Biotechnol. Prog. [JOURNAL]
Sun
200 papers
The combination of oleic acid, linoleic acid, palmitoleic acid, and α-linolenic acid promoted the expansion of NK-92 cells in vitro.
Zhang S, Huang H, Zhang J … +3 more , Zhao Y, Tan WS, Cai H
Biotechnol Prog · 2025 · PMID 39964158 · Publisher ↗
Cell culture medium is an important factor affecting the expansion of NK cells in vitro. As an important component of cell culture medium, lipids participate in various complex physiological activities of cells and signi...
Cell culture medium is an important factor affecting the expansion of NK cells in vitro. As an important component of cell culture medium, lipids participate in various complex physiological activities of cells and significantly affect the expansion of cells. Using NK-92 cells as a model, the lipid metabolism of NK cells in vitro was analyzed, and combined with the kinetic relationship between lipid metabolism and NK cell expansion. Four fatty acids, oleic acid, linoleic acid, palmitoleic acid, and α-linolenic acid, were preliminatively identified as the key lipid combinations. The combination was preliminarily verified on the self-developed serum-free medium. It was found that when the key lipid combination was added according to the concentration in the serum, NK-92 cells expansion reached 188.03 ± 33.34-folds, which was significantly higher than 105.28 ± 13.23-folds in the basic medium. Additionally, NK-92 cells expanded by adding key lipid combinations could maintain cell killing function. Overall, this research provides technical support for the development of NK cell serum-free medium.
Efficient fermentative production of lactodifucotetraose by controlling sequential glycosyltransferase reactions in Escherichia coli.
Moriyama S, Sugita T, Yamashita M
Biotechnol Prog · 2025 · PMID 39912504 · Publisher ↗
Lactodifucotetraose (LDFT) is a human milk oligosaccharide (HMO) that might reduce inflammation in infants. In this study, we established a useful production process of LDFT by engineering two key enzymes, α1,2-fucosyltr...
Lactodifucotetraose (LDFT) is a human milk oligosaccharide (HMO) that might reduce inflammation in infants. In this study, we established a useful production process of LDFT by engineering two key enzymes, α1,2-fucosyltransferase (α1,2-FucT) and α1,3-fucosyltransferase (α1,3-FucT). First, we verified which of 2'-fucosyllactose (2'-FL) or 3-fucosyllactose (3-FL) (mostly unverified) was more useful. We searched for FucTs that functioned efficiently in vivo against the raw material lactose or the two intermediates 2'-FL or 3-FL by external substrate addition to culture medium. We found that α1,2- FucT (HMFT) from Helicobacter mustelae and the N-terminal truncated form of α1,3-FucT from Bacteroides fragilis (BfFucTΔN10) had high potential. 3-FL was not efficiently converted to LDFT, which might be attributed to the low reactivity of HMFT to 3-FL as well as the low uptake efficiency of 3-FL by LacY, as revealed by a growth test with exogenously added FL as the sole carbon source and heterologously expressed intracellular fucosidase. Furthermore, because 3-FL accumulation had a negative impact on cell growth, we avoided the route passing through 3-FL. By adjusting the copy numbers of HMFT and BffucTΔN10, we produced LDFT from lactose predominantly via 2'-FL. Finally, 17.5 g/L of LDFT (with 6.8 g/L 2'-FL and no 3-FL or residual lactose) accumulated in a 3-L fed-batch culture after 77 h. This study reports the detailed analysis of multiple pathways and shows the control of glycosyltransferases can improve the production efficiency of complex HMOs.
Rapid bioreactor process optimization and scale-up for production of a measles vector COVID-19 vaccine candidate.
Hesley DC, Spatafore D, Shingler J … +8 more , McNeely JP, Thompson R, Troutman MC, Baron EKB, Sabia M, Lee CH, Ploeger K, Wagner JM
Biotechnol Prog · 2025 · PMID 39912497 · Publisher ↗
The emergence of SARS-CoV-2 in late 2019 and subsequent worldwide spread and pandemic in 2020 spurred the rapid and agile development of a variety of vaccine candidates. With speed to patients in mind during development...
