Biotechnology Progress最新文献

Scalable process development for rAAV transient transfection production using computational fluid dynamics modeling.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-04-04 DOI: 10.1002/btpr.70028

Jianfa Ou, Yawen Tang, Alexander Williams, Yikun Huang, Roseanna Shimansky, Gianfranco Salinas, Gregory Keil, Jongchan Lee, Michael C Borys, Anurag Khetan

{"title":"Scalable process development for rAAV transient transfection production using computational fluid dynamics modeling.","authors":"Jianfa Ou, Yawen Tang, Alexander Williams, Yikun Huang, Roseanna Shimansky, Gianfranco Salinas, Gregory Keil, Jongchan Lee, Michael C Borys, Anurag Khetan","doi":"10.1002/btpr.70028","DOIUrl":"https://doi.org/10.1002/btpr.70028","url":null,"abstract":"Recombinant adeno-associated virus (rAAV) is a promising delivery vehicle for cell and gene therapies. Upstream development faces challenges like low productivity and inconsistent performance despite advancements. This study presents a scale-up design for robust rAAV production at 250 L scale using a transfection system. Initial process development in shake flasks optimized plasmid ratio to improve rAAV production. However, genome titer decreased by up to 50% in stirred-tank bioreactors, likely due to mechanical shear forces. Stirred-tank bioreactors were modeled with computational fluid dynamics (CFD) by M-STAR (250 mL, 5 L, 50 L) and with empirical correlations by Dynochem (250 L). Hydrodynamics were characterized to provide normalized shear stress across different geometries. The power per unit volume (P/V) of 71 W/m3 was optimal for the 250 mL bioreactor, focusing on cell growth, rAAV genome titer, capsid titer, and full capsid ratio. Based on CFD modeling, a P/V of 20 W/m3 was projected to perform best at 5 and 50 L scales during development, confirmed by comparable genome titer to low shear shake flask culture. A P/V of 15 W/m3 was subsequently projected for final production at the 250 L scale. The negative impact of shear stress could be further mitigated by adding extra Poloxamer-188 as a shear protectant. Additionally, pre-transfection viable cell density (VCD) was identified as a critical attribute. The final process included a 30% fixed-volume dilution of the cell culture along with controlled DNA complexation conditions to improve process robustness. Sequential production at the 250 L scale demonstrated consistent cell growth and rAAV production.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70028"},"PeriodicalIF":2.5,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778856","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

N-1 semi-continuous transient perfusion in shake flask for ultra-high density seeding of CHO cell cultures in benchtop bioreactors.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-04-03 DOI: 10.1002/btpr.70029

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

{"title":"N-1 semi-continuous transient perfusion in shake flask for ultra-high density seeding of CHO cell cultures in benchtop bioreactors.","authors":"Lucas Lemire, Sebastian-Juan Reyes, Yves Durocher, Robert Voyer, Olivier Henry, Phuong Lan Pham","doi":"10.1002/btpr.70029","DOIUrl":"https://doi.org/10.1002/btpr.70029","url":null,"abstract":"One strategy to enhance the production of biological therapeutics is using transient perfusion in the preculture (N-1 stage) to seed the production culture (N stage) at ultra-high cell densities (>10 x 106 viable cells/mL). This very high seeding density improves cell culture performance by shortening the timeline and/or achieving higher final product concentrations. Typically, an N-1 seed train employs bioreactors with alternating tangential flow filtration (ATF) or tangential flow filtration (TFF) perfusion systems or Wave cell bag bioreactor with integrated filtration membrane, which have costs and technical complexity. Here, we propose an alternative method using semi-continuous transient perfusion through media exchange in shake flasks, which is suitable for benchtop-scale intensification process development. Daily media exchange was necessary to prevent nutrient limitations. The observed limitation of maximum viable cell densities (VCD) in various flask sizes was demonstrated to be due to oxygen limitations through the measurements of maximum oxygen transfer rates (OTR) using the sulfite system. By increasing agitation frequency from 200 to 300 RPM, maximum OTR in 500-mL shake flasks was increased by 62.3%, allowing an increase in maximum VCD of 29.6%. However, in 1000-mL shake flasks, an increase in agitation rate resulted in early cell death. After demonstrating that media exchange in shake flasks by centrifugation had no significant impact on cell growth rates, metabolism, and productivity, a benchtop bioreactor was seeded from semi-continuous transient perfusion cell expansion. The ultra-high cell density seeding resulted in a 49.3% increase in space-time-yield (STY) when compared to a standard low seeding density culture.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70029"},"PeriodicalIF":2.5,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143771258","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

