Biotechnology Progress最新文献

Automation of an integrated micro-scale platform for monoclonal antibody process development by incorporation of a depth filter mimic. 单克隆抗体工艺开发集成微尺度平台的自动化，采用深度过滤器模拟。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-10-03 DOI: 10.1002/btpr.70077

Paras Sharma, Petra Sebastian, Lars Robbel, Michael Schmitt, Daniel G Bracewell

{"title":"Automation of an integrated micro-scale platform for monoclonal antibody process development by incorporation of a depth filter mimic.","authors":"Paras Sharma, Petra Sebastian, Lars Robbel, Michael Schmitt, Daniel G Bracewell","doi":"10.1002/btpr.70077","DOIUrl":"https://doi.org/10.1002/btpr.70077","url":null,"abstract":"High throughput process development (HTPD) has been widely adopted for efficient development and optimization of chromatographic operations in monoclonal antibody (mAb) purification. However, the integration of non-chromatographic unit operations, particularly depth filtration following protein A chromatography, which is essential for the removal of process- and product-related impurities prior to the ion exchange chromatography (IEX) operations, remains a challenge due to the absence of commercially available micro-scale depth filtration tools. This limits the integration of this unit operation within the purification sequence, restricting the analysis of process interactions and overall process understanding. In this study, a micro-scale HTPD platform was designed and evaluated to enable integration of a depth filtration mimic, Sartobind® Q anion exchange adsorber, within a mAb purification sequence. This was achieved by translating laboratory-scale protocols to the micro-scale using workflow design tools and executed on an automated liquid handling system. Step yields and impurity clearance were assessed to confirm the equivalence of scale-down. The Sartobind® Q membrane achieved effective removal of host cell DNA (hcDNA), while subsequent IEX operations removed host cell proteins (HCPs) and high molecular weight components (HMWC), meeting target product quality specifications. The platform demonstrated robustness across varying impurity profiles, supporting its applicability for diverse process intermediates. Comparative analysis with laboratory-scale operations confirmed the performance and scalability of the micro-scale system, reducing the total run time by greater than 50%. The integrated HTPD platform offers a resource-efficient, scalable approach for comprehensive mAb purification process development and is suitable for developability assessments during early-stage development.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70077"},"PeriodicalIF":2.5,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145211385","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

Functional CRISPR-Cas9 knockout screening of the genetic determinants of human fibroblast migration propensity. 功能性CRISPR-Cas9基因敲除筛选人成纤维细胞迁移倾向的遗传决定因素。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-10-02 DOI: 10.1002/btpr.70076

Antonio Mazzei, Sebastian Martewicz, Ramin Amiri, Meihua Cui, Nicola Elvassore, Camilla Luni

{"title":"Functional CRISPR-Cas9 knockout screening of the genetic determinants of human fibroblast migration propensity.","authors":"Antonio Mazzei, Sebastian Martewicz, Ramin Amiri, Meihua Cui, Nicola Elvassore, Camilla Luni","doi":"10.1002/btpr.70076","DOIUrl":"https://doi.org/10.1002/btpr.70076","url":null,"abstract":"Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as embryonic development or tumor metastasis. Steps involved in cell migration include cell polarization, formation of membrane protrusions at the cell front side and adhesion disassembly at the rear side, and a general cytoskeletal rearrangement. Overall, it is a complex phenomenon at the interface between mechanical forces and biochemical signaling, with cell-specific and context-specific molecular events acting in the process. Here, we focus on human fibroblast migration induced by a biochemical gradient with an approach that connects the identification of molecular players with the actual mechanical function. We show how to screen for genes and miRNAs involved in migration by the direct integration of a high-throughput gene editing method, the CRISPR-Cas9 knockout pool screening, and a well-established functional assay, the transwell migration assay. Moreover, the screening has been performed after an expansion step aiming at the removal of all the essential genes and miRNAs, so as to identify targets related to the cell migratory ability without affecting other major cellular functions. The results confirm known genes involved in migration, but also highlight new candidates. This work establishes a methodological advancement in the use of CRISPR technology for functional screening and represents a resource for candidate genes and miRNAs playing a role in human fibroblast directional migration under biochemical gradient.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70076"},"PeriodicalIF":2.5,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145205469","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

Scale-up of a monoclonal antibody CHO fed-batch production in stirred tank bioreactors: Effect of hydrodynamic conditions and feeding regimen. 搅拌槽生物反应器中单克隆抗体CHO加料批量生产的扩大：流体动力条件和投料方案的影响。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-09-29 DOI: 10.1002/btpr.70073

