Percy Kampeis

Background/Objectives: Phenethyl isothiocyanate (PEITC), a bioactive compound found in cruciferous vegetables, exhibits potent anti-cancer activity, but its molecular mechanisms in acute lymphoblastic leukemia (ALL) remain poorly defined. This study aimed to investigate the anti-leukemic effects of PEITC. Methods: PEITC cytotoxicity was assessed in human cancer cell lines using resazurin reduction assays. Microarray and Ingenuity Pathway Analysis (IPA) were used to investigate altered molecular signaling pathways. Functional assays were performed to assess DNA damage, cell-cycle progression, apoptosis, autophagy, mitochondrial membrane potential, microtubule organization and endothelial tube formation. Western blotting was performed to examine pathway-related proteins, and preliminary in vivo antitumor activity was evaluated using a CCRF-CEM zebrafish xenograft model. Results: PEITC exerted potent cytotoxicity against CCRF-CEM cells (IC₅₀ = 1.76 ± 0.07 µM). Microarray and IPA analyses predicted inhibition of PI3K/AKT/mTOR signaling, which was further supported by Western blot analysis. Functional assays demonstrated that PEITC induced DNA damage, cell cycle perturbation, mitochondrial dysfunction, and apoptotic and autophagic responses. PEITC also disrupted microtubule organization in GFP-tagged α-tubulin-expressing U2OS cells. Furthermore, PEITC suppressed endothelial tube formation in EA.hy926 cell lines and exhibited preliminary in vivo antitumor activity in a zebrafish xenograft model (n = 10). Conclusions: These findings provide mechanistic insights into the anti-leukemic activity of PEITC and demonstrate its effects on multiple cellular processes associated with leukemia cell survival and proliferation, supporting further investigation of its therapeutic potential in ALL.

Determining the mixing time in a mixing apparatus enables the evaluation of mixing quality and, therefore, represents a valuable tool for characterizing unit operations in process engineering. Mixing is highly relevant in both upstream processes (e.g., bioreactors) and downstream processes (e.g., blending tanks in diafiltration) in biotechnology and pharmaceutics. The aim of combining a colorimetric method with video capture and automated image analysis is to provide a robust, standardized methodology for determining mixing times in transparent bioreactors, such as laboratory-scale glass bioreactors and single-use bioreactors. A Python-based tool for video analysis was developed for this purpose. The Python script provides different mixing indices that enable the evaluation of both global and local mixing times under varying stirring speeds and aeration rates. This approach offers deep insights into the mixing process and enables the identification of heterogeneities. A round-robin study involving members of the DECHEMA Working Group “Single-Use Technologies for Bio-Based Applications” is currently being conducted to enhance the analysis and ensure reproducibility across different laboratories, thereby providing a robust basis for validating computational-fluid dynamics simulations. Initial reproducibility issues were identified and addressed in the standard operating procedure, demonstrating that a framework for automated analysis and improved standardization has been established. Key points: • Mixing-time experiments conducted at different aeration rates and stirring speeds • Automated video analysis for determining mixing times on both global and local scales • Identification of heterogeneities through visualization of local mixing times

Due to a lack of investigated materials for the additive manufacturing of multi-use functional parts in bioprocess engineering, this study aimed to evaluate the influence of multiple autoclaving cycles on the properties of a heat-resistant material (xPeek147) printed with vat photopolymerization. Sample bodies were tested regarding their mechanical properties of tensile strength, elongation at break, and Charpy impact, as well as surface properties of roughness and wettability after up to 50 autoclaving cycles (121 °C, 2 bars, 15 min). The tightness was checked after up to 20 cycles, and accuracy was inspected for manufactured benchmark bodies after up to 10 autoclaving cycles. The reported results showed no significant changes in tensile strength, elongation at break and Charpy impact after 20 cycles, but a significant decrease after 50 autoclaving cycles, accompanied by microcracks in the structure. Regarding the surface properties the material retained its hydrophilicity, and the surface roughness was not affected significantly. No changes in tightness occurred, and the benchmark bodies for dimensional changes showed no process-relevant deviations. Through the investigations, a material for the additive manufacturing of multi-use functional parts for bioprocess engineering was identified. Additionally, a testing method for materials with the same intended application was provided.