The emergence of SARS-CoV-2 in late 2019 and subsequent worldwide spread and pandemic in 2020 spurred the rapid and agile development of a variety of vaccine candidates. With speed to patients in mind during development of measles-vectored vaccine candidate V591, process optimization efforts were made to expand options for raw material sourcing/treatment, enable flexible use of various types of processing equipment, and streamline the overall production process. To that end, both gamma irradiated and heat sterilized microcarriers were tested to expand the supply network for critical process development experiments and manufacturing at a time when worldwide supply chains were strained or disrupted. Single use bioreactors were also evaluated and implemented to reduce experimental turnaround time. Furthermore, to simplify the process and gain additional efficiencies in large scale media preparation, growth and infection media formulations were harmonized with a parallel vaccine development program. These rapid process option evaluations were conducted parallel to critical path scale up, and the combined efforts enabled the rapid demonstration of two full manufacturing scale 2000 L bioreactors less than 6 months after virus seed delivery, culminating in the first large scale measles production process capable of addressing the high dose demands of a pandemic response scenario. Despite subsequent clinical discontinuation of the V591 vaccine candidate, the findings described herein will be useful for enabling rapid and scalable production of other measles-vectored vaccine candidates, oncolytic measles strains, or cell and gene therapies.
Pre-folding purification procedures for inclusion body-derived non-tagged cationic recombinant proteins with multiple disulfide bonds for efficient refolding.
Kimura S, Yamamoto W, Miyamoto A … +2 more , Imamura K, Futami J
Biotechnol Prog · 2025 · PMID 39865388 · Full text
The production of disulfide-containing recombinant proteins often requires refolding of inclusion bodies before purification. A pre-refolding purification step is crucial for effective refolding because impurities in the...
The production of disulfide-containing recombinant proteins often requires refolding of inclusion bodies before purification. A pre-refolding purification step is crucial for effective refolding because impurities in the inclusion bodies interfere with refolding and subsequent purification. This study presents a new pre-refolding procedure using a reversible S-cationization technique for protein solubilization and purification by reversed-phase high performance liquid chromatography. This pre-folding purification step improves refolding yield by effectively removing the refolding inhibitors from contaminates from bacterial inclusion bodies, and reducing proteolytically degraded products. Because this procedure does not require a peptide tag for affinity purification, it is a superior technique to subsequently perform a simplified downstream process wherein the affinity tag needs to be removed. This study reports improved refolding and purification procedure to obtain the highly cationic (pI = 9.25) mouse vascular endothelial cell growth factor (188 amino acids form) that is used as a model protein in our study; this protein shows a homodimeric conformation and possesses multiple disulfides.
Characterization of large transgene integrations in Chinese hamster ovary cells using a bioengineered mammalian transposase.
Marx N, Otte AM, Leitner K … +8 more , Sitepu R, Berger T, Schäpertöns V, Huber CG, Zhu Q, Nema S, Higgins JJ, Borth N
Biotechnol Prog · 2025 · PMID 39846713 · Full text
We present the first use of a bioengineered mammalian transposase system derived from Myotis lucifugus (bMLT) for integration of expression vectors into the CHO genome, focusing on GFP and trastuzumab production. Initial...
We present the first use of a bioengineered mammalian transposase system derived from Myotis lucifugus (bMLT) for integration of expression vectors into the CHO genome, focusing on GFP and trastuzumab production. Initially, CHO-K1 cells are transfected with a GFP reporter and varying amounts of bMLT DNA or mRNA. GFP expression is monitored over 17 weeks without selective pressure. Transfection efficiency shows around 90% GFP-positive cells, but in control cultures GFP expression disappears after 10 days. In contrast, bMLT-treated cultures maintain stable GFP expression, with a dose-dependent integration efficiency of up to 60%. The highest GFP expression per cell is observed with lower bMLT amounts. Next-generation sequencing analysis reveals multiple integration sites, with 85% correctly integrated sequences. Next, CHO-GS cells are transfected with trastuzumab and bMLT DNA or mRNA. Cells are selected in glutamine-free medium with varying methionine sulfoximine (MSX) concentrations. Recovery is faster without MSX, and no difference is observed between bMLT DNA and mRNA transfections. bMLT-treated cultures show a higher percentage of trastuzumab-secreting cells (40%-55%) compared with random integration (0.3%-0.5%). The absence of insulators in the trastuzumab plasmid likely affects selection behavior, as integration in heterochromatic regions results in gene repression. Overall, bMLT-mediated integration proves efficient, generating stable cell pools with high expression profiles without selective pressure. The integration sites' genomic location significantly impacts productivity, with favorable regions supporting higher expression. This method shows promise for the rapid and efficient generation of high-producing cell lines and for rapid evaluation of long-term effects of different cell engineering approaches.
