Biotechnology and Bioengineering最新文献

This study investigates the enzymatic synthesis of amoxicillin, focusing on its kinetic properties and their influence on antibiotic production in a batch-operated enzymatic reactor. The reaction is catalyzed by penicillin G acylase (PGA, E.C.3.5.1.11), which is immobilized on glyoxyl-agarose. The reaction involves the p-hydroxyphenylglycyne methyl ester and 6-aminopenicillanic acid (6-APA) for amoxicillin formation. Under kinetic control, parallel hydrolytic pathways lead to product loss. Two kinetic models were evaluated: one based on Michaelis–Menten kinetics and another incorporating reaction and equilibrium constants for the process steps. Parameter estimation for the models was performed at different concentrations using two mathematical approaches: the Markov chain Monte Carlo (MCMC) method, rooted in Bayesian statistics and characterized as nondeterministic, and genetic algorithm, an evolutionary computation method incorporating crossover, mutation, and selection operators. The relative root mean squared error (rRMSE) was selected as the metric for evaluating the predictive performance of the models. MCMC presented the best results for low ester concentrations, with rRMSE values ranging from 1.48% to 6.10% for the Michaelis–Menten-based model. The mathematical model was validated using data from an enzymatic reactor operating in semi-batch mode, demonstrating a satisfactory capacity to predict the system's dynamic behavior under this operational condition.

Chinese hamster ovary (CHO) cells, widely utilised in biopharmaceutical production, experience various stressors during cell culture that can affect protein expression and folding, particularly within the endoplasmic reticulum (ER). Mild hypothermia is widely employed in CHO cell bioproduction to improve recombinant protein yield and quality; however, its impact on ER-associated pathways, particularly those governing protein folding and stress responses, remains insufficiently characterised. Mass spectrometry-based proteomics allows for the identification and relative quantification of proteins, enabling detailed insights into protein expression, modifications, and functional networks. This study investigates the impact of mild hypothermic conditions (31°C) on the whole cell proteome and ubiquitinated proteome of CHO cells, with a specific focus on ER proteins and ER stress. Using high-resolution mass spectrometry, we conducted a comprehensive proteomic and ubiquitinated proteomic analysis to quantify changes in protein abundance and ubiquitinated peptides under mild hypothermia. The downregulation of several proteins involved in the glycosylation of nascent polypeptides at 31°C, including DDOST, P4HB, PRKSCH and LMAN1, in all cell lines studied suggests that mild hypothermic shock disrupts the cell's normal ability to fold new proteins, leading to ER stress as the misfolded proteins build up. When this is coupled with the maintained cell viability and increased productivity at 31°C, it indicates the ER stress response can mitigate the build-up of misfolded proteins. The differential regulation of the transcription factor eIF2α, downregulated in non-producer cells but upregulated in producer cells at 31°C, suggests that recombinant protein-producing CHO cells possess a more adaptive ER stress response, enabling more efficient function under hypothermic culture conditions. Enhanced ubiquitination of misfolded protein substrates highlights an increased reliance on ER-associated degradation (ERAD) pathways to alleviate proteotoxic stress, as well as the wide range of biological processes that are regulated by ubiquitination as part of the hypothermic stress response. These findings provide new insights into the cellular adaptation mechanisms of CHO cells to mild hypothermia, with implications for optimising bioproduction strategies to improve yield and quality of therapeutic proteins. Our study highlights the importance of understanding the more complex aspects of the proteome and how this additional layer of detail can open new avenues for CHO cell engineering.

Biotechnology products derived from cell lines carry a risk of viral infection. Given this potential risk, an assessment of the virus removal/inactivation that can be achieved by a product's established purification process is required per ICH Q5A guidelines. This viral safety assessment is a critical component of filing an investigational new drug (IND) application for an early-stage product. A viral clearance study must evaluate purification steps in a manufacturing process that are effective in inactivating/removing viruses. The traditional approach to viral clearance studies includes spiking in one virus at a time and quantifying the level of reduction of individual virus by a validated assay for that virus. This paper discusses an approach to spiking in multiple viruses in the same load and quantifying clearance in virus-specific assays to help accelerate the timeline to IND filing for an early-stage product while also reducing support and material requirements for executing viral clearance studies.

