Biotechnology Progress最新文献

{"title":"Multi-omics kinetic analysis of recombinant adeno-associated virus production by plasmid transfection of HEK293 cells","authors":"Min Lu,&nbsp;Zion Lee,&nbsp;Wei-Shou Hu","doi":"10.1002/btpr.3428","DOIUrl":"10.1002/btpr.3428","url":null,"abstract":"<p>Recombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and <i>rep</i> and <i>cap</i> genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics-guided productivity enhancement. Together, multi-omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3428","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139575217","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}

{"title":"Evaluating end-to-end continuous antibody manufacture with column-free capture alternatives from economic, environmental, and robustness perspectives","authors":"Catarina P. G. Neves,&nbsp;Jonathan L. Coffman,&nbsp;Suzanne S. Farid","doi":"10.1002/btpr.3427","DOIUrl":"10.1002/btpr.3427","url":null,"abstract":"<p>Process intensification efforts have renewed interest in the potential of end-to-end continuous manufacture with column-free capture alternatives. This article describes a decisional tool that encompasses mass balance and design equations, process economics, stochastic simulation and multi-criteria decision-making and enables the evaluation of different batch, and continuous flowsheets for monoclonal antibody (mAb) manufacture. The traditional batch process was compared with end-to-end continuous bioprocesses with either protein A capture or column-free capture employing aqueous two-phase extraction or precipitation from economic, environmental, and robustness perspectives. The cost of goods analysis predicted that continuous flowsheets could offer substantial cost savings (~20%–40%) over the batch process at low and medium annual commercial demands (100–500 kg); however, at tonnage demands they resulted in either comparable or higher costs. Comparing the continuous options, the continuous flowsheets with protein A or precipitation yielded similar COG/g values, while aqueous two-phase extraction presented higher costs. The analysis of overall process mass intensities accounting for water and consumables suggested that the continuous flowsheet with protein A would result in the lowest environmental burden. When the economic, environmental, and operational criteria were reconciled using multi-criteria decision-making analysis, the continuous protein A-based flowsheet was found to be the most favorable. A target analysis highlighted the need for process improvements in the following parameters to reduce the manufacturing costs of the continuous column-free capture options below that of protein A: the perfusion volumetric productivity, the harvested cell culture fluid percentage in column-free operations, the column-free step yields along with the implementation of buffer concentrates.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3427","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139575210","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}

{"title":"A custom 3D printed paddlewheel improves growth in flat panel photobioreactor","authors":"Michelle Meagher,&nbsp;Jacob Tamburro,&nbsp;Nanette R. Boyle","doi":"10.1002/btpr.3430","DOIUrl":"10.1002/btpr.3430","url":null,"abstract":"<p>One of the main challenges with using flat panel photobioreactors for algal growth is uneven mixing and settling of cells in corners, especially when bubbling is the only method used for mixing. In order to improve mixing in our flat panel reactor, we designed a custom paddlewheel. Paddlewheels are frequently used in outdoor algae raceway ponds to improve mixing and we are taking advantage of the same principle for mixing in the reactor. The paddlewheel is easily integrated into our PSI FMT150 1-L flat panel photobioreactor and is printed on a 3D printer using high temperature poly lactic acid (HT-PLA). With the inclusion of an annealing step, the paddlewheel is autoclavable. Addition of the paddlewheel in the reactor minimized cell settling and improved algal growth, as evidenced by a nearly 40% increase in oxygen production rates. Nutrient dispersion and utilization in the culture was also improved as evidenced by a corresponding 38% decrease in CO₂ concentration. The paddlewheel device presented here is a cost-effective method for improving algal growth in a flat panel photobioreactor.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139511608","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}

