Biotechnol. Prog. [JOURNAL]
Sun
200 papers
Synthetic modeling: A cell-free approach for faster implementation of Raman spectroscopy in cell culture.
Sanchez C, El Radi H, Gay N … +4 more , Cailletaud J, Grollier K, Thomas F, Gonthiez T
Biotechnol Prog · 2025 · PMID 40126075 · Publisher ↗
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...
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.
5-Aza combined with VPA reprograms human T lineage acute leukemia Jurkat cells into B-cell-like cells by epigenetic activation of PAX5.
Xi W, Zheng G, Chen X … +9 more , Zuo B, Wang W, Li Y, Zhang C, Chu J, Mu X, Wen W, Wang T, Yang AG
Biotechnol Prog · 2025 · PMID 40095298 · Full text
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...
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 CD4T cells following treatment with 5-Aza and VPA. Our results provide a de novo paradigm for the reprogramming of T cells through epigenetic modifications.
Predictive mechanistic model for separation of monoclonal antibody, fab fragment, and aggregate species on multimodal chromatography.
Shekhawat LK, Markle T, Maloisel JL … +1 more , Malmquist G
Biotechnol Prog · 2025 · PMID 40091852 · Publisher ↗
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 o...
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.
Process optimization mitigated the retention loss of an Fc-fusion protein during ultrafiltration/diafiltration.
Yu H, Fei L
Biotechnol Prog · 2025 · PMID 40071869 · Publisher ↗
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...
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.
Improving the expression yield of recombinant adeno-associated virus serotype 2 using dimethyl sulfoxide DMSO as an additive to the triple transient transfection process of HEK293 cells.
Burns A, Ramos-Sono D, Datta S
Biotechnol Prog · 2025 · PMID 40071857 · Publisher ↗
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 embryoni...
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.
Enhancing cryopreservation of human induced pluripotent stem cells: Bottom-up versus conventional freezing geometry.
Teodoro F, El-Guendouz S, Neves R … +3 more , Duarte A, Rodrigues MA, Melo EP
Biotechnol Prog · 2025 · PMID 40071781 · Publisher ↗
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-co...
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.
Enhancing the performance of an in vitro RNA biosensor through iterative design of experiments.
Aw R, Polizzi K
Biotechnol Prog · 2025 · PMID 40071731 · Full text
The quality control of RNA has become increasingly crucial with the rise of mRNA-based vaccines and therapeutics. However, conventional methods such as LC-MS often require specialized equipment and expertise, limiting th...
The quality control of RNA has become increasingly crucial with the rise of mRNA-based vaccines and therapeutics. However, conventional methods such as LC-MS often require specialized equipment and expertise, limiting their applicability to high throughput experiments. Here, we optimize a previously characterized RNA integrity biosensor, that provides a simple colorimetric output, using Design of Experiments (DoE). Through iterative rounds of a Definitive Screening Design (DSD) and experimental validation, we systematically explored different assay conditions to enhance the biosensor's performance. Optimization led to a 4.1-fold increase in dynamic range and reduced RNA concentration requirements by one-third, significantly improving usability. Notable modifications included reducing the concentrations of reporter protein and poly-dT oligonucleotide and increasing DTT concentration, suggesting a reducing environment for optimal functionality. Importantly, the optimized biosensor retained its ability to discriminate between capped and uncapped RNA even at lower RNA concentrations. Overall, our improved biosensor offers enhanced performance and reduced sample requirements, paving the way for rapid, cost-effective RNA quality control in diverse settings, including resource-limited environments.
Development of multiparametric bioprinting method for generation of 3D printed cell-laden structures.
Lipshutz S, Kim Y, Curtis M … +2 more , Friedrich L, Alimperti S
Biotechnol Prog · 2025 · PMID 40071586 · Full text
The organ transplantation field requires new approaches for replacing and regenerating tissues due to the lack of adequate transplant methods. Three-dimensional (3D) extrusion-based bioprinting is a rapid prototyping app...