Lucas Lemire, Sebastian-Juan Reyes, Yves Durocher, Robert Voyer, Olivier Henry, Phuong Lan Pham

{"title":"Scale-up of a monoclonal antibody CHO fed-batch production in stirred tank bioreactors: Effect of hydrodynamic conditions and feeding regimen.","authors":"Lucas Lemire, Sebastian-Juan Reyes, Yves Durocher, Robert Voyer, Olivier Henry, Phuong Lan Pham","doi":"10.1002/btpr.70073","DOIUrl":"https://doi.org/10.1002/btpr.70073","url":null,"abstract":"Key hydrodynamic-related parameters such as volumetric power input (P/V), impeller configuration, aeration strategy, and maximum gas sparge rate, as well as an appropriate feeding strategy, must be carefully selected to improve production yields in bioreactor. In this study, the feeding regimen was found to have an important impact on cell growth and productivity of a cumate-inducible CHO fed-batch cell culture. A low-volume feeding regimen avoided a rapid increase in osmolality, allowing for prolonged cell viability and a 33% increase in volumetric titer compared to the high-volume feeding regimen. Both sparged air and oxygen were used for dissolved oxygen (DO) control, utilizing three levels of airflow rates. An optimum airflow rate of 0.0031 vvm was found to improve cell growth, longevity, and thus final titer. A larger air cap required increased gas flow rates, which led to an earlier cell mortality. Scale-up from 1-L to 10-L bioreactor using constant P/V and air cap volumetric gas flow rate (vvm) allowed for comparable cell growth and productivity. Further investigation of the effect of mixing and aeration was done by maintaining P/V and vvm constant throughout the cell culture, which further improved product titers at 11 days after induction. Our study also demonstrates that keeping a constant volume by removing a culture amount equal to the feed volume added at each sampling event can significantly improve the final volumetric titer. This finding shows the benefit of developing a concentrated feed to reduce the volume increase, which in turn could greatly ease the scale-up task.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70073"},"PeriodicalIF":2.5,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145184491","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

Quantitative variation in the camptothecin produced by diverse endophytic microorganisms of Ophiorrhiza mungos L. 芒戈蛇根不同内生微生物产喜树碱的数量变异。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-09-27 DOI: 10.1002/btpr.70074

Mary Theresa, Arya Nikathil Pradeep, Aswani Ravi, Maya Mathew, Manjusha Premnath, Charuvila T Aravindakumar, Remakanthan Appukuttan, Indu C Nair, Linu Mathew, Sebastian Franz Bender, Marcel G A van der Heijden, Radhakrishnan Edayileveettil Krishnankutty

{"title":"Quantitative variation in the camptothecin produced by diverse endophytic microorganisms of Ophiorrhiza mungos L.","authors":"Mary Theresa, Arya Nikathil Pradeep, Aswani Ravi, Maya Mathew, Manjusha Premnath, Charuvila T Aravindakumar, Remakanthan Appukuttan, Indu C Nair, Linu Mathew, Sebastian Franz Bender, Marcel G A van der Heijden, Radhakrishnan Edayileveettil Krishnankutty","doi":"10.1002/btpr.70074","DOIUrl":"https://doi.org/10.1002/btpr.70074","url":null,"abstract":"Endophytic microorganisms (EMs) residing in medicinal plants form a promising resource of anticancer compounds such as camptothecin (CPT). Given the increasing therapeutic demand for CPT, its sustainable production is of high significance. This study has investigated the EMs isolated from different parts of Ophiorrhiza mungos for the CPT biosynthetic potential. Preliminary screening of EMs for the CPT synthesis was carried out by HPLC analysis of culture extracts, and the HPLC-positive extracts were further confirmed via LC-MS/MS. From a total of 175 EMs screened in the study, 17 strains (14 bacterial and 3 fungal) were found to be CPT producing, with most of them being sourced from the root tissues. Among the bacterial strains, Alcaligenes faecalis subsp. phenolicus S18 exhibited the highest CPT yield (1294.52 μg/L) followed by Bacillus tequilensis (309.02 μg/L). From the fungal strains, Aspergillus sp., S109, S42, and S111 yielded CPT of 22.07, 18.98, and 13.26 μg/L, respectively. Overall, CPT yield among the bacterial producers ranged from 1294.52 to 5.16 μg/L, predominantly from the Bacillus, Acinetobacter, Alcaligenes, and Pseudomonas genera. This study provides the first report on the CPT production by A. faecalis and Aspergillus sp. isolated from O. mungos, and also the first documentation of CPT synthesis in Stenotrophomonas, Fictibacillus, Acinetobacter, and Pseudomonas genera. These findings highlight the potential of novel microbial sources as high-yielding, reliable, and cost-effective alternatives to support commercial CPT production.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70074"},"PeriodicalIF":2.5,"publicationDate":"2025-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145172844","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