{"title":"Transitioning from static to suspension culture system for large-scale production of xeno-free extracellular vesicles derived from mesenchymal stromal cells","authors":"Natália Cristine Dias dos Santos,&nbsp;Paula Bruzadelle-Vieira,&nbsp;Nádia de Cássia Noronha,&nbsp;Amanda Mizukami-Martins,&nbsp;Maristela Delgado Orellana,&nbsp;Maria Vitória L. B. Bentley,&nbsp;Dimas Tadeu Covas,&nbsp;Kamilla Swiech,&nbsp;Kelen Cristina Ribeiro Malmegrim","doi":"10.1002/btpr.3419","DOIUrl":"10.1002/btpr.3419","url":null,"abstract":"<p>Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC-EV in suspension cultures using spinner flasks and human collagen-coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T-flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold-increase was 5.7-fold for the 3D culture system and 4.6-fold for the 2D culture system, signifying a fold-change of 1.2 (calculated as the ratio of fold-increase 3D to fold-increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T-flask cultures. The total cell production in the spinner flask cultures was 5.2-fold higher than that in T-flask cultures. While the EV specific production (particles/cell) in T-flask cultures (4.40 ± 1.21 × 10⁸ particles/mL, <i>p</i> &lt; 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 10⁸ particles/mL, <i>p</i> &lt; 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC-EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC-EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno-free conditions.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139511609","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}

{"title":"Control strategy for biopharmaceutical production by model predictive control","authors":"Touraj Eslami,&nbsp;Alois Jungbauer","doi":"10.1002/btpr.3426","DOIUrl":"10.1002/btpr.3426","url":null,"abstract":"<p>The biopharmaceutical industry is rapidly advancing, driven by the need for cutting-edge technologies to meet the growing demand for life-saving treatments. In this context, Model Predictive Control (MPC) has emerged as a promising solution to address the complexity of modern biopharmaceutical production processes. Its ability to optimize operations and ensure consistent product yields has made it an attractive option for manufacturers in this sector. Furthermore, MPC's alignment with the Process Analytical Technology (PAT) initiative provides an additional layer of assurance, facilitating real-time monitoring and enabling swift adjustments to maintain process integrity. This comprehensive review delves into the various applications of MPC, ranging from robust control to stochastic model predictive control, thereby equipping biotechnologists and process engineers with a powerful toolset. By harnessing the capabilities of MPC, as elucidated in this review, manufacturers can confidently navigate the intricate bioprocessing landscape and unlock this approach's full potential in their production processes.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3426","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139416270","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}

{"title":"Adaptive modeling optimized by the data fusion strategy: Real-time dying cell percentage prediction using capacitance spectroscopy","authors":"Suyang Wu,&nbsp;Stephanie A. Ketcham,&nbsp;Claudia Corredor,&nbsp;Douglas Both,&nbsp;Yuxiang Zhao,&nbsp;James K. Drennen,&nbsp;Carl A. Anderson","doi":"10.1002/btpr.3424","DOIUrl":"10.1002/btpr.3424","url":null,"abstract":"<p>The previous research showcased a partial least squares (PLS) regression model accurately predicting cell death percentages using in-line capacitance spectra. The current study advances the model accuracy through adaptive modeling employing a data fusion approach. This strategy enhances prediction performance by incorporating variables from the Cole–Cole model, conductivity and its derivatives over time, and Mahalanobis distance into the predictor matrix (X-matrix). Firstly, the Cole–Cole model, a mechanistic model with parameters linked to early cell death onset, was integrated to enhance prediction performance. Secondly, the inclusion of conductivity and its derivatives over time in the X-matrix mitigated prediction fluctuations resulting from abrupt conductivity changes during process operations. Thirdly, Mahalanobis distance, depicting spectral changes relative to a reference spectrum from a previous time point, improved model adaptability to independent test sets, thereby enhancing performance. The final data fusion model substantially decreased root-mean squared error of prediction (RMSEP) by around 50%, which is a significant boost in prediction accuracy compared to the prior PLS model. Robustness against reference spectrum selection was confirmed by consistent performance across various time points. In conclusion, this study illustrates that the data fusion strategy substantially enhances the model accuracy compared to the previous model relying solely on capacitance spectra.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139097222","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}

