Biotechnology Progress最新文献

Adaptation of suspension Vero cells to commercially available cell culture media and enhanced production of vaccine-strain mumps virus. 悬浮Vero细胞适应市售细胞培养基和增强疫苗株腮腺炎病毒的生产。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-11 DOI: 10.1002/btpr.88561

Rebecca Hong, Thomas R Blanda, Maria Bednar, Jessica Olson, Elise K B Baron, Daniel Spatafore, Duygu Ercan Oruc, James M Wagner

{"title":"Adaptation of suspension Vero cells to commercially available cell culture media and enhanced production of vaccine-strain mumps virus.","authors":"Rebecca Hong, Thomas R Blanda, Maria Bednar, Jessica Olson, Elise K B Baron, Daniel Spatafore, Duygu Ercan Oruc, James M Wagner","doi":"10.1002/btpr.88561","DOIUrl":"https://doi.org/10.1002/btpr.88561","url":null,"abstract":"Vero cells (referred to as \"Vero\" henceforth) are a commonly used host for the production of a wide variety of vaccines, but due in part to the typically anchorage-dependent nature of the cells, Vero cells also pose multiple challenges during process development, scale-up, and commercialization. The adaptation of Vero to proliferate in a serum-free suspension culture while remaining non-tumorigenic can be a challenging endeavor, and where successful, has often utilized undisclosed and/or complex custom-designed medium formulations. Additional and expanded options for commercially available serum-free media that can support the robust expansion of suspension-adapted Vero cells for viral vaccine manufacturing would be a significant advantage for increasing the industrial adoption of suspension Vero, as virus production can also be highly medium-dependent and sensitive. In this work, we have successfully transitioned suspension-adapted Vero cells from a proprietary medium to a commercially-available base medium (ExCell VPRO) with supplementation of additional commercially-available components, and subsequently infected the resulting ExCell VPRO cell bank lineage with an attenuated vaccine strain Mumps virus. Like many suspension cell line media formulations, off-the-shelf commercially available ExCell VPRO medium contains dextran sulfate, which serves to minimize cell clumping, but is also well-known to directly impact the infectivity of some viruses. Unexpectedly, we have observed that the presence of dextran sulfate in the infection medium increased both culture longevity and infectious virus titers for attenuated Mumps, potentially eliminating the need for a medium exchange between cell growth and infection for a more streamlined and easily scalable attenuated Mumps virus production process.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e88561"},"PeriodicalIF":2.8,"publicationDate":"2026-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149163735","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

Design and scale-up of a mixed-substrate fed-batch strategy for enhanced HPV VLP production in Komagataella phaffii. 设计和扩大混合底物补料批量策略，以增强Komagataella phaffii的HPV VLP生产。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-09 DOI: 10.1002/btpr.88559

Abhilasha K Rani, Nida Khan, Anurag S Rathore

{"title":"Design and scale-up of a mixed-substrate fed-batch strategy for enhanced HPV VLP production in Komagataella phaffii.","authors":"Abhilasha K Rani, Nida Khan, Anurag S Rathore","doi":"10.1002/btpr.88559","DOIUrl":"https://doi.org/10.1002/btpr.88559","url":null,"abstract":"Production of human papillomavirus (HPV) virus-like particles (VLPs) in Komagataella phaffii is often constrained by methanol-associated toxicity and suboptimal feeding strategies during induction. In this study, a microbioreactor-based approach was employed to systematically design and evaluate mixed-substrate fed-batch strategies for enhanced HPV16 L1 VLP production. Four methanol-based feeding compositions, including methanol-only and combinations with glycerol and sorbitol, were investigated under pulse and linear feeding modes. Among the tested conditions, a linear feeding strategy using 70% methanol and 30% glycerol (70 M + 30G) resulted in the highest biomass (DCW: 90.36 g/L) and VLP titer (42 mg/L), corresponding to a two-fold improvement over methanol-only feeding. Enhanced performance was attributed to reduced methanol-induced metabolic stress and improved substrate utilization, leading to more stable dissolved oxygen (DO) profiles and sustained cellular activity. In contrast, pulse feeding resulted in significant DO fluctuations, indicating transient metabolic imbalances and lower overall productivity. The optimized feeding strategy was successfully translated to a 1 L stirred-tank bioreactor, achieving a DCW of 120 g/L and a VLP titer of 49 mg/L, with a ~ 2.3-fold increase compared to the control. The consistency between microscale and bench-scale results demonstrates the predictive capability of microbioreactor systems for fed-batch process development. Overall, this study presents a rational and scalable feeding strategy that improves recombinant VLP production while reducing methanol usage, providing a practical framework for efficient bioprocess design in K. phaffii.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e88559"},"PeriodicalIF":2.8,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149148431","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