Background: Nitrosamines have gained significant attention in the pharmaceutical industry. However, due to the significant daily dosage of large volume parenteral (LVP), the detection limit for these products should be in the ng per liter range, which many published methods cannot achieve. Objective: This study aimed to develop and validate a sensitive LC-MS/MS method for the simultaneous determination of several nitrosamines in LVP drug products. Method: Nitrosamines and related internal standards were separated on a Waters ACQUITY UPLC HSS T3 (100 × 2.1 mm, 1.8 µm) column on a LC-MS/MS system with gradient elution. Prior to the LC injection, a Carbon A solid phase extraction (SPE) column was used to pretreat the test solutions. The mobile phase was composed of 0.1% formic acid in water as mobile phase A, and neat methanol as mobile phase B. Analytes were detected via multiple reaction monitoring (MRM) mode and quantitated against internal reference standards with a quantitation limit of 2.5 ng/L for N-nitrosodimethylamine (NDMA), 0.75 ng/L for other nitrosamine analytes. Results: The LC-MS/MS method was able to separate all analytes of interest by gradient elution within 15 min. The method was validated according to the guidelines described in the International Conference on Harmonization guideline ICH Q2(R2). The LOQ for NDMA is 2.5 ng/L, while for other nitrosamines, it is 0.75 ng/L in peritoneal dialysis and saline matrices. For haemofiltration solution, the LOQ is 1.0 ng/L for NDMA and 0.3 ng/L for other nitrosamines. The RSD% (n = 9) of the recovery did not exceed 25% in the method accuracy evaluation. Comparative testing in three laboratories revealed that all three laboratories are capable of accurately measuring nitrosamine levels at 10 ng/L within the intricate LVP matrix. Conclusions: The LC-MS/MS method for several nitrosamines was successfully developed, validated, and demonstrated to be accurate, robust, and specific. Highlights: The performance of this method can reach single-digit ng/L level in LVP matrices. Comparative testing was conducted in three laboratories located in China, Germany, and the United States to ensure reproducibility of the method.

Zur Optimierung von Zulaufsatzkultur‐Fermentationen von methylotrophen Organismen wird eine Online‐Messmethode vorgestellt, mit der die Methanol‐Konzentration im Medium während einer Fermentation durch ein Spülgaspervaporations‐Prinzip bestimmt werden kann. Im Gegensatz zu anderen Analysemethoden bietet die Messmethode die Möglichkeit, die Substratkonzentration bei Prozessen mit Methanol als zentralem Substrat über eine Regelung auf einem definierten Wert zu halten. Es werden Schwierigkeiten, aber auch deren Überwindung bei der Adaption der Messmethode auf Fermentationsprozesse dargestellt.

A novel in situ magnetic separator is presented in which the separation unit is directly mounted on the bioreactor. Therefore, it is possible to collect the magnetic particles directly from the reaction liquid and transfer them in one step sterile out of the reactor. By consecutive flushing, the particles are concentrated and separated from the rest. The operating parameters were determined and evaluated experimentally and by computational fluid dynamics. The separator was tested in combination with a 5‐L bioreactor with glass flask. Hereby, the accumulation kinetics of the particles were analyzed and used for a simple theoretical model.

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.

Bei der Verwendung der Hochgradienten‐Magnetseparation (HGMS) in der Bioverfahrenstechnik werden meist Schlauchpumpen zur Förderung eingesetzt. Bei konstanter Drehzahl der Pumpe fällt allerdings der Volumenstrom durch den HGMS‐Filter mit der Zeit ab, weil mit zunehmender Filterbeladung der freie Strömungsquerschnitt immer weiter eingeengt wird. Daraus resultiert, dass die Beladung des HGMS‐Filters zeitlich nicht konstant erfolgt. In der vorliegenden Untersuchung wurde der Einsatz eines Ultraschall‐Durchflussmessgeräts für die dynamische Anpassung der Pumpendrehzahl zur Konstanthaltung des Volumenstroms während der Magnetseparation erprobt.