{"title":"Chitosan based hybrid superabsorbent for controlled drug delivery application","authors":"Medha,&nbsp;Sapna Sethi","doi":"10.1002/btpr.3418","DOIUrl":"10.1002/btpr.3418","url":null,"abstract":"<p>In the present study, a hybrid chitosan-alginate superabsorbent is prepared using maleic acid as a cross-linker and acrylamide as a grafting agent using the free radical mechanism. The composite hydrogel shows good swelling capacity along with hemocompatibility and biocompatibility and hence it is utilized as a drug delivery device. The characterization techniques including x-ray diffraction, Fourier transform infrared, x-ray photoelectron spectroscopy, and thermal analysis indicate the successful synthesis of stable hydrogel with rich functionalities. Metformin hydrochloride is used as a model drug which is used to treat diabetes. The drug encapsulation is done using the swelling diffusion method after the synthesis of hydrogel. The release of metformin from the drug-loaded hydrogel at physiological pH highlights the role of non-covalent interactions between the drug and hydrogel. In vitro release studies of Metformin from the drug-loaded hydrogel show higher release profiles at intestinal pH (7.4) compared to stomach pH (1.2). The observed cumulative release is 82.71% at pH 7.4 and 45.67% at pH 1.2 after 10 h. Brunauer–Emmett–Teller analysis reveals the effect of surface area, pore size, and pore volume of hydrogel on the drug release. The drug release from the hybrid chitosan-alginate hydrogel is found to be more sustained in comparison to the pure chitosan hydrogel. For the present drug delivery system, the swelling-controlled release is found to be more dominating than the pH-controlled release. The synthesized hydrogel can be successfully employed as a potential drug delivery system for controlled drug delivery.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139085721","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}

{"title":"Impedance nanobiosensor based on enzyme-conjugated biosynthesized gold nanoparticles for the detection of Gram-positive bacteria","authors":"Sarani Sen,&nbsp;Priyabrata Sarkar","doi":"10.1002/btpr.3421","DOIUrl":"10.1002/btpr.3421","url":null,"abstract":"<p>In this report, gold nanoparticles (GNPS) were synthesized using cell-free extracts of seven different isolates, namely, <i>Pseudomonas aerogenosa</i> CEBP2, <i>Pseudomonas</i> sp. CEBP1, <i>Pseudomonas pseudoalcaligenes</i> CEB1G, <i>Acinetobactor baumani</i> CEBS1, <i>Cuprividus</i> sp. CEB3, <i>Micrococcus luteus</i> CUB12, and <i>Pandoraea</i> sp. CUB2S. The spectroscopic (UV–vis, FTIR, DLS, XRD, EDS) and microscopic (FESEM, TEM) results confirm the reduction of Au³⁺ to Au⁰ in the presence of biomolecules having reducing as well as self-stabilizing activity. In this green synthesis approach, the average particle size of biosynthesized GNPS might vary (4–60 nm) depending on the bacterial species, pH of the media, incubation time, and temperature. In this study, GSH-modified BSGNPs (Au-GSH) have shown antimicrobial activity with better stability against Gram-positive bacteria. After conjugation of lysozyme with Au-GSH (lyso@Au-GSH), the zone of inhibition was enhanced from 12 to 23 mm (Au-GSH). The TEM study shows the spherical GNP (16.65 ± 2.84) turns into a flower-shaped GNP (22.22 ± 3.12) after conjugation with lysozyme due to the formation of the protein corona. Furthermore, the nanobioconjugate (lyso@Au-GSH) was immobilized with Nafion on a glassy carbon electrode to fabricate a label-free impedance biosensor that is highly sensitive to monitor changes in the transducer surface due to biomolecular interactions. The uniquely designed biosensor could selectively detect Gram-positive bacteria in the linear range of 3.0 × 10¹–3 × 10¹⁰ cfu mL⁻¹ with RE &lt;5%. The proposed simplest biosensor exhibited good reproducibility (RSD = 3.1%) and excellent correlation (<i>R</i>² = 0.999) with the standard plate count method, making it suitable for monitoring Gram-positive bacterial contamination in biofluids, food, and environmental samples.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139071351","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}

