Assessing the capabilities of 2D fluorescence monitoring in microtiter plates with data-driven modeling for secondary substrate limitation experiments of Hansenula polymorpha.

BACKGROUND: Non-invasive online fluorescence monitoring in high-throughput microbioreactors is a well-established method to accelerate early-stage bioprocess development. Recently, single-wavelength fluorescence monitoring in microtiter plates was extended to measurements of highly resolved 2D fluorescence spectra, by introducing charge-coupled device (CCD) detectors. Although introductory experiments demonstrated a high potential of the new monitoring technology, an assessment of the capabilities and limits for practical applications is yet to be provided. RESULTS: In this study, three experimental sets introducing secondary substrate limitations of magnesium, potassium, and phosphate to cultivations of a GFP-expressing H. polymorpha strain were conducted. This increased the complexity of the spectral dynamics, which were determined by 2D fluorescence measurements. The metabolic responses upon growth limiting conditions were assessed by monitoring of the oxygen transfer rate and extensive offline sampling. Using only the spectral data, subsequently, partial least-square (PLS) regression models for the key parameters of glycerol, cell dry weight, and pH value were generated. For model calibration, spectral data of only two cultivation conditions were combined with sparse offline sampling data. Applying the models to spectral data of six cultures not used for calibration, resulted in an average relative root-mean-square error (RMSE) of prediction between 6.8 and 6.0%. Thus, while demanding only sparse offline data, the models allowed the estimation of biomass accumulation and glycerol consumption, even in the presence of more or less pronounced secondary substrate limitation. CONCLUSION: For the secondary substrate limitation experiments of this study, the generation of data-driven models allowed a considerable reduction in sampling efforts while also providing process information for unsampled cultures. Therefore, the practical experiments of this study strongly affirm the previously claimed advantages of 2D fluorescence spectroscopy in microtiter plates.

Toward Rapid, Widely Available Autologous CAR-T Cell Therapy - Artificial Intelligence and Automation Enabling the Smart Manufacturing Hospital.

CAR-T cell therapy is a promising treatment for acute leukemia and lymphoma. CAR-T cell therapies take a pioneering role in autologous gene therapy with three EMA-approved products. However, the chance of clinical success remains relatively low as the applicability of CAR-T cell therapy suffers from long, labor-intensive manufacturing and a lack of comprehensive insight into the bioprocess. This leads to high manufacturing costs and limited clinical success, preventing the widespread use of CAR-T cell therapies. New manufacturing approaches are needed to lower costs to improve manufacturing capacity and shorten provision times. Semi-automated devices such as the Miltenyi Prodigy® were developed to reduce hands-on production time. However, these devices are not equipped with the process analytical technology necessary to fully characterize and control the process. An automated AI-driven CAR-T cell manufacturing platform in smart manufacturing hospitals (SMH) is being developed to address these challenges. Automation will increase the cost-effectiveness and robustness of manufacturing. Using Artificial Intelligence (AI) to interpret the data collected on the platform will provide valuable process insights and drive decisions for process optimization. The smart integration of automated CAR-T cell manufacturing platforms into hospitals enables the independent manufacture of autologous CAR-T cell products. In this perspective, we will be discussing current challenges and opportunities of the patient-specific but highly automated, AI-enabled CAR-T cell manufacturing. A first automation concept will be shown, including a system architecture based on current Industry 4.0 approaches for AI integration.

From Single Batch to Mass Production-Automated Platform Design Concept for a Phase II Clinical Trial Tissue Engineered Cartilage Product.

Advanced Therapy Medicinal Products (ATMP) provide promising treatment options particularly for unmet clinical needs, such as progressive and chronic diseases where currently no satisfying treatment exists. Especially from the ATMP subclass of Tissue Engineered Products (TEPs), only a few have yet been translated from an academic setting to clinic and beyond. A reason for low numbers of TEPs in current clinical trials and one main key hurdle for TEPs is the cost and labor-intensive manufacturing process. Manual production steps require experienced personnel, are challenging to standardize and to scale up. Automated manufacturing has the potential to overcome these challenges, toward an increasing cost-effectiveness. One major obstacle for automation is the control and risk prevention of cross contaminations, especially when handling parallel production lines of different patient material. These critical steps necessitate validated effective and efficient cleaning procedures in an automated system. In this perspective, possible technologies, concepts and solutions to existing ATMP manufacturing hurdles are discussed on the example of a late clinical phase II trial TEP. In compliance to Good Manufacturing Practice (GMP) guidelines, we propose a dual arm robot based isolator approach. Our novel concept enables complete process automation for adherent cell culture, and the translation of all manual process steps with standard laboratory equipment. Moreover, we discuss novel solutions for automated cleaning, without the need for human intervention. Consequently, our automation concept offers the unique chance to scale up production while becoming more cost-effective, which will ultimately increase TEP availability to a broader number of patients.

Robotic integration enables autonomous operation of laboratory scale stirred tank bioreactors with model-driven process analysis.

Given its geometric similarity to large-scale production plants and the excellent possibilities for precise process control and monitoring, the classic stirred tank bioreactor (STR) still represents the gold standard for bioprocess development at a laboratory scale. However, compared to microbioreactor technologies, bioreactors often suffer from a low degree of process automation and deriving key performance indicators (KPIs) such as specific rates or yields often requires manual sampling and sample processing. A widely used parallelized STR setup was automated by connecting it to a liquid handling system and controlling it with a custom-made process control system. This allowed for the setup of a flexible modular platform enabling autonomous operation of the bioreactors without any operator present. Multiple unit operations like automated inoculation, sampling, sample processing and analysis, and decision making, for example for automated induction of protein production were implemented to achieve such functionality. The data gained during application studies was used for fitting of bioprocess models to derive relevant KPIs being in good agreement with literature. By combining the capabilities of STRs with the flexibility of liquid handling systems, this platform technology can be applied to a multitude of different bioprocess development pipelines at laboratory scale.

