Biotechnology Progress最新文献

D-Mannitol production was achieved in freshwater Synechocystis sp. PCC6803 via the heterologous expression of mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p) under control of the strong promoter P_trc1. However, only 5.54 mg L⁻¹ of mannitol was found extracellularly after 7 days of cultivation, likely due to insufficient expression of a mutated mtlD lacking a methionine at position 332. This study compared mannitol levels using different promoters (P_trc1, P_psbA2 and P_nrsB) to control the expression of (un)mutated versions of mtlD in Synechocystis with co-expression of m1p. Our data suggest that even without the inducer, the weakest promoter, P_nrsB, can support the expression of an unmutated mtlD in Synechocystis, which leads to 18.2 mg L⁻¹ of mannitol in 7 days without induction. Such titer is already much higher than the first engineered mannitol-producing Synechocystis. When 5 μM nickel sulfate was added to the medium as an inducer, mannitol production could significantly be increased further, up to 92.9 mg L⁻¹ after 7 days of induction, but it partially inhibited growth. Attempts with the other increasingly stronger promoters always failed to express the unmutated mtlD, probably due to the toxicity caused by the accumulation of the intermediate product, mannitol-1-phosphate. These results clearly suggest that the expression level of mtlD is the bottleneck in achieving a high yield of mannitol in Synechocystis, and consequently, that mannitol production can be enhanced by fine-tuning its expression. Future research is needed to identify bottlenecks that hinder mannitol productivity and long-term stability, facilitating the engineering of more efficient mannitol-producing cyanobacterial strains.

Triton X-100 is one of the most widely used detergents for cell lysis in gene therapy product manufacturing. However, due to the aquatic toxicity of the degradation product, Triton X-100 is regulated by the European Chemical Agency as a substance of very high concern. In this study, we aim to identify eco-friendly detergent alternatives to replace Triton X-100. Tween 20, Tween 80, n-Dodecyl-β-D-maltoside (DDM), and Triton CG-110 were tested and compared with Triton X-100. Triton CG-110 demonstrated similar performance during the harvest and subsequent purification process for multiple AAV vectors at different scales. Therefore, Triton CG-110 represents an effective and environmentally safe detergent alternative to Triton X-100.

The inhibition of lignin condensation during biomass pretreatment is crucial for enhancing enzymatic hydrolysis efficiency, since the formation of rigid cross-linked lignin networks hinders cellulose accessibility and enzyme activity. This study investigates the effects of nucleophilic additives, including ascorbic acid (AsA), 2-naphthol (2N), 3-hydroxy-2-naphthoic acid (3H2NA), and 2-naphthol-7-sulfonate (7S2NA), as potential agents to suppress lignin condensation on the phosphoric acid pretreatment of poplar. The phosphoric acid pretreatment demonstrated a remarkable efficacy in the removal of xylan (100%) and lignin (18.06%–31.35%) from poplar, both with and without the inclusion of nucleophilic additives. An enzymatic hydrolysis yield ranging from 71.41% to 100% was achieved with the incorporation of AsA, 2N, 3H2NA, and 7S2NA, compared to a yield of 66.15% for substrates pretreated solely with phosphoric acid. The enhancement in enzymatic hydrolysis yield upon the addition of nucleophilic additives was probably due to the improved cellulose accessibility and the enhanced proportion of cellulose II in the pretreated substrates. The analysis of total phenolic content in the prehydrolysates revealed that 3H2NA and 7S2NA, characterized by their strong hydrophilic groups within their chemical structures, significantly facilitated lignin fractionation during phosphoric acid pretreatment.