Generation of high-expressing transposon-based stable pools to produce (1 + 1) and (2 + 1) bispecific common light chain antibodies 产生高表达的转座子稳定池，产生（1 + 1）和（2 + 1）双特异性普通轻链抗体。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-06-02 DOI: 10.1002/btpr.88517

Sara Ramos, Surya Karunakaran, Thierry Monney, Olivier Bornert, Andreas Reimann, Ferenc Boldog, Michael Dyson, Ankita Srivastava, Jeremy Minshull, Lydia N. Caro

{"title":"Generation of high-expressing transposon-based stable pools to produce (1 + 1) and (2 + 1) bispecific common light chain antibodies","authors":"Sara Ramos, Surya Karunakaran, Thierry Monney, Olivier Bornert, Andreas Reimann, Ferenc Boldog, Michael Dyson, Ankita Srivastava, Jeremy Minshull, Lydia N. Caro","doi":"10.1002/btpr.88517","DOIUrl":"10.1002/btpr.88517","url":null,"abstract":"Bi- and multispecific antibodies can deliver effective protein-based therapeutics by enabling novel biology. Transient expression of these complex asymmetric antibody formats may become a bottleneck in providing sufficient amounts of high-quality material for preclinical discovery and developability assessment to support candidate selection activities. In contrast, transposase-based stable cell line generation is being recognized as a robust and rapid path to enable the evaluation of product quality from stable pool derived material. Transposase-based systems feature a semi-targeted integration of multiple copies of the gene(s) of interest in the host cell's genome resulting in the rapid generation of homogeneous high-expressing (up to g/L range) stable pools after 1–3 weeks of selection phase. Here, we evaluated the Leap-In® transposase technology for the expression of IGI's 1 + 1 and 2 + 1 bispecific, common light chain antibodies, based on IGI's proprietary BEAT® (Bispecific Engagement of Antibodies based on T cell Receptor) platform. To generate stable CHO pools expressing IGI's bispecific molecules, transposon-based constructs expressing the common light chain and the two heavy chain genes were co-transfected with the Leap-In® transposase mRNA. Based on the proportion of the different chain combinations detected in the harvests, their expression levels were further optimized through vector engineering to facilitate heterodimer assembly and maximize productivity. The best pool displayed a titer of 2.33 g/L in 14-day fed-batch cultures, and heterodimer represented 93.8% of the expressed species. The purified antibody displayed 97.4% purity, as determined by SE-HPLC and binding to targets within range, as determined by SPR and cell-based affinity.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148148268","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

Application of commercial ultrafiltration membranes for protein purification by high performance countercurrent membrane purification (HPCMP) 商用超滤膜在高效逆流膜纯化（HPCMP）蛋白中的应用。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-07-07 DOI: 10.1002/btpr.88537

Aylin Mohammadzadehmarandi, Nate Heller, Mohammadreza Ahmadimashhadi, Jon Petrone, Nicholas Marchand, Siavash Darvishmanesh, Ka Zhang, Stefano Menegatti, Andrew L. Zydney