Coupling high-throughput and modeling approaches to streamline early-stage process development for biologics.
Welsh JP, Altern SH, Lyall JY … +5 more , Burgess S, Rauscher MA, Lenhoff AM, Cramer SM, Williams C
Biotechnol Prog · 2025 · PMID 39846604 · Publisher ↗
Platforms have long been implemented for downstream process development of monoclonal antibodies (mAbs) to streamline development and reduce timelines. These platforms are also increasingly being used for other complex b...
Platforms have long been implemented for downstream process development of monoclonal antibodies (mAbs) to streamline development and reduce timelines. These platforms are also increasingly being used for other complex biologics modalities. While development has traditionally been conducted at the lab bench scale in a sequential manner, automated miniaturized and parallelized approaches like RoboColumns and resin plates have also been implemented for chromatographic screening. Additionally, mechanistic modeling for chromatographic separations has also seen increased use for development applications. In this manuscript, we propose a workflow with elements of both high-throughput screening and modeling that provides a streamlined roadmap for early process development. The workflow utilizes automated resin plate screens to both narrow screening conditions and calibrate binding isotherm parameters. Mechanistic models are then used to characterize a robust range of conditions suitable for an early manufacturing process. Miniaturized RoboColumns then confirm the process space, thus completing the development without the use of any traditional lab-scale columns. Case studies demonstrate the utility of this workflow for both cation-exchange (CEX) and multimodal cation-exchange (MMCEX) processes. Process parameter sensitivities across process ranges for the models are compared with typical design-of-experiment (DOE) statistical models. The models are able to predict the mAb product as well as aggregate impurities. This workflow provides a practical method to enable increased process understanding while also reducing timeline and material requirements for development.
Engineering a high-throughput clone for industrial-scale production of long-acting GLP-1 analogue with retained bio-efficacy.
J PKR, Tummuru M, Ramkumar KM
Biotechnol Prog · 2025 · PMID 39846515 · Publisher ↗
Type 2 diabetes mellitus (T2DM) and obesity are critical global health issues with rising incidence rates. Glucagon-like peptide-1 (GLP-1) analogues have emerged as effective treatments due to their ability to regulate b...
Type 2 diabetes mellitus (T2DM) and obesity are critical global health issues with rising incidence rates. Glucagon-like peptide-1 (GLP-1) analogues have emerged as effective treatments due to their ability to regulate blood glucose levels and gastric emptying through central nervous signals involving hypothalamic receptors, such as leptin. To address the short plasma half-life of native GLP-1, a C-16 fatty acid was conjugated to lysine in the GLP-1 analogue sequence to enhance its longevity. This study focuses on engineering a high-throughput clone and evaluation of novel GLP-1 analogues with improved bio-efficacy and production yields. Five plasmid models were created using different N-terminal fusion partners and assessed for hydrophobicity, instability index, and isoelectric point. Three optimal plasmid models were selected based on high-valued hydrophobicity, solubility, and partial solubility. These plasmids were constructed with the pET24a vector, incorporating GLP-1 with fusion tags via recombinant DNA technology and transformed into E. coli BL21 DE3 hosts. The proteins were purified through enzyme digestion and chromatography, resulting in a high-yield peptide. The GLP-1 peptide was conjugated with in-house developed fatty acid compound n-Palmitoyl glutamic acid (n-PGA) and purified using C18 column chromatography, achieving a final product yield of 170-190 mg per liter of fermentation culture. Biological activity was confirmed by cyclic adenosine monophosphate (cAMP) generation and 3 T3 cell differentiation assays, showing a 1.5-fold increase in mRNA gene expression with the clone having n-terminal hydrophobic amino acids, thioredoxin-modified tag, and enterokinase cleavage site, indicating high purity and biological potency of the GLP-1 analogue.
Harnessing cell aggregates for enhanced adeno-associated virus manufacturing: Cultivation strategies and scale-up considerations.