The organ transplantation field requires new approaches for replacing and regenerating tissues due to the lack of adequate transplant methods. Three-dimensional (3D) extrusion-based bioprinting is a rapid prototyping approach that can engineer 3D scaffolds for tissue regeneration applications. In this process, 3D printed cell-based constructs, consisting of biomaterials, growth factors, and cells, are formed by the extrusion of bioinks from nozzles. However, extrusion applies shear stresses to cells, often leading to cellular damage or membrane rupture. To address this limitation, herein, we developed and optimized a 3D bioprinting approach by evaluating the effect of key extrusion-based 3D bioprinting parameters-bioink viscosity, nozzle size, shape, and printing speed-on cell viability. Our results revealed that cells printed in higher-viscosity bioinks, with smaller, cylindrical nozzles, exhibited lower viability due to their exposure to high shear stresses. Translational flow speed had a cell-dependent impact, as different cell types have different sensitivities to the magnitude and duration of shear stress inside the nozzle. Overall, evaluating these parameters could facilitate the development of 3D high-resolution bioprinted constructs for tissue regeneration applications, offering a more efficient alternative to traditional fabrication methods, which are often labor intensive, expensive, and repetitive.
Assessing the probability of clonality achieved by single-cell cloning of CHO cells through cell deposition combined with imaging using distinguishable cells.
Nonet GH, Scut E, Ogawa R … +1 more , Tomic MT
Biotechnol Prog · 2025 · PMID 40034018 · Publisher ↗
Mammalian cell lines used for clinical studies and post-approval production of recombinant DNA-derived biotherapeutics are expected to be derived from a single cell, and regulatory submissions are expected to provide rob...
Mammalian cell lines used for clinical studies and post-approval production of recombinant DNA-derived biotherapeutics are expected to be derived from a single cell, and regulatory submissions are expected to provide robust evidence of monoclonality. Imaged single-cell deposition followed by whole-well imaging using specialized instruments has, in many cell line development labs, replaced the "gold standard" of two rounds of limiting dilution due to its increased speed and the assurance of clonality provided by orthogonal images. However, there is still a lack of information on how the procedures used to define these clonal cell lines perform. Here we use a mixture of two distinguishable Chinese hamster ovary (CHO) cells to document that a greater than 99% probability of clonality can be obtained from our single-cell cloning method that uses our preparation procedures, the VIPS® single-cell deposition instrument, the Cell Metric® whole-well imager, and a comprehensive visual review. Together with the assurance of cell/well images, the determination of the probability of clonality of our VIPS+Cell Metric method provides a strong package of evidence of single-cell derivation of a recombinant CHO cell line.
Efficient expression of recombinant proteins in Bacillus subtilis using a rewired gene circuit of quorum sensing.
Hao W, Yang S, Sheng Y … +4 more , Ye C, Han L, Zhou Z, Cui W
Biotechnol Prog · 2025 · PMID 39968680 · Publisher ↗
Bacillus subtilis is a favored chassis for high productivity of several high value-added product in synthetic biology. Efficient production of recombinant proteins is critical but challenging using this chassis because t...
Bacillus subtilis is a favored chassis for high productivity of several high value-added product in synthetic biology. Efficient production of recombinant proteins is critical but challenging using this chassis because these expression systems in use, such as constitutive and inducible expression systems, demand for coordination of cell growth with production and addition of chemical inducers. These systems compete for intracellular resources with the host, eventually resulting in dysfunction of cell survival. To overcome the problem, in this study, LuxRI quorum sensing (QS) system from Aliivibrio fischeri was functionally reconstituted in B. subtilis for achieving coordinated protein overproduction with cell growth in a cell-density-dependent manner. Furthermore, the output-controlling promoter, P, was engineered through two rounds of evolution, by which we identified four mutants, P22, P47, P56, and P58 that exhibited elevated activity compared to the original P. By incorporating a strong terminator (TB5) downstream of the target gene further enhanced expression level. The expression level of this system surpasses commonly used promoter-based systems in B. subtilis like P43 and P. The LuxRI QS system proves to be a potent platform for recombinant protein overproduction in B. subtilis.
The production of succinate with more CO fixation reactions facilitated by RuBisCO-based engineered Escherichia coli.