N-acetyl-D-mannosamine, a novel additive, effectively reducing high mannose glycosylation of monoclonal antibody without affecting other quality attributes.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-04-02 DOI: 10.1002/btpr.70024

Miaomiao Chai, Hai Shu, Qiancheng Wang, Cong Tian, Linlin Wang, Yinmao Fan, Ruiqiang Sun, Hang Zhou

{"title":"N-acetyl-D-mannosamine, a novel additive, effectively reducing high mannose glycosylation of monoclonal antibody without affecting other quality attributes.","authors":"Miaomiao Chai, Hai Shu, Qiancheng Wang, Cong Tian, Linlin Wang, Yinmao Fan, Ruiqiang Sun, Hang Zhou","doi":"10.1002/btpr.70024","DOIUrl":"https://doi.org/10.1002/btpr.70024","url":null,"abstract":"N-linked glycosylation stands as a pivotal quality attribute for monoclonal antibodies (mAbs), particularly the high mannose (Man5) variant, which significantly influences the pharmacokinetics of mAbs. Traditional approaches to modulate Man5 have frequently resulted in suboptimal outcomes. In this investigation, we introduced a novel additive, N-acetyl-d-mannosamine (ManNAc), which selectively targeted and reduced Man5 without compromising other product quality attributes (PQAs). The study further examined optimal concentrations and timing for the incorporation of ManNAc in the mAbs expression process utilizing CHO-K1 cells within a fed-batch shaker flask culture mode. In the ManNAc titration experiments, we established groups at concentrations of 5, 10, 15, 20, 40, 60, 80, and 100 mM. The findings revealed a concentration-dependent decrease in Man5, with reductions reaching as low as 2.9% from an initial 8.9%. Importantly, cellular growth, metabolism, and other PQAs remained unaffected. Regarding the timing of ManNAc addition, groups were set for days N-1, 0, 5, and 11. The results indicated that ManNAc addition on Day 11 did not affect Man5 levels, whereas earlier additions proved effective. A full factorial design was employed to assess the interplay between ManNAc concentration and addition timing, revealing no significant interaction. Consequently, it is recommended to administer 20-40 mM ManNAc prior to Day 4. The strategy of introducing 20 mM ManNAc on Day 0 has been successfully implemented across 12 clones, achieving an average Man5 reduction of 46%. Collectively, these findings delineate a novel and efficacious strategy for the Man5 modulation, promising enhanced control over this critical quality attribute in mAbs production.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70024"},"PeriodicalIF":2.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762641","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

Continuous glucose feedback control using Raman spectroscopy and deep learning models for biopharmaceutical processes.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-04-02 DOI: 10.1002/btpr.70020

Mohammad Rashedi, Matthew Demers, Hamid Khodabandehlou, Tony Wang, Christopher Garvin, Steve Rianna

{"title":"Continuous glucose feedback control using Raman spectroscopy and deep learning models for biopharmaceutical processes.","authors":"Mohammad Rashedi, Matthew Demers, Hamid Khodabandehlou, Tony Wang, Christopher Garvin, Steve Rianna","doi":"10.1002/btpr.70020","DOIUrl":"https://doi.org/10.1002/btpr.70020","url":null,"abstract":"This study explores the implementation of continuous glucose control strategies in high-consumption, high-complexity cell culture processes using Raman spectroscopy and advanced deep learning models, including convolutional neural networks and variational autoencoder just-in-time learning. By leveraging deep learning-derived process monitoring, the study enhances glucose measurement accuracy and stability, enabling precise control across different glucose set points. This approach allows for a systematic evaluation of glycosylation effects and other critical quality attributes, addressing the impact of glucose variability on product consistency. Continuous glucose control is compared against traditional bolus feeding, demonstrating improved set-point maintenance, reduced high mannose (HM) levels, and enhanced overall titer productivity. To extend these benefits to manufacturing environments where Raman spectroscopy may not be feasible, a continuous glucose calculator (CGC) is developed as a scalable alternative. Experimental validation across multiple cell lines confirmed that both Raman-based and CGC-driven strategies minimized glucose fluctuations, reduced undesirable byproducts, and optimized process yields. These findings highlight the potential of continuous glucose control, combined with deep learning models, to improve bioprocess efficiency and product quality while addressing the challenges of dynamic, high-consumption bioreactor systems.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70020"},"PeriodicalIF":2.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762630","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