Ladd B, Gräslund T, Chotteau V
Biotechnol Prog · 2025 · PMID 39846514 · Full text
The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successf...
The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successfully demonstrated in a process where single cells were first expanded, then cell aggregates were formed by dilution into a different medium 1 day before triple plasmid transfection was conducted. An alternative approach for the STR inoculation using a seed taken from a high cell density perfusion (HCDP) culture was also investigated. It was, however, found that the spent medium of the HCDP inhibited the transfection of HEK293T cell aggregates, which was confirmed when testing with single-cell suspension culture. The formation of aggregates in shaken multi-well plates was also investigated to develop a screening system using the average power input as a scale-down criterion, which revealed that cell aggregates could be generated in 12-well plates, however with a larger size than in a STR. Taking into account the reported higher rAAV production of adherent cells in comparison with single cells for triple-plasmid transfection, HEK293T cell aggregates can possibly surpass single-cell suspension in space-time rAAV yield. The formation of HEK293T cell aggregates in a STR system offers a promising approach for scaling up and intensifying rAAV production by triple-plasmid transfection, in comparison with traditional 2D scale-up methods.
In-line prediction of viability and viable cell density through machine learning-based soft sensor modeling and an integrated systems approach: An industrially relevant PAT case study.
Suman S, Murr M, Crowe J … +5 more , Holt S, Morris J, Yongky A, McElearney K, Bolton G
Biotechnol Prog · 2025 · PMID 39846513 · Publisher ↗
The biopharmaceutical industry is shifting toward employing digital analytical tools for improved understanding of systems biology data and production of quality products. The implementation of these technologies can str...
The biopharmaceutical industry is shifting toward employing digital analytical tools for improved understanding of systems biology data and production of quality products. The implementation of these technologies can streamline the manufacturing process by enabling faster responses, reducing manual measurements, and building continuous and automated capabilities. This study discusses the use of soft sensor models for prediction of viability and viable cell density (VCD) in CHO cell culture processes by using in-line optical density and permittivity sensors. A significant innovation of this study is the development of a simplified empirical model and adoption of an integrated systems approach for in-line viability prediction. The initial evaluation of this viability model demonstrated promising accuracy with 96% of the residuals within a ±5% error limit and a Final Day mean absolute percentage error of ≤5% across various scales and process conditions. This model was integrated with a VCD prediction model utilizing Gaussian Process Regressor with Matern Kernel (nu = 0.5), selected from over a hundred advanced machine learning techniques. This VCD prediction model had an R of 0.92 with 89% predictions within ±10% error and significantly outperformed the commonly used partial least squares regression models. The results validated the use of these models for real-time in-line prediction of viability and VCD and highlighted the potential to substantially reduce reliance on labor-intensive discrete offline measurements. The integration of these innovative technologies aligns with regulatory guidelines and establishes a foundation for further advancements in the biomanufacturing industry, promising improved process control, efficiency, and compliance with quality standards.
Process intensification of the baculovirus expression vector system using a perfusion process with a low multiplicity of infection at high cell concentrations.
Altenburg JJ, Juarez-Garza BE, van Keimpema J … +5 more , van Oosten L, Pijlman GP, van Oers MM, Wijffels RH, Martens DE
Biotechnol Prog · 2025 · PMID 39846509 · Full text
The emergence of new viruses and the spread of existing pathogens necessitate efficient vaccine production methods. The baculovirus expression vector system (BEVS) is an efficient and scalable system for subunit and viru...
The emergence of new viruses and the spread of existing pathogens necessitate efficient vaccine production methods. The baculovirus expression vector system (BEVS) is an efficient and scalable system for subunit and virus-like particle vaccine production and gene therapy vectors. However, current production processes are often limited to low cell concentrations (1-4 × 10 cells/mL) in fed-batch mode. To improve the volumetric productivity of the BEVS, a medium exchange strategy was investigated. Screening experiments were performed to test baculovirus (expressing green fluorescent protein; GFP) infection and productivity of insect cell cultures infected at high cell concentration (1-2 × 10 cells/mL), showing that infection at high cell concentrations was possible with medium exchange. Next, duplicate perfusion runs with baculovirus infection were performed using a cell concentration upon infection (CCI) of 1.2 × 10 cells/mL and a multiplicity of infection (MOI) of 0.01, reaching a maximum viable cell concentration of 2.8 × 10 cells/mL and a maximum GFP production of 263 mg/L. The volumetric productivity of these perfusion runs was 4.8 times higher than for reference batch processes with a CCI of 3 × 10 cells/mL and an MOI of 1. These results demonstrate that process intensification can be achieved for the BEVS by implementing perfusion, resulting in a higher volumetric productivity.