Secondary feed filtration and storage conditions influence trace element availability and process performance at 2000 L scale. 二次进料过滤和贮存条件影响2000l规模下微量元素的利用率和工艺性能。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-09-25 DOI: 10.1002/btpr.70071

Abhinav R Jain, Juan Sebastian Reyes, Cuijuan Yuan, Qing Zhao, Ruiqiang Sun, Hang Zhou, Vikram Sisodiya, Yashas Rajendra

{"title":"Secondary feed filtration and storage conditions influence trace element availability and process performance at 2000 L scale.","authors":"Abhinav R Jain, Juan Sebastian Reyes, Cuijuan Yuan, Qing Zhao, Ruiqiang Sun, Hang Zhou, Vikram Sisodiya, Yashas Rajendra","doi":"10.1002/btpr.70071","DOIUrl":"https://doi.org/10.1002/btpr.70071","url":null,"abstract":"Achieving consistent CHO cell culture performance during process scale-up is critical but often challenged by subtle changes in operational parameters. This study investigates how differences in feed media filtration and storage during scale-up can impact CHO cell culture performance. A 70% reduction in titer and a 25% drop in peak viable cell density (VCD) were observed at 2000 L scale. Root cause analysis revealed that the secondary filtration of feed media was likely a contributing factor. Trace element analysis confirmed significant copper(II) ions (Cu2+) loss in feed media at 2000 L, likely due to precipitation during storage and subsequent removal by secondary sterile filtration. This resulted in continued lactate accumulation and reduced titer. Feed storage conditions had an impact on Cu2+ stability, with room temperature storage accelerating Cu2+ loss when compared to storage at 2 to 8°C. By eliminating the secondary filtration step and optimizing feed media storage conditions, process performance was successfully restored at 2000 L scale, matching smaller scale performance. This study highlights how feed filtration and storage critically affect micronutrient stability and availability during scale-up. While secondary filtration may be used for additional microbial control, it can inadvertently alter feed composition, affecting cell metabolism and productivity. Thorough evaluation of feed stability, filtration, and storage strategies is therefore key to ensuring consistent bioreactor performance across scales.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70071"},"PeriodicalIF":2.5,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145136399","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

An economically cost-effective production medium optimization of Bacillus subtilis Ö-4-68: A potential probiotic for aquaculture. 经济高效的枯草芽孢杆菌生产培养基优化Ö-4-68：一种潜在的水产养殖益生菌。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-09-24 DOI: 10.1002/btpr.70070

Sezen Demirhan-Yazıcı, Kemal Karaca, Ayşe Nalbantsoy, Rengin Eltem

{"title":"An economically cost-effective production medium optimization of Bacillus subtilis Ö-4-68: A potential probiotic for aquaculture.","authors":"Sezen Demirhan-Yazıcı, Kemal Karaca, Ayşe Nalbantsoy, Rengin Eltem","doi":"10.1002/btpr.70070","DOIUrl":"https://doi.org/10.1002/btpr.70070","url":null,"abstract":"Probiotic use has become more important in aquaculture for healthy and sustainable output. In particular, Bacillus spp. have emerged as effective probiotic agents, improving gut health, enhancing the immune system, promoting growth, and providing protection against pathogens in fish. Therefore, the application of Bacillus in aquaculture offers a strategic approach to increasing productivity while reducing the reliance on antibiotics. In this study, the antibacterial activities of Bacillus isolates, whose probiotic properties will be determined, against test bacteria that are fish pathogens such as Aeromonas hydrophila, Vibrio anguillarum, Lactococcus garvieae, and Yersinia ruckeri were determined by using cross-streak method and agar well diffusion methods. Then, antibiotic resistances of 75 isolates determined to have antibacterial activity were screened against 9 different antibiotics by the agar disc diffusion method. Gastric juice (pH 2.5) tolerance of 55 isolates determined to be sensitive to antibiotics was examined, and the tolerance of 13 isolates to gastric juice was determined. Optimum growth characteristics at acidic pH, surface hydrophobicity, bile tolerance, and protease, amylase, lipase, and cellulase activities, hemolytic activities, coagulase activities, bacterial adhesion abilities, and biofilm production properties of these isolates were determined. As a result, Bacillus subtilis Ö-4-68, with the best probiotic properties, was selected from the examined isolates, and production medium optimization was carried out with laboratory scale statistical experiment design (Response Surface Methodology, RSM) for high amount of biomass production. As a result of the trials, an economical cost-effective production medium content with high biomass production was determined.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70070"},"PeriodicalIF":2.5,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145129977","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