Zhou X, Li L, Sun S … +2 more , Xiong P, Liu X
Biotechnol Prog · 2025 · PMID 39968675 · Publisher ↗
Redesigning metabolic pathways to enhance the efficiency of carbon fixation during chemical biosynthesis is a promising approach for achieving cleaner and greener production of multi-carbon compounds. In this study, we e...
Redesigning metabolic pathways to enhance the efficiency of carbon fixation during chemical biosynthesis is a promising approach for achieving cleaner and greener production of multi-carbon compounds. In this study, we established a model of cell growth in Escherichia coli that is dependent on the RuBisCO-Prk pathway by regulating its central metabolism. This rewiring ensures that growth depends on RuBisCO's carboxylation, allowing heterotrophic growth to rely on carbon fixation. This model was verified by detecting the growth curve, and it was used to screen four RuBisCO genes, of which the gene from Rhodospirillum rubrum ATCC 11170 serves as a growth advantage for E.coli. In addition, this model was applied to construct an efficient succinate biosynthetic pathway that can produce two moles of succinate from one mole of xylose and three moles of CO. Compared to conventional succinate biosynthesis, this strategy has a CO fixation capacity that is 1.5 times greater. Furthermore, to optimize succinate production, various approaches were employed, including the optimization of key enzymes, substrate transport, and the supply of inorganic carbon. The resulting strain was capable of producing succinate at a level of 2.09 ± 0.14 g/L, which is nearly 22.4 times that of the original strain. In conclusion, this study was developed for the production of two moles of succinate by implementing three moles of carbon fixation reactions and demonstrated the feasibility of various optimization strategies in biological carbon fixation.
Enhancing real-time cell culture process monitoring through the integration of advanced machine learning techniques: A comparative analysis of Raman and capacitance spectroscopies.
Xu F, Pinto N, Zhou G … +1 more , Ahuja S
Biotechnol Prog · 2025 · PMID 39968672 · Publisher ↗
Machine learning (ML) techniques have emerged as an important tool improving the capabilities of online process monitoring and control in cell culture process for biopharmaceutical manufacturing. A variety of advanced ML...
Machine learning (ML) techniques have emerged as an important tool improving the capabilities of online process monitoring and control in cell culture process for biopharmaceutical manufacturing. A variety of advanced ML algorithms have been evaluated in this study for cell growth monitoring using spectroscopic tools, including Raman and capacitance spectroscopies. While viable cell density can be monitored real-time in the cell culture process, online monitoring of cell viability has not been well established. A thorough comparison between the advanced ML techniques and traditional linear regression method (e.g., Partial Least Square regression) reveals a significant improvement in accuracy with the leading ML algorithms (e.g., 31.7% with Random Forest regressor), addressing the unmet need of continuous monitoring viability in a real time fashion. Both Raman and capacitance spectroscopies have demonstrated success in viability monitoring, with Raman exhibiting superior accuracy compared to capacitance. In addition, the developed methods have shown better accuracy in a relatively higher viability range (>90%), suggesting a great potential for early fault detection during cell culture manufacturing. Further study using ML techniques for VCD monitoring also showed an increased accuracy (27.3% with Raman spectroscopy) compared to traditional linear modeling. The successful integration of ML techniques not only amplifies the potential of process monitoring but also makes possible the development of advanced process control strategies for optimized operations and maximized efficiency.
Microcontact printing of lectin self-assembled monolayers for arbovirus detection.
Lucena RPS, Silva-Junior AG, Frías IAM … +5 more , Gil LHV, Cordeiro MT, El Salhi AE, Andrade CAS, Oliveira MDL
Biotechnol Prog · 2025 · PMID 39968671 · Publisher ↗
Arboviruses significantly burden public health in Brazil, constituting a constant challenge for health authorities. The diagnosis and, consequently, clinical management and the reporting of arbovirus infections in region...