Highly iterated palindrome 1 sequence improves Synechococcus sp. PCC 7002 transformation efficiencies in a homology- and methylation-dependent manner.
Kamoku C, Nielsen DR
Biotechnol Prog · 2025 · PMID 39846505 · Publisher ↗
The ability to precisely engineer cyanobacterial metabolism first requires the ability to efficiently deliver engineered DNA constructs. Here, we investigate how natural transformation efficiencies in Synechococcus sp. P...
The ability to precisely engineer cyanobacterial metabolism first requires the ability to efficiently deliver engineered DNA constructs. Here, we investigate how natural transformation efficiencies in Synechococcus sp. PCC 7002 can be greatly improved by leveraging the native and abundant cyanobacterial Highly Iterated Palindrome 1 (HIP1) sequence. While including at least one homologous HIP1 site within the homology arms of an integrating plasmid increased integration efficiency by up to 7-fold, methylation of those sites by HIP1 methyltransferase (encoded by slr0214 from Synechococcus sp. PCC 6803) boosted this to greater than a 100-fold improvement overall. Non-homologous HIP1 sites also improved transformation efficiencies of both integrating and replicating episomal plasmids (by up to 60- and 9-fold, respectively), but only if methylated. The collective data further reveal that HIP1 does not function as part of a native restriction enzyme system in PCC 7002, but rather may improve transformation efficiency via an alternative mechanism(s), occurring prior to and/or during homologous recombination. Future studies are needed, however, to more clearly elucidate the specific role of HIP1 during natural transformation of cyanobacteria.
Insights into the morphology-productivity relationship of filamentous fungi through small-scale cultivation and automated microscopy of Thermothelomyces thermophilus.
Rohr K, Geinitz B, Seiffarth J … +7 more , Anbarani A, Bernauer S, Moch M, Tenhaef J, Wiechert W, Nöh K, Oldiges M
Biotechnol Prog · 2025 · PMID 39846493 · Full text
Filamentous fungi are a cornerstone in the biotechnological production of enzymes, proteins, and organic acids. However, challenges in understanding and controlling the relationship between morphology and productivity ca...
Filamentous fungi are a cornerstone in the biotechnological production of enzymes, proteins, and organic acids. However, challenges in understanding and controlling the relationship between morphology and productivity can limit their application. This study addresses these challenges using Thermothelomyces thermophilus, a promising thermophilic fungus known for the production of thermostable enzymes. We investigated the effects of environmental conditions on fungal morphology and enzyme production using a combination of microbioreactor cultivation, automated liquid handling, and automated microscopy. Specifically, batch and fed batch cultivations were performed at different pH levels and glucose feeding rates to study their effects on secretory phytase production, fungal growth, and morphology. Results from batch cultivations revealed a two-fold higher phytase activity at pH 5.5 compared to pH 6.5, with notably smaller fungal fragments at the end of cultivation. Conversely, fed batch cultivations at a feeding rate of 1 g (l h) glucose showed a 1.6-fold higher enzyme activity at pH 5.5, accompanied by much larger fungal aggregates throughout the feeding phase. These findings suggest that large aggregates are associated with high productivity; however, their breakdown further enhances enzyme release, increasing activity in the supernatant. This study not only provides insights on the morphology-productivity relationship of T. thermophilus, but also demonstrates the efficacy of integrating microbioreactors with automated microscopy. This methodology represents a significant advance in the field of fungal biotechnology, paving the way for more efficient industrial bioprocesses.
In silico optimization of a challenging bispecific antibody chromatography step.
Bencze Z, Hahn T, Kornmann H … +2 more , Graf P, Trunzer T
Biotechnol Prog · 2025 · PMID 39846490 · Publisher ↗
Mechanistic modeling of chromatographic steps is an effective tool in biopharma process development that enhances process understanding and accelerates optimization efforts and subsequent risk assessment. A relatively ne...
