Biotechnol. Prog. [JOURNAL]
Sun
200 papers
Use of LC-MS to characterize host cell protein removal during depth filtration.
Chu LK, Borujeni EE, Xu X … +1 more , Zydney AL
Biotechnol Prog · 2025 · PMID 40406926 · Full text
The removal of host cell proteins (HCPs) is crucial in biopharmaceutical production, as residual impurities can impact product safety and efficacy. While a number of studies have demonstrated that depth filtration can pr...
The removal of host cell proteins (HCPs) is crucial in biopharmaceutical production, as residual impurities can impact product safety and efficacy. While a number of studies have demonstrated that depth filtration can provide significant HCP removal, there is little information on its effectiveness in removing specific HCPs. This study examines the application of liquid chromatography-mass spectrometry (LC-MS) to track HCP removal during depth filtration, providing a detailed analysis of HCP behavior with two commercial depth filters. Our findings reveal significant variability in HCP breakthrough behavior, with transmission patterns showing minimal correlation with either the protein isoelectric point or hydrophobicity, highlighting the unique behavior of individual HCPs. Both the X0SP and X0HC depth filters achieved almost complete removal of Lipoprotein Lipase, and the X0SP filter also effectively removed Lysosomal Acid Lipase (LAL), both known to degrade polysorbate in monoclonal antibody formulations. However, neither filter provided significant removal of Alpha-enolase, Carboxypeptidase D, Glutathione S-transferase, or Phospholipase B-like 2. The X0SP filter showed equal or better removal for 18 out of 20 problematic HCPs, with greater HCP removal seen at lower conductivity. This work provides a detailed framework for understanding and optimizing depth filtration processes, offering insights into the effectiveness of depth filters for removal of problematic HCPs.
Minimizing batch-to-batch variability of a live virus vaccine by process analytical technologies.
Forrester K, Blanda TR, Trauger M … +2 more , Thompson R, Templeton N
Biotechnol Prog · 2025 · PMID 40401434 · Full text
For bioprocesses producing live virus, such as enterovirus Coxsackievirus A21, viral titer (infectivity basis) decay rates can exceed 30% within a day. Consequently, harvest timing is paramount. To optimize titer at harv...
For bioprocesses producing live virus, such as enterovirus Coxsackievirus A21, viral titer (infectivity basis) decay rates can exceed 30% within a day. Consequently, harvest timing is paramount. To optimize titer at harvest, a continuous viral product titer model was generated to elucidate kinetics. The model leveraged experimentally determined viable cell density, cell-specific viral productivity, and viral specific decay rates. Next, three separate online process analytical technology (PAT) harvest triggers were developed to predict maximal viral titer. Finally, the PAT harvest triggers were tested alongside traditional time-based harvests. The harvest triggers utilized common bioprocessing tools - dissolved oxygen (DO) and capacitance probes - to track DO and viable cell volume (VCV) and derived a third parameter, cell-specific oxygen uptake rate. Harvesting with PAT triggers allowed for significantly improved batch-to-batch consistency. The standard deviation of harvest yield was reduced by 41% (DO), 56% (OUR) and 71% (capacitance) as compared to the industry standard time-based harvest. Even when a process deviation in inoculated cell density occurred, causing a significant shift in viral titer kinetics, the PAT harvest triggers yielded greater than 87% of peak titer. By comparison, the time-based harvest yielded 16%.
Special issue on integrated continuous biomanufacturing.
Walther J
Biotechnol Prog · 2026 · PMID 40396366 · Publisher ↗
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Transcriptomic functional characterization of recombinant adeno-associated virus producing cell line adapted to suspension-growth.
Kuo HJ, Srinivasan P, Lin YC … +4 more , Lu M, Rungkittikhun C, Zhang Q, Hu WS
Biotechnol Prog · 2025 · PMID 40396307 · Full text
Recombinant adeno-associated virus (rAAV) is a widely used delivery vehicle in gene therapy. A scalable production technology is essential for its wide clinical applications. We have taken a synthetic biology approach to...
Recombinant adeno-associated virus (rAAV) is a widely used delivery vehicle in gene therapy. A scalable production technology is essential for its wide clinical applications. We have taken a synthetic biology approach to generate HEK293-based cell lines which harbor integrated genetic elements encoding essential AAV and adenoviral helper components and can be induced to produce rAAV. Through cycles of cell line enhancement, a high rAAV productivity could be achieved. The cell lines, like their parental HEK293, grew adherently. For scalable production, cell cultivation in suspension is highly desirable. A producer cell line GX6B was adapted to suspension growth in serum-free medium (named GX6Bs). However, it had substantially reduced virus titer. Returning GX6Bs cells to adherent culture conditions using adherent medium and cultured stationarily brought the productivity back to close to the level of adherent GX6B. A survey of the transcriptome revealed that induction and rAAV production elicited a wide range of cellular changes in various functional classes, including host immune defense response and nucleosome organization. The response was more subdued in suspension-growing GX6Bs. Upon reverting to adherent growth, the cellular transcriptome change regained its vigor to be more similar to that seen in GX6B. The GX6Bs maintained in suspension serum-free conditions were then reverted to the adherent culture medium but under an agitated culture environment to keep suspension growth for rAAV production. The productivity returned to within 25%-50% of GX6B. This work demonstrated the feasibility of the suspension culture of synthetic cell lines for the expansion and production of rAAV.