Scalable production and biophysical characterization of an enzyme cocktail derived from human red blood cells. 从人红细胞中提取的鸡尾酒酶的规模化生产和生物物理特性。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-09-19 DOI: 10.1002/btpr.70072

Mohd Asim Khan, Griffin J Beyer, Naomi Goosby, Lilly Ortiz, Andre F Palmer

{"title":"Scalable production and biophysical characterization of an enzyme cocktail derived from human red blood cells.","authors":"Mohd Asim Khan, Griffin J Beyer, Naomi Goosby, Lilly Ortiz, Andre F Palmer","doi":"10.1002/btpr.70072","DOIUrl":"https://doi.org/10.1002/btpr.70072","url":null,"abstract":"Red blood cells (RBCs) play a critical role in oxygen and carbon dioxide transport, which is facilitated by RBC-encapsulated hemoglobin (Hb) and carbonic anhydrase (CA). In addition, RBCs are constantly exposed to oxidative stress due to the intracellular reactive oxygen species (ROS) generated during Hb auto-oxidation. Antioxidant enzymes within RBCs, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx), counteract ROS generation to protect the RBC from oxidative stress. Therefore, this study presents a scaled-up method to extract an enzyme cocktail from lysed human RBCs, enriched with the major RBC enzymes with minimal Hb contamination. Using ethanol-chloroform precipitation and multiple biophysical analyses (SDS-PAGE, SEC-HPLC, MALDI-TOF, and LC-MS/MS), the RBC enzymes were successfully separated from Hb in the hemolysate. The purified enzyme cocktail exhibited minimal Hb contamination and retained a significant amount of CA, and antioxidative enzymes like SOD and CAT. Therefore, this scalable RBC enzyme purification method provides an efficient approach for isolating RBC enzymes with broad biomedical relevance.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70072"},"PeriodicalIF":2.5,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145085167","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

Exploring the design space for Triton X-100 substitutes in viral inactivation applications. 探索Triton X-100替代品在病毒灭活应用中的设计空间。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-09-03 DOI: 10.1002/btpr.70069

Yuqi Du, Shanshan Wu

{"title":"Exploring the design space for Triton X-100 substitutes in viral inactivation applications.","authors":"Yuqi Du, Shanshan Wu","doi":"10.1002/btpr.70069","DOIUrl":"10.1002/btpr.70069","url":null,"abstract":"The urgent need to replace the European-prohibited Triton X-100 in biomanufacturing has been hindered by insufficient data on alternative detergents' minimum effective concentrations (MECs) and process robustness in viral inactivation. This study makes systematic research including: (1) Establishment of MECs for novel Triton X-100 substitutes (TXR-1/VIS/13-S9/C16) achieving effective inactivation of Xenotropic murine leukemia virus and Pseudorabies virus (log10 reduction factor >4) across diverse CHO harvest fluids; (2) Demonstration of broad-spectrum efficacy against various viruses, with TXR-1/VIS/13-S9 maintaining effective inactivation for Bovine viral diarrhea virus, Vesicular stomatitis virus, Baculovirus, and Herpes simplex virus type 1; (3) Identification of PS20's material-dependent inactivation dynamics, establishing standalone parameters (4 h at 37°C) that achieve equivalent viral inactivation to traditional tri(n-butyl)phosphate -combined methods without requiring lipase activity-a paradigm shift in detergent application. Crucially, process optimization revealed that extending exposure time (1-4 h) enhanced PS20/PS80 efficacy more effectively than two fold concentration increases, providing cost-effective solutions. These findings deliver broader design spaces for implementing eco-friendly detergents while ensuring compliance with EMA/ICH viral safety standards.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70069"},"PeriodicalIF":2.5,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144940714","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