Cryopreservation practices in clinical and preclinical iPSC-based cell therapies: Current challenges and future directions.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-04-02 DOI: 10.1002/btpr.70031

Michael Dobruskin, Geoffrey Toner, Ronald Kander

引用次数: 0

Enhancement of D-mannitol production by fine-tuned expression of mannitol-1-phosphate dehydrogenase in Synechocystis sp. PCC6803.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-04-02 DOI: 10.1002/btpr.70027

Wenyang Wu, Wei Du, Klaas J Hellingwerf, Filipe Dos Branco Dos Santos

{"title":"Enhancement of D-mannitol production by fine-tuned expression of mannitol-1-phosphate dehydrogenase in Synechocystis sp. PCC6803.","authors":"Wenyang Wu, Wei Du, Klaas J Hellingwerf, Filipe Dos Branco Dos Santos","doi":"10.1002/btpr.70027","DOIUrl":"https://doi.org/10.1002/btpr.70027","url":null,"abstract":"D-Mannitol production was achieved in freshwater Synechocystis sp. PCC6803 via the heterologous expression of mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p) under control of the strong promoter Ptrc1. However, only 5.54 mg L-1 of mannitol was found extracellularly after 7 days of cultivation, likely due to insufficient expression of a mutated mtlD lacking a methionine at position 332. This study compared mannitol levels using different promoters (Ptrc1, PpsbA2 and PnrsB) to control the expression of (un)mutated versions of mtlD in Synechocystis with co-expression of m1p. Our data suggest that even without the inducer, the weakest promoter, PnrsB, can support the expression of an unmutated mtlD in Synechocystis, which leads to 18.2 mg L-1 of mannitol in 7 days without induction. Such titer is already much higher than the first engineered mannitol-producing Synechocystis. When 5 μM nickel sulfate was added to the medium as an inducer, mannitol production could significantly be increased further, up to 92.9 mg L-1 after 7 days of induction, but it partially inhibited growth. Attempts with the other increasingly stronger promoters always failed to express the unmutated mtlD, probably due to the toxicity caused by the accumulation of the intermediate product, mannitol-1-phosphate. These results clearly suggest that the expression level of mtlD is the bottleneck in achieving a high yield of mannitol in Synechocystis, and consequently, that mannitol production can be enhanced by fine-tuning its expression. Future research is needed to identify bottlenecks that hinder mannitol productivity and long-term stability, facilitating the engineering of more efficient mannitol-producing cyanobacterial strains.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70027"},"PeriodicalIF":2.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762638","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

Triton CG-110 as an alternative for cell lysis in manufacturing of adeno-associated virus-based gene therapy.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-03-26 DOI: 10.1002/btpr.70025

Yixuan Ming, Tianyi Zhou, Bin Lu, Yemaiza Ojeda-Lassalle, Pasquale Valerio, Lu Wang, Mi Jin

引用次数: 0

Effect of nucleophilic additives on phosphoric acid pretreatment of lignocelluloses.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-03-25 DOI: 10.1002/btpr.70026

Xin Tan, Xuan Wu, Wei Wang, Jiale An, Qin Zhang, Song Tang, Bangxiang He, Chenhuan Lai, Yequan Sheng

{"title":"Effect of nucleophilic additives on phosphoric acid pretreatment of lignocelluloses.","authors":"Xin Tan, Xuan Wu, Wei Wang, Jiale An, Qin Zhang, Song Tang, Bangxiang He, Chenhuan Lai, Yequan Sheng","doi":"10.1002/btpr.70026","DOIUrl":"https://doi.org/10.1002/btpr.70026","url":null,"abstract":"The inhibition of lignin condensation during biomass pretreatment is crucial for enhancing enzymatic hydrolysis efficiency, since the formation of rigid cross-linked lignin networks hinders cellulose accessibility and enzyme activity. This study investigates the effects of nucleophilic additives, including ascorbic acid (AsA), 2-naphthol (2N), 3-hydroxy-2-naphthoic acid (3H2NA), and 2-naphthol-7-sulfonate (7S2NA), as potential agents to suppress lignin condensation on the phosphoric acid pretreatment of poplar. The phosphoric acid pretreatment demonstrated a remarkable efficacy in the removal of xylan (100%) and lignin (18.06%-31.35%) from poplar, both with and without the inclusion of nucleophilic additives. An enzymatic hydrolysis yield ranging from 71.41% to 100% was achieved with the incorporation of AsA, 2N, 3H2NA, and 7S2NA, compared to a yield of 66.15% for substrates pretreated solely with phosphoric acid. The enhancement in enzymatic hydrolysis yield upon the addition of nucleophilic additives was probably due to the improved cellulose accessibility and the enhanced proportion of cellulose II in the pretreated substrates. The analysis of total phenolic content in the prehydrolysates revealed that 3H2NA and 7S2NA, characterized by their strong hydrophilic groups within their chemical structures, significantly facilitated lignin fractionation during phosphoric acid pretreatment.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70026"},"PeriodicalIF":2.5,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699201","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

