Biotechnology Progress最新文献_第3页

pH and conductivity transients during elution of IgG from protein A columns.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-18 DOI: 10.1002/btpr.3534

Rainer Hahn, Lukas Berger, Jürgen Beck, Giorgio Carta

{"title":"pH and conductivity transients during elution of IgG from protein A columns.","authors":"Rainer Hahn, Lukas Berger, Jürgen Beck, Giorgio Carta","doi":"10.1002/btpr.3534","DOIUrl":"https://doi.org/10.1002/btpr.3534","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3534"},"PeriodicalIF":2.5,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439855","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

The combination of oleic acid, linoleic acid, palmitoleic acid, and α-linolenic acid promoted the expansion of NK-92 cells in vitro.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-18 DOI: 10.1002/btpr.70000

Shumin Zhang, Huimin Huang, Jingwei Zhang, Yuanyuan Zhao, Wen-Song Tan, Haibo Cai

{"title":"The combination of oleic acid, linoleic acid, palmitoleic acid, and α-linolenic acid promoted the expansion of NK-92 cells in vitro.","authors":"Shumin Zhang, Huimin Huang, Jingwei Zhang, Yuanyuan Zhao, Wen-Song Tan, Haibo Cai","doi":"10.1002/btpr.70000","DOIUrl":"https://doi.org/10.1002/btpr.70000","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70000"},"PeriodicalIF":2.5,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439857","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

Flocculation-based clarification for production of protein therapeutics in Pichia pastoris: Recombinant human serum albumin as a case study.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-18 DOI: 10.1002/btpr.70001

Vishwanath Hebbi, Jashwant Kumar, Anurag S Rathore

{"title":"Flocculation-based clarification for production of protein therapeutics in Pichia pastoris: Recombinant human serum albumin as a case study.","authors":"Vishwanath Hebbi, Jashwant Kumar, Anurag S Rathore","doi":"10.1002/btpr.70001","DOIUrl":"https://doi.org/10.1002/btpr.70001","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70001"},"PeriodicalIF":2.5,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439850","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

Mechanistic insights into hypoxia-induced metabolic reprogramming in colorectal cancer through genome-scale modeling.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-18 DOI: 10.1002/btpr.70002

Sonal Omer, Subasree Sridhar, D Karunagaran, G K Suraishkumar

{"title":"Mechanistic insights into hypoxia-induced metabolic reprogramming in colorectal cancer through genome-scale modeling.","authors":"Sonal Omer, Subasree Sridhar, D Karunagaran, G K Suraishkumar","doi":"10.1002/btpr.70002","DOIUrl":"https://doi.org/10.1002/btpr.70002","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70002"},"PeriodicalIF":2.5,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439853","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

Efficient fermentative production of lactodifucotetraose by controlling sequential glycosyltransferase reactions in Escherichia coli.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-06 DOI: 10.1002/btpr.70010

Shu Moriyama, Tomotoshi Sugita, Makoto Yamashita

{"title":"Efficient fermentative production of lactodifucotetraose by controlling sequential glycosyltransferase reactions in Escherichia coli.","authors":"Shu Moriyama, Tomotoshi Sugita, Makoto Yamashita","doi":"10.1002/btpr.70010","DOIUrl":"https://doi.org/10.1002/btpr.70010","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70010"},"PeriodicalIF":2.5,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254546","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

Rapid bioreactor process optimization and scale-up for production of a measles vector COVID-19 vaccine candidate.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-06 DOI: 10.1002/btpr.70004

David C Hesley, Daniel Spatafore, Jillian Shingler, Joshua P McNeely, Rachel Thompson, Matthew C Troutman, Elise K B Baron, Megan Sabia, Christopher H Lee, Kristin Ploeger, James M Wagner

{"title":"Rapid bioreactor process optimization and scale-up for production of a measles vector COVID-19 vaccine candidate.","authors":"David C Hesley, Daniel Spatafore, Jillian Shingler, Joshua P McNeely, Rachel Thompson, Matthew C Troutman, Elise K B Baron, Megan Sabia, Christopher H Lee, Kristin Ploeger, James M Wagner","doi":"10.1002/btpr.70004","DOIUrl":"https://doi.org/10.1002/btpr.70004","url":null,"abstract":"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.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70004"},"PeriodicalIF":2.5,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254547","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

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