Changes in serotype or genetic payload of recombinant adeno associated virus (rAAVs) gene therapies require adapting the transfection conditions of the upstream HEK293 cultivations. This study adopts an iterative model-based experiment design approach, where increasing data availability is leveraged to evolve models of different complexity. Initial models based on data from shaker flask runs guided the design of the first round at Ambr250 scale. With Ambr250 data becoming available, hybrid models capturing process state evolutions and historical models incorporating these evolutions to predict rAAV titer, were developed. These models were then combined into a full model approach, which was utilized within a Bayesian Optimization framework for the design of a second round of Ambr250 scale runs. The iterative approach was tested across different projects applying transfer learning to enhance the predictive power and improve the subsequent optimization. The approach was benchmarked against a statistical Design of Experiment method. The results show that the model-based experiment design consistently (and across projects) produces higher rAAV titer values than the benchmark approach (Project C: 4.4% or 7.0% increases in titer values relative to the response surface modeling approach for ELISA and ddPCR, respectively; Project D: 32.4% or 10.9% increases in titer values relative to the standard DoE-screening pick for ELISA and ddPCR, respectively), effectively optimizing the transfection mixture composition. The combination of propagation and historical models, augmented by transfer learning and an ever-increasing amount of data, enhanced the process design workflow, contributing to improved rAAV production through efficient transfection strategies.

Monitoring cell culture is crucial for gaining a deeper understanding of processes and ensuring the production of safe and high-quality products. The capability to measure in real time several parameters of interest can be achieved with Raman spectroscopy. However, before using Raman spectroscopy to monitor a specific process, a calibration phase is required to develop chemometric models that correlate Raman spectra with the target parameters. It is mandatory to conduct this phase with multiple batches to build robust models that account for biological variability. This model building phase can be time-consuming and require a lot of resources. The industry is actively seeking solutions to simplify and expedite this step without compromising accuracy. Moreover, the current approach has limitations regarding changing cell culture media, celllines, or process scale. The novel synthetic model approach provides a significant gain of time and resources for the calibration phase, which is reduced to just a few days. The methodology involves using cell-free samples of cell culture media that are spiked with various concentrations of target compounds. The results indicate that the innovative approach enables accurate measurement for glucose and lactate parameters in real process conditions comparable to a standard modeling methodology.

Epigenetic regulation plays an important role in cell fate reprogramming. Here, we found that inhibitors of epigenetic modifiers, including VPA, TSA, and 5-Aza-2'-deoxycytidine, can induce phenotypic transformation from Jurkat cells into B-cell-like cells. When Jurkat cells were treated with 5-Aza combined with VPA, B cell and stem cell marker expression was observed. These gene expression pattern changes were most remarkable in the optimized B cell induction conditions provided by the cocultured and genetically modified murine bone marrow OP9 cells. In such conditions, Jurkat cells were endowed with the ability to secrete B cell cytokines, and B lymphocyte-related genes and pathways were activated. In studying the mechanism underlying Jurkat cell reprogramming by 5-Aza and VPA, we found that PAX5, the key transcription factor regulating B cell development, was significantly upregulated. Treatment with 5-Aza and VPA inhibited the methylation of CpG islands and upregulated the acetylated H3K9 modification in the PAX5 promoter region, respectively, thus epigenetically activating the expression of PAX5 and promoting the reprogramming of Jurkat cells. Similar reprogramming results were also observed in primary CD4⁺T cells following treatment with 5-Aza and VPA. Our results provide a de novo paradigm for the reprogramming of T cells through epigenetic modifications.

The specific selectivities offered by multimodal ligands drive the increased application of multimodal chromatography in the purification of complex new “multispecific” antibodies, which requires improved understanding of the protein-multimodal ligand interaction mechanism. In the present study, a mechanistic model is developed to predict monoclonal antibody (mAb1)-Fab fragment (Fab) and heterogeneous aggregates separation on Capto™ MMC ImpRes multimodal resin based on the general rate model coupled with the proposed preferential interaction (PI) analysis-based Langmuir non-linear binding model. The model input value of binding parameters is obtained from Perkin et al. developed PI model, fit to the characteristic ‘U’-shaped curve for isocratic retention factors of mAb1, Fab, and aggregates as a function of NaCl salt concentrations. The model successfully simulates mAb1 and Fab elution peaks, whereas in the absence of deconvoluted peaks of heterogeneous aggregates, aggregates are modeled as a single species, giving satisfactory prediction of elution peak position, describing the average of the multiple (majority as double peaks) aggregate elution peaks. The physical significance of model estimated binding parameters is obtained from model estimated total number of released counter salt ions and water molecules for each species during binding, found to be consistent with their isocratic retention data. The underlying mechanism of double peak elution of aggregates during linear gradient elution was investigated based on mechanistic model estimated equilibrium constant. The proposed predictive mechanistic model was successfully validated by predicting mAb1, Fab, and aggregates elution peaks for the multimodal column operated in hydrophobic interaction mode and can be successfully implemented for process development.