Iterative hybrid model based optimization of rAAV production.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-03-24 DOI: 10.1002/btpr.70006

Claudio Müller, Gerald Siegwart, Susanne Heider, Michael Sokolov, Angela Botros, Alexandra Umprecht, Moritz von Stosch, Mariano Nicolas Cruz Bournazou

{"title":"Iterative hybrid model based optimization of rAAV production.","authors":"Claudio Müller, Gerald Siegwart, Susanne Heider, Michael Sokolov, Angela Botros, Alexandra Umprecht, Moritz von Stosch, Mariano Nicolas Cruz Bournazou","doi":"10.1002/btpr.70006","DOIUrl":"https://doi.org/10.1002/btpr.70006","url":null,"abstract":"Changes in serotype or genetic payload of recombinant adeno associated virus (rAAVs) gene therapies require adapting the transfection conditions of the upstream HEK293 cultivations. This study adopts an iterative model-based experiment design approach, where increasing data availability is leveraged to evolve models of different complexity. Initial models based on data from shaker flask runs guided the design of the first round at Ambr250 scale. With Ambr250 data becoming available, hybrid models capturing process state evolutions and historical models incorporating these evolutions to predict rAAV titer, were developed. These models were then combined into a full model approach, which was utilized within a Bayesian Optimization framework for the design of a second round of Ambr250 scale runs. The iterative approach was tested across different projects applying transfer learning to enhance the predictive power and improve the subsequent optimization. The approach was benchmarked against a statistical Design of Experiment method. The results show that the model-based experiment design consistently (and across projects) produces higher rAAV titer values than the benchmark approach (Project C: 4.4% or 7.0% increases in titer values relative to the response surface modeling approach for ELISA and ddPCR, respectively; Project D: 32.4% or 10.9% increases in titer values relative to the standard DoE-screening pick for ELISA and ddPCR, respectively), effectively optimizing the transfection mixture composition. The combination of propagation and historical models, augmented by transfer learning and an ever-increasing amount of data, enhanced the process design workflow, contributing to improved rAAV production through efficient transfection strategies.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70006"},"PeriodicalIF":2.5,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699206","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

Synthetic modeling: A cell-free approach for faster implementation of Raman spectroscopy in cell culture.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-03-24 DOI: 10.1002/btpr.70018

Célia Sanchez, Hadi El Radi, Nathan Gay, Johan Cailletaud, Kévin Grollier, Fabrice Thomas, Thierry Gonthiez

{"title":"Synthetic modeling: A cell-free approach for faster implementation of Raman spectroscopy in cell culture.","authors":"Célia Sanchez, Hadi El Radi, Nathan Gay, Johan Cailletaud, Kévin Grollier, Fabrice Thomas, Thierry Gonthiez","doi":"10.1002/btpr.70018","DOIUrl":"https://doi.org/10.1002/btpr.70018","url":null,"abstract":"Monitoring cell culture is crucial for gaining a deeper understanding of processes and ensuring the production of safe and high-quality products. The capability to measure in real time several parameters of interest can be achieved with Raman spectroscopy. However, before using Raman spectroscopy to monitor a specific process, a calibration phase is required to develop chemometric models that correlate Raman spectra with the target parameters. It is mandatory to conduct this phase with multiple batches to build robust models that account for biological variability. This model building phase can be time-consuming and require a lot of resources. The industry is actively seeking solutions to simplify and expedite this step without compromising accuracy. Moreover, the current approach has limitations regarding changing cell culture media, celllines, or process scale. The novel synthetic model approach provides a significant gain of time and resources for the calibration phase, which is reduced to just a few days. The methodology involves using cell-free samples of cell culture media that are spiked with various concentrations of target compounds. The results indicate that the innovative approach enables accurate measurement for glucose and lactate parameters in real process conditions comparable to a standard modeling methodology.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e70018"},"PeriodicalIF":2.5,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143690733","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