{"title":"Application of commercial ultrafiltration membranes for protein purification by high performance countercurrent membrane purification (HPCMP)","authors":"Aylin Mohammadzadehmarandi, Nate Heller, Mohammadreza Ahmadimashhadi, Jon Petrone, Nicholas Marchand, Siavash Darvishmanesh, Ka Zhang, Stefano Menegatti, Andrew L. Zydney","doi":"10.1002/btpr.88537","DOIUrl":"10.1002/btpr.88537","url":null,"abstract":"High performance countercurrent membrane purification (HPCMP) has emerged as a promising diffusive separation strategy for the purification of recombinant proteins and monoclonal antibodies (mAbs). However, prior studies employed dialysis membranes, which are designed for diffusive separations but have not been validated for biopharmaceutical manufacturing. This study evaluates the ability of commercial ultrafiltration (UF) membranes, engineered for pressure-driven filtration, to support highly selective diffusive separations. Scanning electron microscopy (SEM) analysis of the membrane cross-sections revealed marked morphological differences between UF and dialysis membranes. Yet, transport studies using polydisperse dextrans and model proteins demonstrated that UF membranes can deliver exceptional diffusive separation selectivity. In particular, a 100 kDa polysulfone membrane (Cytiva) provided >150-fold diffusive selectivity between bovine serum albumin (BSA) and immunoglobulin G (IgG). When utilized for mAb separation from a clarified CHO cell culture fluid, this membrane achieved >98% product yield, near-complete removal of low molecular weight species, and significant reduction in both DNA and host cell protein (HCP) levels. These results demonstrate that commercial UF membranes that are validated for bioprocessing applications can be repurposed for HPCMP, enabling scalable, continuous downstream processing of biotherapeutics.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.88537","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148403453","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

Mechanistic deconvolution of BSA size variants by constrained Raman pseudo-Voigt hard modeling during anion-exchange chromatography 阴离子交换色谱中约束Raman伪voigt硬模拟的BSA尺寸变化机制反褶积。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-06-25 DOI: 10.1002/btpr.88534

Jakob Heyer-Müller, Robin Schiemer, Lars Robbel, Michael Schmitt, Jürgen Hubbuch

{"title":"Mechanistic deconvolution of BSA size variants by constrained Raman pseudo-Voigt hard modeling during anion-exchange chromatography","authors":"Jakob Heyer-Müller, Robin Schiemer, Lars Robbel, Michael Schmitt, Jürgen Hubbuch","doi":"10.1002/btpr.88534","DOIUrl":"10.1002/btpr.88534","url":null,"abstract":"In biopharmaceutical manufacturing, protein aggregation is a critical quality attribute, necessitating rapid and reliable analytical strategies during downstream processes like anion-exchange chromatography (AEX). While Raman spectroscopy enables continuous monitoring of protein secondary structure, standard data-driven regression models struggle to decouple intrinsic structural changes from gradient-induced solvent and buffer drifts under dynamic chromatographic conditions. Addressing this methodological gap, this study establishes a constrained pseudo-Voigt hard modeling framework for the mechanistic deconvolution of bovine serum albumin (BSA) size variants during in-line Raman monitoring of AEX processes. By explicitly defining a parametric background model to capture salt-induced spectral drift, the methodology effectively isolates matrix variations from genuine protein-specific signals. The constrained hard model was applied to 285 in-line spectra across diverse chromatographic conditions, achieving reconstruction fidelity while maintaining stable, physically interpretable component identities. The mechanistically derived Amide I center of mass emerged as a robust, aggregation-sensitive descriptor that preserves structural information despite strong concentration dynamics. Furthermore, the extracted spectral features demonstrated strong predictive performance for monomer concentration and acceptable accuracy for high molecular weight components. Collectively, these results demonstrate that constrained spectral hard modeling provides a highly interpretable, robust, and calibration-light alternative to classical partial least squares approaches for the real-time monitoring of protein size variants.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.88534","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148325967","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

