Biotechnology Progress最新文献_第4页

Pre-folding purification procedures for inclusion body-derived non-tagged cationic recombinant proteins with multiple disulfide bonds for efficient refolding.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-25 DOI: 10.1002/btpr.3532

Shuichiro Kimura, Wataru Yamamoto, Ai Miyamoto, Koreyoshi Imamura, Junichiro Futami

{"title":"Pre-folding purification procedures for inclusion body-derived non-tagged cationic recombinant proteins with multiple disulfide bonds for efficient refolding.","authors":"Shuichiro Kimura, Wataru Yamamoto, Ai Miyamoto, Koreyoshi Imamura, Junichiro Futami","doi":"10.1002/btpr.3532","DOIUrl":"https://doi.org/10.1002/btpr.3532","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3532"},"PeriodicalIF":2.5,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143045604","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Harnessing cell aggregates for enhanced adeno-associated virus manufacturing: Cultivation strategies and scale-up considerations.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3522

Brian Ladd, Torbjörn Gräslund, Véronique Chotteau

{"title":"Harnessing cell aggregates for enhanced adeno-associated virus manufacturing: Cultivation strategies and scale-up considerations.","authors":"Brian Ladd, Torbjörn Gräslund, Véronique Chotteau","doi":"10.1002/btpr.3522","DOIUrl":"https://doi.org/10.1002/btpr.3522","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3522"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022042","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

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.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3520

Sneha Suman, Michaela Murr, Jacob Crowe, Spencer Holt, Jakob Morris, Andrew Yongky, Kyle McElearney, Glen Bolton

{"title":"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.","authors":"Sneha Suman, Michaela Murr, Jacob Crowe, Spencer Holt, Jakob Morris, Andrew Yongky, Kyle McElearney, Glen Bolton","doi":"10.1002/btpr.3520","DOIUrl":"https://doi.org/10.1002/btpr.3520","url":null,"abstract":"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 R2 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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3520"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022047","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Characterization of large transgene integrations in Chinese hamster ovary cells using a bioengineered mammalian transposase.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3524

Nicolas Marx, Anna-Maria Otte, Klaus Leitner, Rehmadanta Sitepu, Thomas Berger, Veronika Schäpertöns, Christian G Huber, Quan Zhu, Sandeep Nema, Joseph J Higgins, Nicole Borth

{"title":"Characterization of large transgene integrations in Chinese hamster ovary cells using a bioengineered mammalian transposase.","authors":"Nicolas Marx, Anna-Maria Otte, Klaus Leitner, Rehmadanta Sitepu, Thomas Berger, Veronika Schäpertöns, Christian G Huber, Quan Zhu, Sandeep Nema, Joseph J Higgins, Nicole Borth","doi":"10.1002/btpr.3524","DOIUrl":"https://doi.org/10.1002/btpr.3524","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3524"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Engineering a high-throughput clone for industrial-scale production of long-acting GLP-1 analogue with retained bio-efficacy.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3529

Praveen Kumar Reddy J, Murali Tummuru, Kunka Mohanram Ramkumar

{"title":"Engineering a high-throughput clone for industrial-scale production of long-acting GLP-1 analogue with retained bio-efficacy.","authors":"Praveen Kumar Reddy J, Murali Tummuru, Kunka Mohanram Ramkumar","doi":"10.1002/btpr.3529","DOIUrl":"https://doi.org/10.1002/btpr.3529","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3529"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

In silico optimization of a challenging bispecific antibody chromatography step.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3531

Zsófia Bencze, Tobias Hahn, Henri Kornmann, Pia Graf, Tatjana Trunzer

{"title":"In silico optimization of a challenging bispecific antibody chromatography step.","authors":"Zsófia Bencze, Tobias Hahn, Henri Kornmann, Pia Graf, Tatjana Trunzer","doi":"10.1002/btpr.3531","DOIUrl":"https://doi.org/10.1002/btpr.3531","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3531"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A method for facile production of variable lymphocyte receptors using SHuffle Escherichia coli.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3530

Elizabeth A Appelt, James B Thoden, Candis Dancy, Hannah D Bachmeier, Seth A Gehrke, Melanie A Staffenson, Ivan Rayment, Moriah E Katt, Hazel M Holden, Eric V Shusta

{"title":"A method for facile production of variable lymphocyte receptors using SHuffle Escherichia coli.","authors":"Elizabeth A Appelt, James B Thoden, Candis Dancy, Hannah D Bachmeier, Seth A Gehrke, Melanie A Staffenson, Ivan Rayment, Moriah E Katt, Hazel M Holden, Eric V Shusta","doi":"10.1002/btpr.3530","DOIUrl":"10.1002/btpr.3530","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3530"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Coupling high-throughput and modeling approaches to streamline early-stage process development for biologics.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3523

John P Welsh, Scott H Altern, Jessica Y Lyall, Sean Burgess, Michael A Rauscher, Abraham M Lenhoff, Steven M Cramer, Chris Williams

{"title":"Coupling high-throughput and modeling approaches to streamline early-stage process development for biologics.","authors":"John P Welsh, Scott H Altern, Jessica Y Lyall, Sean Burgess, Michael A Rauscher, Abraham M Lenhoff, Steven M Cramer, Chris Williams","doi":"10.1002/btpr.3523","DOIUrl":"https://doi.org/10.1002/btpr.3523","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3523"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Insights into the morphology-productivity relationship of filamentous fungi through small-scale cultivation and automated microscopy of Thermothelomyces thermophilus.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3528

Katja Rohr, Bertram Geinitz, Johannes Seiffarth, Aydin Anbarani, Sören Bernauer, Matthias Moch, Julia Tenhaef, Wolfgang Wiechert, Katharina Nöh, Marco Oldiges

{"title":"Insights into the morphology-productivity relationship of filamentous fungi through small-scale cultivation and automated microscopy of Thermothelomyces thermophilus.","authors":"Katja Rohr, Bertram Geinitz, Johannes Seiffarth, Aydin Anbarani, Sören Bernauer, Matthias Moch, Julia Tenhaef, Wolfgang Wiechert, Katharina Nöh, Marco Oldiges","doi":"10.1002/btpr.3528","DOIUrl":"https://doi.org/10.1002/btpr.3528","url":null,"abstract":"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)-1 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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3528"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Process intensification of the baculovirus expression vector system using a perfusion process with a low multiplicity of infection at high cell concentrations.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3527

Jort J Altenburg, Brenda E Juarez-Garza, Jelle van Keimpema, Linda van Oosten, Gorben P Pijlman, Monique M van Oers, René H Wijffels, Dirk E Martens

{"title":"Process intensification of the baculovirus expression vector system using a perfusion process with a low multiplicity of infection at high cell concentrations.","authors":"Jort J Altenburg, Brenda E Juarez-Garza, Jelle van Keimpema, Linda van Oosten, Gorben P Pijlman, Monique M van Oers, René H Wijffels, Dirk E Martens","doi":"10.1002/btpr.3527","DOIUrl":"https://doi.org/10.1002/btpr.3527","url":null,"abstract":"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 × 106 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 × 107 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 × 107 cells/mL and a multiplicity of infection (MOI) of 0.01, reaching a maximum viable cell concentration of 2.8 × 107 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 × 106 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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3527"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0