Engineering in Life Sciences最新文献

The growing demand for large quantities of high-purity lentiviral vectors (LVs) has driven the development of scalable, cost-effective purification strategies. Membrane chromatography is particularly well-suited for purifying large biological entities like LVs due to its low mass transfer resistance and minimal back pressure. Among these technologies, anion exchange (AEX) chromatography serves as a key unit operation during the intermediate purification stage of industrial-scale bioprocesses. Sartobind Convec D, a weak AEX membrane with reduced ligand density, lack of hydrogel grafting, and adjusted pore size distribution, was specifically designed for LVs capture. While AEX is commonly employed immediately after clarification to capture LVs, incorporating a tangential flow filtration (TFF) step beforehand can offer several advantages, including product concentration, buffer exchange, and removal of low-molecular-weight impurities. Hydrosart High-Performance TFF membranes, composed of regenerated cellulose, are characterized by high flux rates and are marketed as well-suited for viral vector purification. This study investigated the integration of these two technologies into intermediate purification workflows for LVs. Specifically, the impact of introducing a TFF step prior to AEX chromatography (TFF-AEX workflow) was compared to a process utilizing only AEX chromatography following harvest and clarification (AEX-only workflow). In the AEX-only workflow, Sartobind Convec D membranes were used directly after clarification to capture LVs. These membrane adsorbers can accommodate high flow rates, making them suitable for early downstream processing. In the TFF-AEX workflow, clarified LVs harvests were first concentrated and diafiltrated using Hydrosart TFF membranes, allowing for product enrichment, buffer conductivity adjustment, and removal of smaller contaminants prior to AEX loading. As a result, the TFF-AEX workflow demonstrated improved dynamic binding capacity and enhanced overall impurity removal compared to the AEX-only approach.

In vitro measurement of protein diffusion within matrices that simulate the subcutaneous (SQ) environment is of interest, given that protein-based therapeutics formulated for SQ injection comprise the largest class of biologics. To mimic the in vivo transport of a biologic from the SQ injection site through the extracellular matrix (ECM), in vitro diffusion assays typically utilize hyaluronic acid (HA) matrices, as it is the principal component of ECM. However, broad utility has been hampered by inherent lot-to-lot variability in commercially sourced HA, wherein key properties that impact protein diffusion (for example, molecular weight distribution and viscosity) differ across lots, even when nominal molecular weights are identical, making it challenging to compare results across matrices prepared from different HA lots. To address this gap, we report a facile approach wherein binary HA blends generated from individual HA matrices derived from distinct HA lots are functionally equivalent with respect to protein diffusion, that is, the diffusion of a representative set of proteins matches that in a previously reported single HA lot-derived matrix that served as a representative reference. Taken altogether, our protocols enable preparing blended HA matrices with consistent diffusion properties, enabling the use of in vitro assays that leverage this capability.

Practical application: The measurement of in vitro diffusion of IgG-type proteins enables calculation of diffusion coefficients that could help to guide the formulation of protein-based therapeutics, administered by subcutaneous (SQ) injection, and used for treating a range of diseases, including cancer. The side-by-side comparison of these proteins over a period of time provides confirmation of consistency of properties when in vitro hyaluronic acid matrices, within which injected protein diffusion is measured, are also consistent. However, their broad utility has been hindered by the inherent variability of commercial sources of HA used to make-up matrices that simulate the SQ environment in a predictable manner. Our research addresses this gap by defining an approach (validated with rheological and diffusion measurements) that facilitates the preparation of blended matrices from different lots of HA. The resulting matrix properties enable reliable measurement of protein diffusion from one lot to the next.

The clinical translation of CRISPR genome-editing therapies is often hindered by inefficient delivery of the CRISPR-Cas RNA-protein complex into target cells. The most widely used CRISPR-Cas9 system poses a significant challenge for efficient delivery into cells due to its large size (∼1.4 kDa). Recently reported compact Cas proteins, such as Cas12f (552 Da), Cas12k (639 Da), and Cas12m (596 Da) represent attractive alternatives as cargoes for delivery. In this brief research report, we employ efficient delivery vectors to evaluate the efficiency of cellular uptake of a compact Cas protein (Cas12f) compared to the widely used larger Cas9 in human cells. Our findings demonstrate that compact Cas proteins may facilitate more efficient cellular penetration and delivery, making them a promising alternative for the development of CRISPR-based therapies.

Practical Application:

Our study demonstrates that compact Cas proteins significantly enhance cellular uptake compared to larger Cas proteins. This improved uptake efficiency suggests that compact Cas proteins could be more effective for clinical application, where size constraints and delivery efficiency are critical challenges. Combined with the optimization and refinement of the editing efficiencies of compact Cas systems, our study provokes further exploration of compact Cas proteins in various therapeutic contexts to advance the development of more efficient CRISPR-based therapies.

Microbial bioprocessing is a key technology for the production of a wide range of biomolecules, including proteins, enzymes, antibiotics, and other bioactive compounds. In recent years, there has been an increasing interest in using microfluidic platforms for bioprocessing, due to the ability to precisely control and manipulate fluids at the microscale. Microfluidics offers a transformative platform for the manufacturing of biomolecules intended for clinical applications by addressing key technical challenges in scalability, precision, reproducibility, and the ability to study complex biological systems. In this review, various methods used to fabricate microfluidic platforms and the current state-of-the-art in the synthesis/production of biopharmaceuticals, polymers, bioactive compounds, and real-time monitoring in microscale bioprocesses are discussed. Additionally, the future trends and directions are highlighted. Overall, we envisage the utilization of microfluidic platforms to advance the field of microbial bioprocessing and applications in the biomedical field.