A straightforward approach for converting existing batch production to an integrated continuous manufacturing suite by adopting membrane-based technology 采用基于膜的技术将现有的批量生产转换为集成的连续制造套件的直接方法。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-06-10 DOI: 10.1002/btpr.88523

Mingyue Fang, Ce Shi, Anmin Zhou, Huilin Zhu, Hang Zhou, Sherry Gu, Weichang Zhou

{"title":"A straightforward approach for converting existing batch production to an integrated continuous manufacturing suite by adopting membrane-based technology","authors":"Mingyue Fang, Ce Shi, Anmin Zhou, Huilin Zhu, Hang Zhou, Sherry Gu, Weichang Zhou","doi":"10.1002/btpr.88523","DOIUrl":"10.1002/btpr.88523","url":null,"abstract":"The rapidly developing antibody market poses challenges to the production cost and efficiency, which can be addressed through continuous process. However, a hurdle of high capital investment remains a concern especially for the company with existing fed-batch capacity. This study proposes a straightforward approach to convert existing batch production suites into continuous ones using membrane-based technology. A conceptual validation was conducted at a 100 L pilot scale, and a head-to-head comparison was carried out. The results demonstrated that Protein A membrane chromatography could achieve a productivity 6.3 times higher than that of periodic counter-current chromatography (PCC), without requiring the purchase of any dedicated equipment. Using Q membrane, compared with traditional column anion exchange chromatography, can achieve a 16-fold increase in load capacity and a 25-fold increase in productivity. Finally, VF-UF/DF (Virus Filtration-Ultrafiltration/diafiltration) linkage process was designed, resulting in a 20% reduction in process time. During the 23-day upstream perfusion and 15-day downstream processing, the cumulative titer reached 51.0 g/L, with an 83.1% yield in downstream processing. With downstream process time shortening from 4 to 1.5 days, the process capacity could be improved by 8 times using the existing batch production suites and the same facility area. PATs, including ultra-performance liquid chromatography, Raman spectroscopy, FlowVPX, and an inline turbidity meter, were integrated to monitor process CQAs (Critical Quality Attributes): monomer purity, protein concentration, and turbidity, to enable efficient process control in the future.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148216274","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

Advanced glucose control strategies leveraging Raman spectroscopy for optimized mammalian cell culture manufacturing 利用拉曼光谱优化哺乳动物细胞培养制造的先进葡萄糖控制策略。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-06-29 DOI: 10.1002/btpr.88531

Matthew Banner, Daniel Ray, Irfan Ahanger, Fabio Zurlo, Noah Hitchcock, Rajesh Mistry, Kasia Kozakowska-McDonnell, Matthew Cheeks, Richard Turner, Jonathan Welsh, Suzanne S. Farid, Michael Thomas, Stephen Goldrick

{"title":"Advanced glucose control strategies leveraging Raman spectroscopy for optimized mammalian cell culture manufacturing","authors":"Matthew Banner, Daniel Ray, Irfan Ahanger, Fabio Zurlo, Noah Hitchcock, Rajesh Mistry, Kasia Kozakowska-McDonnell, Matthew Cheeks, Richard Turner, Jonathan Welsh, Suzanne S. Farid, Michael Thomas, Stephen Goldrick","doi":"10.1002/btpr.88531","DOIUrl":"10.1002/btpr.88531","url":null,"abstract":"Maintaining consistent quality in the manufacturing of biotherapeutic proteins in mammalian cell culture is challenging, with unplanned deviations causing inconsistencies and potential batch failure. Current methods for monitoring and controlling critical process parameters (CPPs) rely on slow, labor-intensive offline analyses. This is particularly problematic in upstream manufacturing, where infrequent measurements hinder real-time CPP monitoring and result in suboptimal control. This study developed and implemented a robust, standardized framework that integrates Raman spectroscopy with real-time machine learning models and bioreactor control via OPC-UA, enabling seamless communication and effective control of mammalian cell culture CPPs. High-accuracy ML models (R2 >0.92) enabled real-time monitoring of glucose, lactate, viable cell density, and antibody titre. The glucose model was integrated into an automated process analytical technology (PAT) control strategy to maintain glucose at a predefined setpoint. The PAT strategy was compared to a manual bolus glucose control under high and low glucose feeding regimes. The PAT control approach observed an increase in biotherapeutic product titre by up to 35% and reduced glycation by up to 27%. These results indicated that both glucose setpoints and fluctuations impacted cell culture performance. In conclusion, this study highlighted the value of PAT tools in automating control loops, consequently improving process performance with enhanced productivity and quality.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.88531","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148350474","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