Arboviruses significantly burden public health in Brazil, constituting a constant challenge for health authorities. The diagnosis and, consequently, clinical management and the reporting of arbovirus infections in regions where multiple arboviruses coexist are complex processes. Herein, we report the development of a new electrochemical biosensor based on Concanavalin A (ConA) to identify carbohydrate patterns in the viral structure of Dengue 3 (DENV-3), Zika (ZIKV) and Chikungunya (CHIKV) viruses. The biorecognition of arboviruses was carried out through functionalization with 4-aminophenylacetic acid (CMA) on poly (ethylene terephthalate) (PET) substrate coated with a gold layer combining microcontact printing (μCP). Bovine serum albumin (BSA) was used after ConA immobilization to block binding to nonspecific sites. Subsequently, the interaction between ConA and arbovirus was characterized by standard atomic force microscopy (AFM), fluorescence microscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Fluorescent imaging was conducted to confirm the occurrence of the DENV-3, ZIKV, and CHIKV detection processes. The obtained results demonstrated the success of the biosensor (CMA-ConA-BSA) manufactured on a PET substrate using μCP for detecting medically significant arboviruses. RCT values showed an increase in impedimetric response total of the system after exposition to DENV-3 (RCT = 68.82 kΩ) and a lower recognition to CHIKV (RCT = 44.44 kΩ). The present biosensor platform reveals the applicability of the ConA lectin in the viral biorecognition process based on flexible biosensors for differential detection of DENV-3, ZIKV, and CHIKV. ConA-based electrochemical biosensor provide high selectivity, real-time detection, and low volumes of analytes.
Detergent/surfactant retention during ultrafiltration in the formulation of biotherapeutics.
Chu LK, Du Z, Billups M … +2 more , Oh HJ, Zydney AL
Biotechnol Prog · 2025 · PMID 39968661 · Full text
Surfactants like polysorbate (Tween®) are commonly used as excipients in the production of monoclonal antibodies and other recombinant proteins. The retention behavior of these excipients in the final ultrafiltration ste...
Surfactants like polysorbate (Tween®) are commonly used as excipients in the production of monoclonal antibodies and other recombinant proteins. The retention behavior of these excipients in the final ultrafiltration step can be difficult to predict due to the presence of both monomers and micelles. This study examined the retention of polysorbate during ultrafiltration through cellulose and polyethersulfone membranes with nominal molecular weight cutoffs of 10, 30, and 100 kDa. Novel flux stepping experiments were performed to examine the effects of concentration polarization on surfactant transmission. Polysorbate 20 transmission through the 30 kDa membrane was a strong function of the surfactant concentration, decreasing from nearly 100% for a 2.5 mg/L solution to <10% for a 50 mg/L solution due to high retention of the micelles. Polysorbate transmission was lower for the polyethersulfone membrane due to polysorbate adsorption. A simple mathematical model was developed to describe the polysorbate transmission accounting for the effects of concentration polarization as well as the presence of surfactant monomers and micelles. Model calculations were in good agreement with the experimental data, providing a framework for the analysis and design of ultrafiltration/diafiltration processes for biopharmaceutical formulations containing surfactants.
A synthetic platform for developing recombinant adeno-associated virus type 8 producer cell lines.
Lin YC, Kuo HJ, Lu M … +3 more , Mahl T, Aslanidi G, Hu WS
Biotechnol Prog · 2025 · PMID 39968660 · Full text
Recombinant adeno-associated virus (rAAV) is one of the most widely used viral vectors for gene therapy. It is used in very high doses for the treatment of many diseases, making large-scale production for clinical applic...
Recombinant adeno-associated virus (rAAV) is one of the most widely used viral vectors for gene therapy. It is used in very high doses for the treatment of many diseases, making large-scale production for clinical applications challenging. We have established a synthetic biology-based platform to construct stable production cell lines, which can be induced to produce rAAV2. In this study, we extended our cell line construction pipelines for rAAV2 to rAAV8, a serotype whose tropism makes it attractive for gene delivery in multiple tissues. The Genome Module, encoding the rAAV2 genome, and Replication Modules, containing Rep68, DBP and E4orf6 coding sequences, originally used for rAAV2 were retained, but the Packaging Module was modified to replace the AAV2 intron-less cap gene (VP123) with that of AAV8. These three genetic modules were integrated into HEK293 genome to generate four rAAV8 producer cell lines VH1-4, which all produced rAAV8 upon induction. Their productivity was similar to the initial rAAV2 producer cell lines GX2/6 constructed using the same pipeline, but was much lower than conventional triple plasmid transfection. We identified Cap protein production and capsid formation as a potential limiting factor, just as we observed in GX2/6. By integrating more copies of AAV8 VP123 into VH3 clone, the encapsidated rAAV8 titer increased 20-fold to a level comparable to triple transfection. By tuning induction conditions to modulate capsid production, the full particle content could be elevated. This study demonstrated that our rAAV producer cell line development platform is robust and applicable to different AAV serotypes.