Need for speed: evaluation of dilute and shoot-mass spectrometry for accelerated metabolic phenotyping in bioprocess development.

With the utilization of small-scale and highly parallelized cultivation platforms embedded in laboratory robotics, microbial phenotyping and bioprocess development have been substantially accelerated, thus generating a bottleneck in bioanalytical bioprocess sample analytics. While microscale cultivation platforms allow the monitoring of typical process parameters, only limited information about product and by-product formation is provided without comprehensive analytics. The use of liquid chromatography mass spectrometry can provide such a comprehensive and quantitative insight, but is often limited by analysis runtime and throughput. In this study, we developed and evaluated six methods for amino acid quantification based on two strong cation exchanger columns and a dilute and shoot approach in hyphenation with either a triple-quadrupole or a quadrupole time-of-flight mass spectrometer. Isotope dilution mass spectrometry with 13C15N labeled amino acids was used to correct for matrix effects. The versatility of the methods for metabolite profiling studies of microbial cultivation supernatants is confirmed by a detailed method validation study. The methods using chromatography columns showed a linear range of approx. 4 orders of magnitude, sufficient response factors, and low quantification limits (7-443 nM) for single analytes. Overall, relative standard deviation was comparable for all analytes, with < 8% and < 11% for unbuffered and buffered media, respectively. The dilute and shoot methods with an analysis time of 1 min provided similar performance but showed a factor of up to 35 times higher throughput. The performance and applicability of the dilute and shoot method are demonstrated using a library of Corynebacterium glutamicum strains producing L-histidine, obtained from random mutagenesis, which were cultivated in a microscale cultivation platform.

Uncover aldehydes in biomass hydrolyzates: disproportionation of aldehydes in alkaline solution and subsequent measurement using an automated HPAEC-PAD method.

High-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) was used for developing a method for identifying and quantifying aldehydes in biomass hydrolyzates. This method was optimized to the requirements of HPAEC-PAD in order to allow for a simultaneous determination of aldehydes by respective Cannizzaro alcohols. To this end, sodium hydroxide concentration (0.1 to 5.0 mol/L), temperature (30 to 40 °C), and reaction time (0 to 24 h) were investigated for sufficient and reproducible disproportionation of the biomass-derived aldehydes. The optimized method for aldehyde disproportionation and subsequent measurement are 1 mol/L sodium hydroxide, 40 °C, and 1 h reaction time. The detection limits resulting from this method are lower than 68.55 mg/L and the sensitivity above 0.024 (nC min)/(mg/L) for 3,4-dimethoxybenzaldehyde. Linearity for aldehyde calibration always exceeded 0.98. Thus, HPAEC-PAD analysis allows for the quantification of biomass-derived compounds from all natural polymers and, therefore, it has exemplarily been used to quantify aldehyde concentration of beech wood, orange peel, and algae biomass hydrolyzates. Graphical abstract.

A comparison of behavioral parent training programs for fathers of children with attention-deficit/hyperactivity disorder.

Few behavioral parent training (BPT) treatment studies for attention-deficit/hyperactivity disorder (ADHD) have included and measured outcomes with fathers. In this study, fathers were randomly assigned to attend a standard BPT program or the Coaching Our Acting-Out Children: Heightening Essential Skills (COACHES) program. The COACHES program included BPT plus sports skills training for the children and parent-child interactions in the context of a soccer game. Groups did not differ at baseline, and father ratings of treatment outcome indicated improvement at posttreatment for both groups on measures of child behavior. There was no significant difference between groups on ADHD-related measures of child outcome. However, at posttreatment, fathers who participated in the COACHES program rated children as more improved, and they were significantly more engaged in the treatment process (e.g., greater attendance and arrival on time at sessions, more homework completion, greater consumer satisfaction). The implications for these findings and father-related treatment efforts are discussed.

Enhancing traditional behavioral parent training for single mothers of children with ADHD.

Behavioral parent training is an efficacious treatment for attention-deficit/hyperactivity disorder (ADHD). However, single-mother households are at high risk for poor outcomes during and following behavioral parent training. This study randomly assigned cohorts of 120 single mothers of children (ages 5-12 years) with ADHD to a waitlist control group, a traditional behavioral parent training program, or an enhanced behavioral parent training program -- the Strategies to Enhance Positive Parenting (STEPP) program. Intent-to-treat analysis demonstrated benefits of participating in behavioral parent training, in general, and the STEPP program more specifically at immediate posttreatment on child and parental functioning. Moreover, the STEPP program resulted in increased engagement to treatment. However, results indicated that behavioral parent training does not normalize behavior for most children and treatment gains are not maintained.

A pilot study of the feasibility and efficacy of the Strategies to Enhance Positive Parenting (STEPP) program for single mothers of children with ADHD.

OBJECTIVE: The Strategies to Enhance Positive Parenting (STEPP) program was developed to address putative factors related to poor engagement in and outcomes following traditional behavioral parent training (BPT) for single mothers of children diagnosed with ADHD. METHOD: Twelve single mothers of children with ADHD were enrolled in an initial investigation of the feasibility and preliminary efficacy of the 9-week STEPP program. RESULTS: Results indicated that the STEPP program was effective in reducing problematic child behavior and improving parental stress and psychopathology at posttreatment. The STEPP program resulted in high rates of treatment attendance and completion and consumer satisfaction with the program. However, results also indicated that the STEPP program did not improve childrens' overall psychosocial impairment and resulted in small effect size findings across measures. CONCLUSION: The results of the pilot study are encouraging but indicate a need to improve the potency and delivery of certain aspects of the STEPP program.