{"title":"Adapting virus filtration to continuous processing: Effects of product and process variability on filtration performance","authors":"Julie Kozaili,&nbsp;William Rayfield,&nbsp;Adrian Gospodarek,&nbsp;Mark Brower,&nbsp;Daniel Strauss","doi":"10.1002/btpr.3407","DOIUrl":"10.1002/btpr.3407","url":null,"abstract":"<p>Virus filtration (VF) is an important unit operation in the manufacture of biotherapeutics that provides robust removal of potential virus contaminants. Small virus removal can be impacted by the low operating pressures and potential depressurization events that are often associated with continuous operations where increased operational flexibility for higher loading at low flux and low pressure is required. In this study, we evaluated the impact of low flux (7 LMH) and pressure interruptions on minute virus of mice (MVM) removal. We used long-term filtrations conducted to a target throughput of 1000 L/m² with four different monoclonal antibodies on small-scale hollow fiber virus filters with a hydrophilic modified polyvinylidene fluoride membrane. These conditions are certainly challenging for any VF operation and ensuring robust viral clearance under such conditions is critical to the design and implementation of continuous VF. Planova BioEX filters effectively removed MVM at 4 log or greater when run continuously for up to 6 days. Interestingly, pressure increases associated with filter fouling over the duration of long-term filtrations were shown to be reflective of load material variability and could be remediated by implementation of an inline prefilter. Pressure interruptions had minimal impact on overall MVM logarithmic reduction value. Effective virus removal was achieved with pressure increases being largely product-specific, which demonstrates the capability of the virus filter to remove virus independent of pressure increases that are expected to occur with increased protein load.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3407","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139037403","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}

{"title":"Effect of pH, NaCl concentration, and mAb concentration of feed solution on the filterability of Planova™ 20N and Planova™ BioEX","authors":"Chie Hashikawa-Muto,&nbsp;Yoshiro Yokoyama,&nbsp;Ryo Hamamoto,&nbsp;Kazuya Kobayashi,&nbsp;Yumiko Masuda,&nbsp;Koichi Nonaka","doi":"10.1002/btpr.3420","DOIUrl":"10.1002/btpr.3420","url":null,"abstract":"<p>Virus filtration is one of the most important steps in ensuring viral safety during the purification of monoclonal antibodies (mAbs) and other biotherapeutics derived from mammalian cell cultures. Regarding the various virus retentive filters, including Planova filters, a great deal of data has been reported on the virus retention capability and its mechanism. Along with the virus retention capability, filterability is a key performance indicator for designing a robust and high-throughput virus filtration step. In order to obtain higher filterability, optimization of the feed solution conditions, and filter selection is essential; however, limited data are available regarding the filtration characteristics of Planova filters. Furthermore, for Planova 20N and Planova BioEX, the virus retention characteristics were reported to differ due to their respective membrane materials and layer structures. Whether these filters differ in their filtration characteristics is an interesting question, but no comparative evaluations have been reported. In this study, the filterability of the two filters was investigated and compared using 15 feed mAb solutions of a single mAb selected by design of experiments with different combinations of pH, NaCl concentration, and mAb concentration. The filterability of Planova 20N was affected not only by the feed solution viscosity, but also by the mAb aggregate content of the feed mAb solution and mAb-membrane electrostatic interactions. In contrast, the filterability of Planova BioEX decreased under some buffer conditions. These findings and the established design spaces of these filters provide valuable insights into the process optimization of virus filtration.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3420","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139037404","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}