Improving outcomes in intensified processing via optimization of the cell line development workflow.
Balassi V, Otto M, Kretzmer C … +6 more , Petersen A, McLaurin C, Mahadevan J, Gustin J, Borgschulte T, Razafsky D
Biotechnol Prog · 2025 · PMID 39968655 · Full text
As the industry continues to explore the benefits of continuous and intensified manufacturing, it is important to assure that the cell line development (CLD) workflows in practice today are well suited to generate clones...
As the industry continues to explore the benefits of continuous and intensified manufacturing, it is important to assure that the cell line development (CLD) workflows in practice today are well suited to generate clones that meet the unique challenges associated with these processes. Most cell lines used in intensified processes are currently developed using traditional fed-batch CLD workflows followed by adaptation of these cell lines to perfusion processes. This method maybe suboptimal as fed-batch CLD workflows select clones which produce high volumetric titers irrespective of cell growth rate and specific productivity (qP). Although sufficient for fed-batch processes, performance of cells derived from this traditional CLD workflow may not be maintained in perfusion processes, where an intricate balance of performance parameters is needed. Until now, a thorough investigation into the effect of the CLD workflow on top clone performance in perfusion processes has not been conducted. Here, we show how the CLD workflow impacts cell performance in both fed-batch and perfusion processes, emphasizing the advantages of adopting a perfusion-specific CLD workflow which includes the use of medium specially designed for expansion and production in a perfusion setting, scale-down models which more accurately simulate perfusion process, and the adoption of perfusion-specific cell line selection criteria. Together, this results in the development of more efficient cell lines, fit for continuous and intensified processing.
Recent trends in biotechnological production, engineering, and applications of lysophospholipases.
Nazir A, Sajjad M
Biotechnol Prog · 2025 · PMID 39968651 · Publisher ↗
Oil degumming process involves the removal of gums, which is required to improve the physicochemical and storage properties of the vegetable oils. Degumming of oils can be carried out by using chemicals, membranes (polym...
Oil degumming process involves the removal of gums, which is required to improve the physicochemical and storage properties of the vegetable oils. Degumming of oils can be carried out by using chemicals, membranes (polymeric, inorganic, and ceramic), or enzymes, for example, phospholipases. Phospholipases are enzymes of tremendous significance in the degumming process as they convert gums to fatty acids and lipophilic substances. They provide a cost-effective and safe alternative to other degumming processes without affecting the oil yield. Lysophospholipases (LPLs) are highly valuable tools for degumming vegetable oils. LPLs can hydrolyze fatty acyl ester bonds of phosphatidylcholine at the sn-1 and sn-2 positions of glycerol moiety. In addition, they have the ability to catalyze hydrolysis lysophospholipids' ester bond either at sn-1 or sn-2 position. In this review, biotechnological production and biochemical characteristics of LPLs from three domains of life are highlighted. In comparison to bacterial and eukaryotic LPLs, archaeal LPLs were found to be active at high temperatures. Broad substrate specificity and thermostability of archaeal LPLs make them ideal candidates for the industrial degumming of oils. However, improvement of activity and substrate specificity of archaeal LPLs is required for enhancing their industrial utility. In the current review, various protein-engineering approaches (directed evolution, rational design, site-saturation mutagenesis, and fusion technology) as well as in silico tools have been discussed to increase the commercial significance of LPLs.
Mechanistic insights into hypoxia-induced metabolic reprogramming in colorectal cancer through genome-scale modeling.
Omer S, Sridhar S, Karunagaran D … +1 more , Suraishkumar GK
Biotechnol Prog · 2025 · PMID 39964193 · Publisher ↗
The hypoxic colorectal cancer (CRC) microenvironment is a complex niche. Hence, in vivo, the metabolism occurring in the cancer cell is not fully known due to difficulties in estimating metabolic fluxes and metabolite ex...