One of the widely used techniques for producing recombinant adeno-associated virus serotype 2 (rAAV2) particles, as viral vectors for gene therapy applications, is the triple transient (TT) transfection of human embryonic kidney 293 (HEK293) cells. It is desirable to optimize this transfection process for more efficient manufacturing of rAAV viral vectors for gene therapy purposes. We examined the application of dimethyl sulfoxide (DMSO) as an additive to this transfection technique to improve the expression yield of rAAV2 particles with HEK293 cells in adherent and suspension cell culture modalities. This assistance by DMSO should increase the trafficking of plasmid DNA (pDNA) through the cell membrane, and thus, increase the viral titer of rAAV2 full capsids at the time of harvesting the cell culture. The study demonstrated that DMSO as an additive for the TT transfection process led to an 8.2-fold increase in the expression yield of full AAV2 capsids using HEK293 cells in adherent cell culture modality, and also led to a 4.0-fold increase in the expression yield of full AAV2 capsids using HEK293 cells in suspension cell culture modality. There are no reported studies on the application of DMSO as an additive to the TT transfection process of HEK293 cells for the production of AAV particles. This is a novel, simple, and inexpensive method to improve the yield of rAAV2 full capsids with the TT transfection process of HEK293 cells, using a well-known cryoprotectant agent (CPA), as an additive to this transfection process.

Induced pluripotent stem cells (iPSCs) hold large potential in regenerative medicine due to their pluripotency and unlimited self-renewal capacity without the ethical issues of embryonic stem cells. To provide quality-controlled iPSCs for clinical therapies, it is essential to develop safe cryopreservation protocols for long-term storage, preferably amenable to scale-up and automation. We have compared the impact of two different freezing geometries (bottom-up and conventional radial freezing) on the viability and differentiation potential of human iPSCs. Our results demonstrate that bottom-up freezing under optimized conditions significantly increases iPSC viability, up to 9% for cell membrane integrity and up to 21% for cell metabolic state, compared to conventional freezing. The improvement achieved for bottom-up versus conventional freezing was maintained after scale-up from cryogenic vials to 30 mL bags, highlighting its potential for clinical applications. These findings show that bottom-up freezing can offer a more controlled and scalable cryopreservation strategy for iPSCs, promoting their application in regenerative medicine.

In the downstream processing of antibody-based therapeutics, ultrafiltration/diafiltration (UF/DF) is commonly applied for concentration and buffer exchange in the final formulation. For a given molecule, various factors such as membrane type, feed flux, and transmembrane pressure (TMP) can significantly influence the performance of UF/DF, impacting yield, buffer exchange efficiency, and product quality. Conventional membrane pore size selection is based on product molecular weight to ensure high retention. While working on an Fc-fusion protein, we found that the pH of load material had a critical effect on the retention of the molecule due to conformational changes at different pH values, as evidenced by the size-exclusion chromatography (SEC). Meanwhile, optimization of the UF/DF process underscored the importance of concentration polarization to protein retention. Approaches to reduce concentration polarization, such as increasing feed flux and lowering TMP, resulted in less protein loss in the permeate stream. High retention of this Fc-fusion protein during the UF/DF step can be achieved not only by utilizing a 5 kDa membrane but also by employing a 10 kDa membrane with optimized process parameters such as load conditions, feed flux, and TMP. These observations provide important insights on the factors impacting protein retention beyond the molecular weight cutoff (MWCO) of UF/DF membrane.