Biotechnology Progress最新文献_第8页

Improving outcomes in intensified processing via optimization of the cell line development workflow 通过优化细胞系开发工作流程，改善强化处理的结果。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-19 DOI: 10.1002/btpr.70003

Vincent Balassi, Mary Otto, Corey Kretzmer, Amber Petersen, Channing McLaurin, Jana Mahadevan, Jason Gustin, Trissa Borgschulte, David Razafsky

{"title":"Improving outcomes in intensified processing via optimization of the cell line development workflow","authors":"Vincent Balassi, Mary Otto, Corey Kretzmer, Amber Petersen, Channing McLaurin, Jana Mahadevan, Jason Gustin, Trissa Borgschulte, David Razafsky","doi":"10.1002/btpr.70003","DOIUrl":"10.1002/btpr.70003","url":null,"abstract":"As the industry continues to explore the benefits of continuous and intensified manufacturing, it is important to assure that the cell line development (CLD) workflows in practice today are well suited to generate clones that meet the unique challenges associated with these processes. Most cell lines used in intensified processes are currently developed using traditional fed-batch CLD workflows followed by adaptation of these cell lines to perfusion processes. This method maybe suboptimal as fed-batch CLD workflows select clones which produce high volumetric titers irrespective of cell growth rate and specific productivity (qP). Although sufficient for fed-batch processes, performance of cells derived from this traditional CLD workflow may not be maintained in perfusion processes, where an intricate balance of performance parameters is needed. Until now, a thorough investigation into the effect of the CLD workflow on top clone performance in perfusion processes has not been conducted. Here, we show how the CLD workflow impacts cell performance in both fed-batch and perfusion processes, emphasizing the advantages of adopting a perfusion-specific CLD workflow which includes the use of medium specially designed for expansion and production in a perfusion setting, scale-down models which more accurately simulate perfusion process, and the adoption of perfusion-specific cell line selection criteria. Together, this results in the development of more efficient cell lines, fit for continuous and intensified processing.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"41 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.70003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143448015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enhancing real-time cell culture process monitoring through the integration of advanced machine learning techniques: A comparative analysis of Raman and capacitance spectroscopies 通过集成先进的机器学习技术增强实时细胞培养过程监测：拉曼光谱和电容光谱的比较分析。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-19 DOI: 10.1002/btpr.70013

Feng Xu, Nuno Pinto, George Zhou, Sanjeev Ahuja

{"title":"Enhancing real-time cell culture process monitoring through the integration of advanced machine learning techniques: A comparative analysis of Raman and capacitance spectroscopies","authors":"Feng Xu, Nuno Pinto, George Zhou, Sanjeev Ahuja","doi":"10.1002/btpr.70013","DOIUrl":"10.1002/btpr.70013","url":null,"abstract":"Machine learning (ML) techniques have emerged as an important tool improving the capabilities of online process monitoring and control in cell culture process for biopharmaceutical manufacturing. A variety of advanced ML algorithms have been evaluated in this study for cell growth monitoring using spectroscopic tools, including Raman and capacitance spectroscopies. While viable cell density can be monitored real-time in the cell culture process, online monitoring of cell viability has not been well established. A thorough comparison between the advanced ML techniques and traditional linear regression method (e.g., Partial Least Square regression) reveals a significant improvement in accuracy with the leading ML algorithms (e.g., 31.7% with Random Forest regressor), addressing the unmet need of continuous monitoring viability in a real time fashion. Both Raman and capacitance spectroscopies have demonstrated success in viability monitoring, with Raman exhibiting superior accuracy compared to capacitance. In addition, the developed methods have shown better accuracy in a relatively higher viability range (>90%), suggesting a great potential for early fault detection during cell culture manufacturing. Further study using ML techniques for VCD monitoring also showed an increased accuracy (27.3% with Raman spectroscopy) compared to traditional linear modeling. The successful integration of ML techniques not only amplifies the potential of process monitoring but also makes possible the development of advanced process control strategies for optimized operations and maximized efficiency.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"41 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143448013","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 production of succinate with more CO2 fixation reactions facilitated by RuBisCO-based engineered Escherichia coli 基于rubisco的工程大肠杆菌促进了更多CO2固定反应的琥珀酸盐生产。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-02-19 DOI: 10.1002/btpr.70015

Xiuyuan Zhou, Linqing Li, Shengjie Sun, Peng Xiong, Xiutao Liu

{"title":"The production of succinate with more CO2 fixation reactions facilitated by RuBisCO-based engineered Escherichia coli","authors":"Xiuyuan Zhou, Linqing Li, Shengjie Sun, Peng Xiong, Xiutao Liu","doi":"10.1002/btpr.70015","DOIUrl":"10.1002/btpr.70015","url":null,"abstract":"Redesigning metabolic pathways to enhance the efficiency of carbon fixation during chemical biosynthesis is a promising approach for achieving cleaner and greener production of multi-carbon compounds. In this study, we established a model of cell growth in Escherichia coli that is dependent on the RuBisCO-Prk pathway by regulating its central metabolism. This rewiring ensures that growth depends on RuBisCO's carboxylation, allowing heterotrophic growth to rely on carbon fixation. This model was verified by detecting the growth curve, and it was used to screen four RuBisCO genes, of which the gene from Rhodospirillum rubrum ATCC 11170 serves as a growth advantage for E.coli. In addition, this model was applied to construct an efficient succinate biosynthetic pathway that can produce two moles of succinate from one mole of xylose and three moles of CO2. Compared to conventional succinate biosynthesis, this strategy has a CO2 fixation capacity that is 1.5 times greater. Furthermore, to optimize succinate production, various approaches were employed, including the optimization of key enzymes, substrate transport, and the supply of inorganic carbon. The resulting strain was capable of producing succinate at a level of 2.09 ± 0.14 g/L, which is nearly 22.4 times that of the original strain. In conclusion, this study was developed for the production of two moles of succinate by implementing three moles of carbon fixation reactions and demonstrated the feasibility of various optimization strategies in biological carbon fixation.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"41 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143447639","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

pH and conductivity transients during elution of IgG from protein A columns 从A蛋白柱中洗脱IgG时的pH值和电导率变化。

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":"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":"41 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3534","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":"OA","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 油酸、亚油酸、棕榈油酸和α-亚麻酸联合作用可促进NK-92细胞体外扩增。

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":"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":"41 3","pages":""},"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 基于絮凝澄清的毕赤酵母蛋白治疗剂生产：重组人血清白蛋白为例研究。

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":"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":"41 3","pages":""},"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 通过基因组尺度模型对低氧诱导的结直肠癌代谢重编程的机制见解。

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":"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":"41 3","pages":""},"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 通过控制顺序糖基转移酶反应在大肠杆菌中有效发酵生产乳二糖四糖。

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":"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":"41 3","pages":""},"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 快速优化生物反应器工艺并扩大生产麻疹媒介COVID-19候选疫苗。

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 III, 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 III, 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":"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":"41 3","pages":""},"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 包涵体衍生的具有多个二硫键的非标记阳离子重组蛋白的折叠前纯化程序，用于高效的再折叠。

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":"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":"41 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3532","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0