Expression cassettes are usually integrated into the genome of this methylotrophic yeast. This manuscript describes a method for fast and reliable gene copy number determinations for P. pastoris expression strains. We believe that gene copy number determinations are important for all researchers working with P. pastoris and also many other research groups using similar gene integration techniques for the transformation of other yeasts. The described method uses real-time PCR to quantify the integrated expression cassettes. Similar methods were employed previously for other host systems such as animal and plant cells but no such method comparing different detection methods and describing details for yeast analysis by quantitative PCR is known to us, especially for methylotrophic yeasts such as P. pastoris. Neglecting gene copy numbers can easily lead to false interpretations of experimental results from codon optimization or promoter studies and co-expression of helper proteins as demonstrated in an application example, which is also described here.

Random tag insertions by Transposon Integration mediated Mutagenesis (TIM).

Transposon Integration mediated Mutagenesis (TIM) is a broadly applicable tool for protein engineering. This method combines random integration of modified bacteriophage Mu transposons with their subsequent defined excision employing type IIS restriction endonuclease AarI. TIM enables deletion or insertion of an arbitrary number of bases at random positions, insertion of functional sequence tags at random positions, replacing randomly selected triplets by a specific codon (e.g. scanning) and site-saturation mutagenesis. As a proof of concept a transposon named GeneOpenerAarIKan was designed and employed to introduce 6xHis tags randomly into the esterase EstC from Burkholderia gladioli. A TIM library was screened with colony based assays for clones with an integrated 6xHis tag and for clones exhibiting esterase activity. The employed strategy enables the isolation of randomly tagged active enzymes in single mutagenesis experiments.