Mechanistic modeling of chromatographic steps is an effective tool in biopharma process development that enhances process understanding and accelerates optimization efforts and subsequent risk assessment. A relatively new model for ion exchange chromatography is the colloidal particle adsorption (CPA) formalism, which promises improved separation of material and molecule-specific parameters. This case study demonstrates a straightforward CPA modeling workflow to describe an ion exchange chromatography polishing step of a knobs-into-holes construct bispecific antibody molecule. An adapted Yamamoto method was used to calculate charge and equilibrium parameters at three pH values. The remaining model parameters, binding kinetics, and effective mass transfer coefficients were determined via inverse fitting. The model was created from six experiments in total, tested on model parameter uncertainty, and evaluated on its power to predict changes in the biomolecule's retention behavior when variations in elution salt concentration occur. Finally, a three-step-gradient experiment was optimized, separating the desired bispecific antibody from its low and high molecular weight impurities, achieving a monomer yield of 68% and purity of 96%. Testing the model against a different load composition demonstrated its ability to extrapolate. An in silico one-factor-at-time and two-parameter screening of the optimized method identified the salt concentration to elute weaker binding impurities as a critical process attribute, while deviations in the buffer pH had a minor influence.
A method for facile production of variable lymphocyte receptors using SHuffle Escherichia coli.
Appelt EA, Thoden JB, Dancy C … +7 more , Bachmeier HD, Gehrke SA, Staffenson MA, Rayment I, Katt ME, Holden HM, Shusta EV
Biotechnol Prog · 2025 · PMID 39846486 · Full text
Variable lymphocyte receptors (VLRs) are the antigen receptors of jawless vertebrates such as lamprey. VLRs are of growing biotechnological interest for their ability to bind certain antigenic targets with higher affinit...
Variable lymphocyte receptors (VLRs) are the antigen receptors of jawless vertebrates such as lamprey. VLRs are of growing biotechnological interest for their ability to bind certain antigenic targets with higher affinity than traditional immunoglobulins. However, VLRs are disulfide-bonded proteins that are often challenging to produce requiring genetic modifications, fusion partners, non-scalable host cell lines or inclusion body formation and refolding. As a potential VLR expression platform option, the SHuffle Escherichia coli strain has been genetically altered to allow cytoplasmic disulfide bond formation by mutations to thioredoxin reductase (trxB) and glutathione reductase (gor) to create an oxidative cytoplasm. Furthermore, the SHuffle strain expresses disulfide bond isomerase DsbC in the cytoplasm to promote correct disulfide bond pairing. Here, we demonstrate that the SHuffle strain can produce high yield VLRs with titers ranging from 2 to 32 mg of VLR per liter of SHuffle culture. Three VLRs (P1C10, RBC36, VLRA.R2.1) were expressed in SHuffle E. coli and the products were compared directly to those generated using the Rosetta E. coli strain. All VLRs were validated for correct sequence, purity, and activity. For all VLRs, SHuffle E. coli produced 2-9 times more soluble VLRs than Rosetta E. coli. Furthermore, the soluble protein fraction was 2-6 times greater in SHuffle E. coli than Rosetta E. coli for all VLRs. Overall, these results suggest that the E. coli SHuffle strain is a convenient and effective expression system for producing large amounts of VLRs.
Stipulations of cell and gene therapy and the ties to biomanufacturing.
Allisha J, Das J, Dunnigan T … +2 more , Sharfstein ST, Datta P
Biotechnol Prog · 2025 · PMID 39846483 · Publisher ↗
Cell and gene therapy (CGT) products are emerging and innovative biopharmaceuticals that hold promise for treating diseases that are otherwise beyond the scope of conventional medicines. The evolution of CGT from a resea...
Cell and gene therapy (CGT) products are emerging and innovative biopharmaceuticals that hold promise for treating diseases that are otherwise beyond the scope of conventional medicines. The evolution of CGT from a research idea to a promising therapeutic product is due to the complementary advancements across various scientific disciplines. First, the innovations and advancements in gene editing and delivery technology have provided fundamental tools to manipulate genes and cells for therapeutic pursuits. Second, advancements in applied and translational research, including how clinical trials are designed, performed, evaluated, and analyzed, have transformed the technology into a potential therapeutic product. Third, advancements in scaling up the production of CGT products have been critical in delivering the product for preclinical studies, clinical trials, and approved treatments. In parallel, regulatory requirements have continuously evolved, with lessons learned from translational studies and biomanufacturing. These combined efforts have transformed CGT products from a promising concept into a reality with the potential to treat a wide range of diseases. However, continued R&D and regulatory oversight are crucial to further improve the safety, efficacy, and accessibility of CGT products.