Elucidation of cell culture impacts on hydroxylysine levels in monoclonal antibodies using high-throughput analytical quantification and media components. 利用高通量分析定量和培养基成分阐明细胞培养对单克隆抗体中羟赖氨酸水平的影响。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-08-28 DOI: 10.1002/btpr.70068

Roli Kargupta, Shannon Rivera, Brent Kochert, Kyle Devenney, Daniel Donelly, Tariq Atieh, Fang Li, Jessica Pan, Daya Patel, Venkata Tayi, Gaurav Chauhan, Rebecca Chmielowski, Susan J Abbondanzo

{"title":"Elucidation of cell culture impacts on hydroxylysine levels in monoclonal antibodies using high-throughput analytical quantification and media components.","authors":"Roli Kargupta, Shannon Rivera, Brent Kochert, Kyle Devenney, Daniel Donelly, Tariq Atieh, Fang Li, Jessica Pan, Daya Patel, Venkata Tayi, Gaurav Chauhan, Rebecca Chmielowski, Susan J Abbondanzo","doi":"10.1002/btpr.70068","DOIUrl":"https://doi.org/10.1002/btpr.70068","url":null,"abstract":"Hydroxylysine (Hyl) is a post-translational hydroxyl modification of lysine that is not commonly observed at very high levels and thus is not usually considered a product quality attribute (PQA). Post-translation modifications (PTMs) are considered potential PQAs when elevated levels are observed - requiring monitoring and investigation. In a recent monoclonal antibody expression using Media A, Hyl levels were observed at ~20%-35%. At such elevated percentage levels, Hyl was considered a PQA - triggering a root-cause investigation in the upstream activities like cell culture conditions and media components. Initial detection of the Hyl modification originated from non-quantitative, intact mass analysis with confirmation of site-location determined by peptide mapping. Through the root-cause investigation, it was determined that levels of Hyl were underestimated by ~10-fold using tryptic peptide mapping analysis without inclusion of miscleaved peptides. The analytical procedure was revised from trypsin-digestion to IdeS-digestion, a reduced mass analysis, to accurately and rapidly quantify Hyl levels of investigational samples. Proprietary Media B was utilized to reduce the Hyl level by 2-fold to ~10%-15%. Further investigation into the media and feed components determined that increasing concentration of Fe(III) content decreased Hyl levels. Supplementation of Fe(III) served as a robust mitigation strategy of Hyl reduction in upstream process. Media B was used to scale up to a 500 L bioreactor while maintaining the lower Hyl level. The analytical and cell culture methods developed in this study can be leveraged to detect and tune Hyl levels.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70068"},"PeriodicalIF":2.5,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144940777","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

Production of recombinant methionine-containing elastin-like polypeptides in a fermenter using ECPM1 medium. 用ECPM1培养基在发酵罐中生产含重组蛋氨酸的弹性蛋白样多肽。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-08-22 DOI: 10.1002/btpr.70057

Alice Delhaes, Laure Bataille, Myriam Médéric, Sébastien Lecommandoux, Elisabeth Garanger

{"title":"Production of recombinant methionine-containing elastin-like polypeptides in a fermenter using ECPM1 medium.","authors":"Alice Delhaes, Laure Bataille, Myriam Médéric, Sébastien Lecommandoux, Elisabeth Garanger","doi":"10.1002/btpr.70057","DOIUrl":"https://doi.org/10.1002/btpr.70057","url":null,"abstract":"Elastin-like polypeptides (ELPs) are recombinant protein-like polymers whose macromolecular structure can be precisely controlled through genetic manipulation of their sequence and length. Their lower critical solution temperature (LCST) phase behavior facilitates purification via chromatography-free techniques and can be explored for self-assembly. As a result, ELPs are extensively investigated for diverse biological, biomedical, and biotechnological applications. So far, ELPs have mostly been isolated from bacteria grown in flasks or fermenters containing complex media that only yield limited amounts of biomass. We herein explored the use of the semi-defined ECPM1 medium, known to limit the accumulation of toxic metabolites and rich in glycerol as a low energy carbon source, to produce ELPs of different chain lengths and containing oxidation-sensitive methionine residues. We report the optimized bioproduction using ECPM1 of ELP[M1V3-n] with n = 20, 40, 80 in a fermenter in good yields and confirm their intact protein sequence using various chemical characterization techniques.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70057"},"PeriodicalIF":2.5,"publicationDate":"2025-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144940686","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