The hypoxic colorectal cancer (CRC) microenvironment is a complex niche. Hence, in vivo, the metabolism occurring in the cancer cell is not fully known due to difficulties in estimating metabolic fluxes and metabolite exchanges. Genome-scale metabolic modeling helps estimate such metabolic fluxes to gain insights into the metabolic behavior of individual cancer cell types under various tumor microenvironments (TME). We developed a simplified approach to apply proteomics data-based enzyme usage constraints and integrated reactive species (RS) reactions in a context-specific genome-scale metabolic model (GSMM) of HCT116, a CRC cell line. The combined modeling approach reproduced several phenotypes of HCT116 under hypoxia such as the Warburg effect. The integration of the RS module with the hypoxic HCT116 context-specific GSMM highlighted the hypoxia-mediated dysregulation occurring in important metabolic pathways such as hyaluronan metabolism in which 80% of the reactions from the total reactions corresponding to this metabolic pathway were dysregulated. Similarly, 23% of reactions in the urea cycle, 26% of reactions in eicosanoid metabolism and 38% of reactions in glyoxylate and dicarboxylate metabolism were dysregulated.
Flocculation-based clarification for production of protein therapeutics in Pichia pastoris: Recombinant human serum albumin as a case study.
Hebbi V, Kumar J, Rathore AS
Biotechnol Prog · 2025 · PMID 39964173 · Publisher ↗
Pichia pastoris has been used as an expression system for multiple biotherapeutic products due to the unique advantages it offers with respect to cell density, protein titer, extracellular expression, and other such adva...
Pichia pastoris has been used as an expression system for multiple biotherapeutic products due to the unique advantages it offers with respect to cell density, protein titer, extracellular expression, and other such advantages. However, clarification of cell broth presents a significant challenge, primarily due to the high cell density (up to 50% W/V). Additionally, the abundance of host cell proteins complicates secondary clarification, impacting subsequent chromatographic, and filtration steps. In this study, a flocculation-based cell clarification method has been developed for the primary recovery of protein therapeutic products from Pichia broth. Human serum albumin (HSA) has been used as a case study. Unlike polymer-based flocculants, which introduce challenges in process clearance, the proposed method employs process-compatible salts. The approach has been designed and optimized using Quality by Design (QbD) principles, achieving a clarification efficiency with up to 90% recovery and a reduction of host cell proteins by up to 30%. The proposed methodology would be applicable to other biotherapeutic applications involving protein production in P. pastoris.
pH and conductivity transients during elution of IgG from protein A columns.
Hahn R, Berger L, Beck J … +1 more , Carta G
Biotechnol Prog · 2025 · PMID 39964169 · Full text
The evolution of pH and conductivity waves during elution of IgG from protein A columns is studied for the resin MabSelect PrismA as well as other commercial resins using glycine and acetate buffers as eluents. The effec...
The evolution of pH and conductivity waves during elution of IgG from protein A columns is studied for the resin MabSelect PrismA as well as other commercial resins using glycine and acetate buffers as eluents. The effects of buffer composition, IgG load, and residence time are explored. For glycine buffers, conductivity and pH waves travel through the column at different speeds, with the conductivity wave emerging in the column void volume and the pH waves emerging in 1 to 6 column volumes (CV) dependent on the buffer composition. The pH effluent temporarily overshoots the feed value, followed by a sharp drop as the pH approaches the eluent pH. For these conditions, elution of IgG is delayed and, at high loads, most of the IgG loaded elutes from the column at high pH values. Complex pH profiles, involving overshoots and delays between conductivity and pH waves are also observed when eluting with dilute sodium acetate (50 mM) or with acetic acid, but to a lesser extent. No overshoots or delays are observed for more concentrated sodium acetate (100 mM). A mechanistic model is developed to predict elution with glycine buffers. Model predictions agree with the experimental trends. In particular, the model shows that when eluting a partially loaded column, IgG desorbed near the column entrance is re-adsorbed downstream of the pH front eventually emerging at high concentration and high pH. The model can be used to design optimized buffers and predict the pH of the IgG elution pool.
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