A design space for the filtration of challenging monoclonal antibodies using Planova™ S20N, a new regenerated cellulose virus removal filter.
Inoue A, Murakami S, Futamura A … +2 more , Watanabe N, Masuda Y
Biotechnol Prog · 2025 · PMID 39846448 · Full text
Virus removal by filtration is a crucial step in ensuring the safety of therapeutic antibodies and other biopharmaceutical products by mitigating the risk of endogenous and adventitious viral contamination. However, ther...
Virus removal by filtration is a crucial step in ensuring the safety of therapeutic antibodies and other biopharmaceutical products by mitigating the risk of endogenous and adventitious viral contamination. However, there are monoclonal antibodies (mAb) that are difficult to filter effectively using virus removal filters (i.e., challenging mAbs), necessitating the creation of guidelines for designing suitable filtration conditions for these challenging molecules. This study presents a filtration design space for filtration conditions using a new regenerated cellulose membrane filter with a representative challenging mAb. The filter demonstrated that filtration throughput is adaptable across a wide range of conditions for low to medium mAb concentrations, indicating its suitability for introduction into platform processes for related biopharmaceutical products.
Sensitivity of bulk electrical impedance spectroscopy (bio-capacitance) probes to cell and culture properties: Study on CHO cell cultures.
Salimi E, Absalan S, Robitaille J … +4 more , Montes J, Butler M, Thomson D, Bridges G
Biotechnol Prog · 2025 · PMID 39723484 · Full text
Bulk electrical impedance spectroscopy (bio-capacitance) probes, hold significant promise for real-time cell monitoring in bioprocesses. Focusing on Chinese hamster ovary (CHO) cells, we present a sensitivity analysis fr...
Bulk electrical impedance spectroscopy (bio-capacitance) probes, hold significant promise for real-time cell monitoring in bioprocesses. Focusing on Chinese hamster ovary (CHO) cells, we present a sensitivity analysis framework to assess the impact of cell and culture properties on the complex permittivity spectrum, ε, and its associated parameters, permittivity increment, Δε, critical frequency, f, and Cole-Cole parameter, α, measured by bio-capacitance probes. Our sensitivity analysis showed that Δε is highly sensitive to cell size and concentration, making it suitable for estimating biovolume during the exponential growth phase, whereas f provides information about cumulative changes in cell size, membrane permittivity, and cytoplasm conductivity during the transition to death phase. The analysis indicated that specific information about cell membrane permittivity or internal conductivity cannot be extracted from ε spectrum. Based on the sensitivity analysis, we proposed two alternative parameters for monitoring cells in bioprocesses: Δε and Δε/Δε, using measurements at 300 kHz, 1 MHz, and 10 MHz. Δε is suitable for estimating viable cell density during the exponential growth phase due to its lower sensitivity to cell size. Δε/Δε can replace f due to similar sensitivities to cell size and dielectric properties. These frequencies are within most bio-capacitance probes' optimal operation range, eliminating the need for low-frequency electrode polarization and high-frequency stray capacitances corrections. Experimental measurements on CHO cells confirmed the results of sensitivity analysis.
Online monitored characterization of Phocaeicola vulgatus for organic acid production using anaerobic microtiter plate cultivations.
Keitel L, Schick B, Pohen G … +2 more , Yordanov S, Büchs J
Biotechnol Prog · 2025 · PMID 39704382 · Full text
Phocaeicola vulgatus (formerly Bacteroides vulgatus), an anaerobic gut bacterium, produces several organic acids. Research on P. vulgatus is still in its infancy. However, a detailed understanding of P. vulgatus growth a...