In-line Raman spectroscopy real-time glucose prediction method for commercial pneumococcal vaccine drug substance fermentation manufacturing process control 在线拉曼光谱实时葡萄糖预测方法用于商品肺炎球菌疫苗原料药发酵生产过程控制。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-06-21 DOI: 10.1002/btpr.88532

Griffin P. Thomas, Brian C. Marks, Barbara B. Alvarado-Hernandez, Matthew Woodling, James O'Connor, Roberto Ortiz, John Higgins, Jon Shanter

{"title":"In-line Raman spectroscopy real-time glucose prediction method for commercial pneumococcal vaccine drug substance fermentation manufacturing process control","authors":"Griffin P. Thomas, Brian C. Marks, Barbara B. Alvarado-Hernandez, Matthew Woodling, James O'Connor, Roberto Ortiz, John Higgins, Jon Shanter","doi":"10.1002/btpr.88532","DOIUrl":"10.1002/btpr.88532","url":null,"abstract":"Traditional off-line analysis methods to quantify residual metabolite concentrations in a drug substance fermentation process consume valuable time, require costly resources, and demand potentially hazardous manual operations when working with pathogenic biological organisms. This case study focuses on the successful method development and validation of Raman spectroscopy as an in-line process analytical technology (PAT) to replace the existing off-line assay for real-time glucose quantification. The value and feasibility of Raman spectroscopy as an in situ PAT method are recognized and demonstrated as an enabling technology suited to mitigate challenges fundamental to lab-scale process development and commercial manufacturing. Herein, application of chemometric techniques and multivariate analysis of Raman spectral data is described to develop a partial least squares model capable of predicting real-time and accurate glucose concentrations with a root mean square error of prediction of 1.2 g/L. Through this work, the predictive Raman PAT glucose quantification method was validated to make process decisions in a commercially licensed Pneumococcal vaccine drug substance manufacturing facility for the first time at our company. The method was integral to the process control strategy and applied to accurately and robustly indicate the glucose concentration target ± 2 g/L for triggering subsequent process operations. Several advantages were realized through this work including advanced process control, reduced operating costs, and improved safety posture.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.88532","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148293367","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

Purification and concentration of model viruses using single-pass tangential flow filtration 用单次切向流过滤纯化和浓缩模型病毒。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-07-04 DOI: 10.1002/btpr.88521

Luis Alberto Mejía-Manzano, Lynn Manchester, Grace Wallis, Kan Wang, Bruce P. Lee, Michael Betenbaugh, Caryn L. Heldt