Sterile filtration is an essential step in the production of lipid nanoparticles (LNPs) used in mRNA vaccines and therapeutics. The overlap between the particle size of the LNPs (typically 50–200 nm) and the pore size of the sterilizing-grade membranes (rated at 0.2 µm) complicates the design and operation of the sterile filtration process. The objective of this study was to characterize the pore size distribution, fouling behavior, and capacity of different sterilizing-grade membranes and prefilters for LNP filtration. Mercury intrusion porosimetry revealed significant variability in pore size distributions across several sterilizing-grade membranes despite their consistent 0.2 µm rating, with LNP filtration capacity strongly correlated with the experimentally observed mean pore size. Dual-layer membranes, including the 0.8/0.2 µm Sartopore 2 XLG, significantly enhanced LNP filtration capacity and reduced fouling due to their integrated prefilter layer. Prefilter membranes with pore sizes in the 400–800 nm range provided the greatest enhancement in LNP filtration capacity. These findings highlight that for a given LNP formulation, the filtration capacity is strongly influenced by sterilizing-grade filter selection and specifically by the membrane pore size distribution and morphology, which are generally not published by filter manufacturers.

To enable adeno-associated viral vectors (AAV) to achieve their maximum potential, next-generation manufacturing processes must be developed to make gene therapies more affordable and accessible. This study focused on the design of two different intensified AAV downstream manufacturing processes at bench and pilot scale. Novel clarification methods were studied at bench scale, including the use of BioOptimal™ MF-SL tangential flow microfilters for continuous removal of cell debris. Membrane adsorbers were used for further clarification, including DNA removal. Single pass tangential flow filtration (SPTFF) was implemented at bench scale by feeding the clarified cell lysate (CCL) into two Pellicon XL50 cassettes with 100 kDa regenerated cellulose membranes. At pilot scale, a multi-membrane staged SPTFF module was designed to concentrate 10 L of AAV CCL. Both SPTFF systems provided 12X inline volumetric concentration with AAV yield > 99% after an appropriate buffer chase. Host cell protein removal was 48% and 37% for the bench and pilot scale processes, respectively. As an initial proof-of-concept, an integrated process was developed at pilot-scale which linked clarification, SPTFF, and affinity chromatography. The integrated process offered an 81% reduction in total operating time (due to the reduced volume of load material for the affinity column after preconcentration by SPTFF), 36% improvement in affinity resin utilization (due to the higher AAV concentration in the column load), and an estimated 10% reduction in raw material costs. These improvements translated to an 8.5-fold increase in overall productivity compared to an equivalent batch process, underscoring the potential for SPTFF to intensify large-scale AAV downstream processing.

Cellulose-active Lytic Polysaccharide Monooxygenases (LPMO) facilitate plant cell wall deconstruction by attacking ordered regions of cellulose. In vitro, reductants (e.g., ascorbic acid) reduce LPMOs to Cu(I)-LPMO, and hydrogen peroxide (H₂O₂) serves as co-substrate for oxidative cleavage of cellulose glycosidic bonds. Super-resolution single-molecule imaging by total internal reflection fluorescence microscopy was used to visualize and enumerate binding events of fluorescently-labeled Thermothielavioides terrestris AA9E (TtAA9E) on highly ordered cellulose fibrils in oxygen-scavenging buffer systems. In the glucose oxidase/catalase (GODCAT) system, oxygen is converted to H₂O₂, then removed by catalase. Adding ascorbic acid to the GODCAT system promoted rapid binding to cellulose by TtAA9E. In contrast, absent both oxygen and H₂O₂ in the protocatechuic acid/protocatechuate 3,4-dioxygenase (PCA/PCD) oxygen-scavenging system, adding ascorbic acid nearly eliminated cellulose binding by TtAA9E. Our results suggest that in the GODCAT system, TtAA9Es are reduced by ascorbic acid and activated by H₂O₂, facilitating binding to cellulose. In the PCA/PCD system, reduced TtAA9Es are not activated due to the lack of H₂O₂, suggesting that reduced Cu(I)-TtAA9E cannot bind to cellulose without H₂O₂. Notably, in the PCA/PCD system with ascorbic acid, oxidized sugar release initially lagged but was observed at longer reaction times, suggesting that H₂O₂ could be a limiting reactant generated in situ as oxygen becomes absorbed into solution. Binding durations of LPMO to cellulose were independent of experimental conditions: ( 82% ± 6%) of cellulose-bound LPMOs resided briefly for 14 ± 2.5 s, while 16% ± 5% of the bound enzymes remained for 60 ± 9 s.