Menthol, a natural organic compound and the primary component of mint, exhibits diverse biological activities, including analgesic, anti-inflammatory, antibacterial, neuroprotective, and anticancer effects. The chemical modification of menthol, through processes such as esterification and amination, further enhances these activities, expanding its potential applications in drug development, agriculture, and food preservation. This review explores the structure-activity relationships (SAR) of menthol and its derivatives, emphasizing the significance of molecular modifications in enhancing their pharmacological effects. Research indicates that menthol and its derivatives can improve drug permeation, reduce inflammation, enhance memory, and even target cancer cells through various mechanisms. In addition, we examine the safety and pharmacokinetics of menthol and its derivatives to better understand their clinical potential. Although significant progress has been made in preclinical models, further research is necessary to fully elucidate their mechanisms of action and optimize their therapeutic efficacy in clinical settings. Continued innovation in drug delivery technologies and the development of novel menthol derivatives present promising prospects for future therapeutic applications.

Optical spectroscopic techniques have been successfully employed in bioprocessing as process analytical technology for real-time process monitoring in numerous applications. The implementation of spectroscopy-based PAT techniques commonly necessitates the generation of representative process data used for calibration and validation of multivariate statistical models for analyzing the sample composition in real-time. To automate the generation of such data, we present a novel assembly of a commercially available chromatography system in combination with a Raman spectrometer for fast and accurate acquisition of Raman spectra. Using the ultra-/diafiltration (UF/DF) process as a case study, our methodology involved the preparation of representative calibration and validation mixtures of phosphate and citrate buffer and lysozyme as a model protein. Chemometric PLS models were calibrated and validated using these datasets, and applied to in-line recorded Raman spectra during a UF/DF experiment. The primary results demonstrated that the novel assembly provides robust and precise offline measurement of Raman spectra, which directly compare with in-line record data. The chemometric PLS models showed good alignment in calibration and validation datasets (R2 and Q2), and could be used to simultaneously monitor the buffer and protein concentrations in real-time during UF/DF. This study provides a simple, commercially available setup for automated acquisition of Raman spectra and demonstrates its straightforward application to bioprocess monitoring.

RETRACTION: E.M. Abed, F. Yazdian, A.A. Sepahi, and B. Rasekh, “Synthesis and Evaluation of PCL/Chitosan/CQD-Fe Magnetic Nanocomposite for Wound Healing: Emphasis on Gene Expression,” Engineering in Life Sciences 25, no. 1 (2025): e202400038, https://doi.org/10.1002/elsc.202400038.

The above article, published online on 19 January 2025 in Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the journal Editor-in-Chief, Ralf Takors; and Wiley-VCH GmbH. Following an investigation by the publisher, the parties have concluded that this article was accepted solely on the basis of a compromised peer review process. Therefore, the article must be retracted.

Celosia argentea is an undervalued crop that shows potential for production enhancement due to elevated leaf nutrient accumulative ability. By investigating propagation using various in vitro culture systems, thidiazuron (TDZ)-supplemented nutrient media enhanced yield from 10 plants per explant in semi-solid medium, to 27 under continuous immersion in liquid media in recipient for automated temporary immersion (RITA) bioreactors, to 63 under temporary immersion in liquid media in a balloon-type bubble bioreactor (BTBB). TDZ in the BTBB system also increased shoot biomass and subsequent nutrient content relative to TDZ-free media in ex vitro plants. Ex vitro plants originating from both continuous and temporary media immersion in BTBBs outperformed those in all other culture systems in accumulating leaf Mg, Fe, Ca and Zn to meet the recommended dietary allowance for males and females. The genotypic variance and genetic advance of the mean at 5% selection intensity varied for each nutrient per culture system, with and without TDZ. Selective breeding at 5% selection intensity would improve leaf nutrient content but is specific to the culture system and the presence of TDZ. This is the first study to use liquid-based bioreactor systems for C. argentea propagation thereby providing new opportunities to upscale plant production for high nutrient-accumulating genotypes.

Practical application: This study establishes a commercially viable protocol for the large-scale clonal propagation of Celosia argentea, a nutrient-rich, fast growing leafy vegetable with untapped agronomic value. Using temporary immersion bioreactors and thidiazuron-supplemented media, the system delivers up to 63 plants per explant, more than 6-fold the yield of conventional methods, while significantly boosting leaf biomass and nutrient content (Mg, Ca, Fe, Zn). These results position C. argentea as a functional crop for health-focused markets and ready-to-cook vegetable lines. The low-input cultivation needs and rapid production cycle (8 weeks in vitro, 8 weeks ex vitro) make it ideal for high-turnover commercial nurseries, contract growers, and vertical farming operations. The systems reproducibility and high heritability of nutritional traits further support selective breeding programs for premium-value cultivars. This propagation platform offers agribusinesses a scalable entry point into the expanding market for nutrient-dense indigenous vegetables with health and wellness appeal.