Phocaeicola vulgatus (formerly Bacteroides vulgatus), an anaerobic gut bacterium, produces several organic acids. Research on P. vulgatus is still in its infancy. However, a detailed understanding of P. vulgatus growth and metabolism is essential for its assessment as an organic acid producer. Media variations, including different initial glucose and NHCl concentrations and osmolalities, are significant means to yield higher organic acid titers. Furthermore, examining different nitrogen and carbon sources is important to evaluate the potential of P. vulgatus for growth on renewable resources. Cultivations were performed in an in-house built device for anaerobic online-monitoring of fluorescence and scattered light in microtiter plates. Results revealed that the highest organic acid concentrations were reached while using galactose, glucose, or xylose as a carbon source, high osmolalities, and 0.25 g L NHCl. In addition, the organic acid composition changed with changing carbon and nitrogen sources. P. vulgatus was successfully further characterized, thereby contributing to a faster characterization of other anaerobic strains and paving the way for anaerobic organic acid production.
High-sensitivity real-time monitoring of pH and respiration activity unveils metabolic dynamics in shake flask cultures.
Sarikaya B, Günster K, Grebe LA … +4 more , Forsten E, Hürter K, Büchs J, Magnus J
Biotechnol Prog · 2025 · PMID 39668734 · Full text
Erlenmeyer shake flasks are widely used during the first steps of bioprocess development. Despite their broad application in academia and industry, shake flasks usually lack standardized and user-friendly online monitori...
Erlenmeyer shake flasks are widely used during the first steps of bioprocess development. Despite their broad application in academia and industry, shake flasks usually lack standardized and user-friendly online monitoring techniques. In this work, the pH and Respiratory Activity MOnitoring System (pH-RAMOS) for the non-invasive online measurement of the oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and pH in up to eight parallel shake flasks under sterile conditions is presented. The OTR and CTR are quasi-continuously measured in the headspace of the shake flasks using dedicated oxygen and carbon dioxide sensors, enabling precise respiratory quotient (RQ) evaluation. Self-adhesive pH sensor spots are used for the high-frequent real-time pH monitoring of the culture. These prototype pH sensor spots stand out due to their simple sterilizability and subsequent one-point calibration in the cultivation medium. The long-term stability of the pH sensor spots was assessed in a 28-day long abiotic experiment. The novel pH-RAMOS was validated with different eukaryotic and prokaryotic microorganisms, such as Ogataea polymorpha, Ustilago trichophora, and Vibrio natriegens. The combination of online OTR, CTR, RQ, and pH signals allowed for identifying various metabolic phenomena, such as oxygen limitations, substrate limitations, diauxies, and the production or consumption of specific compounds, based on their degree of reduction or change of pH. The high-frequent and sensitive pH-monitoring was particularly advantageous for registering subtle and transient metabolic phenomena.
Electrospun gelatin/hyaluronic acid nanofibers as a platform for uric acid delivery to neural tissue.
Street RM, Kung FH, Beringer LT … +3 more , Amchin DB, Firestein BL, Schauer CL
Biotechnol Prog · 2025 · PMID 39523662 · Publisher ↗
Uric acid (UA) is an antioxidant that has been reported to be a neuroprotective compound for injuries and diseases, and specifically, diseases of the central nervous system. However, uric acid is highly insoluble in aque...
Uric acid (UA) is an antioxidant that has been reported to be a neuroprotective compound for injuries and diseases, and specifically, diseases of the central nervous system. However, uric acid is highly insoluble in aqueous solutions, and high levels in the serum lead to gout, which limits its use in humans. Here, we develop a novel drug delivery platform that will release uric acid in a sustained manner for application to neural tissue. We demonstrate that one-step incorporation of UA into an electrospun gelatin/hyaluronic acid nanofiber mat results in controlled release of UA in culture medium. Taking a unique approach, we made solutions of 12% gelatin and 1% hyaluronic acid in a formic acid solvent and added UA for production of nanofiber mats. We then dehydrothermally crosslinked the mats and tested for release of UA into physiological cell culture medium. To test whether the mats have any detrimental effects on healthy nervous system tissue, we cultured spinal cord explants on the mats extended and assessed extensions from the explants. We observed that comparable numbers and lengths of dendrites are extended from the spinal cord tissue, regardless of the amount UA content in the mats. Our results suggest that electrospun gelatin/hyaluronic acid nanofibers can be used as a platform for sustained uric acid delivery to neural tissue without detrimental effects.
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