{"title":"Purification and concentration of model viruses using single-pass tangential flow filtration","authors":"Luis Alberto Mejía-Manzano, Lynn Manchester, Grace Wallis, Kan Wang, Bruce P. Lee, Michael Betenbaugh, Caryn L. Heldt","doi":"10.1002/btpr.88521","DOIUrl":"10.1002/btpr.88521","url":null,"abstract":"The vaccine and viral vector industry is growing at an accelerated rate. To improve harvest and purification processes, the development of continuous membrane-based operations, such as normal flow filtration (NFF) and single pass tangential flow filtration (SPTFF) for concentration were explored. This work was conducted using two model viruses, non-enveloped porcine parvovirus (PPV) and enveloped Suid herpesvirus (SuHV). The viruses are in the same family as the gene therapy vectors adeno associated virus and herpes simplex virus, respectively. SPTFF design started with batch TFF for membrane selection. Hollow fiber membranes with a 100 and 300 kDa molecular weight cut off were defined for PPV and SuHV SPTFF operations, respectively. The SPTFF runs for PPV did not provide any concentration of the virus and low protein and DNA removal, unlike batch TFF. Two hollow fiber membranes run at 10 mL/min and 2 psi were the best condition for SuHV concentration, with approximately 100-fold titer concentration and protein and DNA removal of 37% ± 5% and 32% ± 8%, respectively. This concentration was superior to the batch TFF and indicated a strong dependence on flow rate and transmembrane pressure. For NFF, filters selection and performance tests were carried out for NFF of PPV and SuHV, as well as cleaning protocols for hollow fiber membranes. The ultimate goal is to integrate this work into the continuous purification of viral vectors produced in mammalian cell cultures to reduce costs and increase throughput.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.88521","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148381721","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

Targeting Warburg effect in Chinese hamster ovary cell culture with pyruvate dehydrogenase kinase inhibitors 丙酮酸脱氢酶激酶抑制剂在中国仓鼠卵巢细胞培养中的靶向Warburg效应。

IF 2.8 3区生物学

Biotechnology Progress Pub Date : 2026-10-05 Epub Date: 2026-07-08 DOI: 10.1002/btpr.88536

Geetanjali Basavaraj Hubli, Richa Katiyar, Vrinda Kapoor, Girish H. Rajacharya, Anurag S. Rathore

{"title":"Targeting Warburg effect in Chinese hamster ovary cell culture with pyruvate dehydrogenase kinase inhibitors","authors":"Geetanjali Basavaraj Hubli, Richa Katiyar, Vrinda Kapoor, Girish H. Rajacharya, Anurag S. Rathore","doi":"10.1002/btpr.88536","DOIUrl":"10.1002/btpr.88536","url":null,"abstract":"Production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is affected by the “Warburg effect” which is characterized by high lactate accumulation. Therefore, the present study explores the use of pyruvate dehydrogenase kinase (PDK) inhibitors dichloroacetate (DCA), metformin (M), (S)-3,3,3-trifluoro-2-hydroxy-2-methyl propionic acid (TPP), and 4-phenylbutyrate (PB) to counteract this metabolic bottleneck. To achieve optimal mAb yields, individual inhibitors (0.5–10 mM) were screened, followed by evaluation of their combinatorial effects. A combination of 5 mM DCA and 1 mM TPP (DCA + TPP) revealed ~2-fold enhancement in mAb yield (1.2 g/L) under fed-batch conditions, accompanied by a marked reduction in lactate accumulation as compared to control. The optimized DCA + TPP combination also exhibited 15%–20% enhancement in the mAb yield as compared to a single PDK inhibitor that is, DCA (5 mM) and TPP (1.0 mM) at the same concentration. Metabolic analysis revealed a 19.5-fold increase in pyruvate flux and a 2-fold increase in intracellular acetyl-CoA. The critical product quality attributes exhibited insignificant impact on charge variant (<3%), aggregation, and glycosylation of mAb. The amino acid consumption profiles predominantly showed depletion of glycine and tryptophan, whereas threonine and aspartic acid were primarily consumed in the control. Proteomic analysis exhibited upregulation and downregulation of various proteins, which significantly contribute to mAb's yield. Validation in Trastuzumab-producing CHO-S cells confirmed a 30% increase in mAb yield and a 10% improvement in cell density. These outcomes demonstrate that the combination of DCA + TPP is a promising metabolic intervention to mitigate the Warburg effect and enhance recombinant protein production in mammalian systems.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 5","pages":""},"PeriodicalIF":2.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148410084","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