Accelerating IND-enabling toxicology studies using protein products from stable pools or pools of clones in Chinese hamster ovary cells.
Jiang Y, Jiang L, Yang Z … +10 more , Liu X, Wang Y, Ying M, Huang H, Xu Y, Zhou H, Huang J, Gu X, Zhou W, Huang Y
Biotechnol Prog · 2025 · PMID 40384562 · Publisher ↗
In recent years, accelerating Chemistry, Manufacturing, and Controls (CMC) workflows for clinical entry has become a critical focus in biologics development. Advances in the development of cell lines, cell culture proces...
In recent years, accelerating Chemistry, Manufacturing, and Controls (CMC) workflows for clinical entry has become a critical focus in biologics development. Advances in the development of cell lines, cell culture processes, and analytical technologies have enabled the generation of more homogeneous stable pool populations with increased productivity. Leveraging the experience gained from the COVID-19 product development, the strategic use of stable cell pools or a pool of clones for early-stage non-GMP material generation and process development has proven transformative in significantly reducing the CMC timeline to investigational new drug (IND). This study provides a comprehensive comparison of bioprocess performance and product quality attributes of materials produced from stable pools or a pool of clones (toxicology study materials) versus those from clonally derived cells (GMP clinical batches) across six First-in-Human (FIH) programs involving mAbs, bsAb, and Fc-fusion proteins. The results demonstrate a strong alignment and the feasibility of using protein materials from stable pools or a pool of clones in toxicology studies. In conclusion, utilizing non-clonal CHO cell-derived material for preclinical studies offers a strategic approach that can be broadly applied to complex molecules across various disease areas, even under standard regulatory filings, accelerating the path to clinical trials.
Enhanced cell-specific productivity through delayed supplementation of antioxidants in intensified processes.
Wu S, Lu YA, Devenney K … +3 more , Kotzbauer E, Lee K, Tayi VS
Biotechnol Prog · 2025 · PMID 40326050 · Publisher ↗
Antioxidant supplementation to serum-free culture media is a common strategy to enhance productivity through oxidative stress alleviation. In this study, it was hypothesized that certain antioxidants can improve the spec...
Antioxidant supplementation to serum-free culture media is a common strategy to enhance productivity through oxidative stress alleviation. In this study, it was hypothesized that certain antioxidants can improve the specific productivity of a CHO-GS cell line expressing a bi-specific antibody. A fed-batch (FB) screening study investigated several antioxidants and revealed rosmarinic acid (RoA) and retinyl acetate (RAc), to a lesser extent, improved cell productivity. Contrary to the previous literature reports, the addition of RoA and/or RAc resulted in slower cell growth and reduced peak viable cell density, counteracting the enhanced specific productivity. We hypothesized that supplementing RoA/RAc after the exponential growth phase would increase titer through enhanced specific productivity without substantially impeding cell growth. This hypothesis was tested in three different ways: (1) supplementing RoA/RAc to the feed, rather than the basal media, in the FB process; (2) implementing the intensified fed-batch (iFB) process mode which started with high seeding VCD, bypassing the exponential cell growth phase; (3) supplementing RoA/RAc to the production phase perfusion media, rather than the growth phase perfusion media, in the perfusion-based continuous manufacturing (CM) process. All three methods were proven effective in titer improvement, which supported the hypothesis. Additionally, RoA/RAc significantly impacted product quality, with variations depending on the process mode and components. Overall, their supplementation led to decreased N-glycan mannose percentage and increased product fragmentation and aggregation. These changes do not fully align with the previous reports, highlighting that the supplementation strategy needs to be evaluated carefully based on cell line and expressed molecule type.
Techno-economic analysis of membrane-based continuous capture chromatography platforms for large-scale antibody production.
Romero JJ, Jenkins EW, Birtwistle MR … +1 more , Husson SM
Biotechnol Prog · 2025 · PMID 40270480 · Full text
Continuous manufacturing platforms and membrane chromatography are process technologies with the potential to reduce production costs and minimize process variability in monoclonal antibody production. This study present...
Continuous manufacturing platforms and membrane chromatography are process technologies with the potential to reduce production costs and minimize process variability in monoclonal antibody production. This study presents a simulation and optimization framework to perform techno-economic analyses of these strategies. Multi-objective optimization was used to compare batch and continuous multicolumn operating modes and membrane and resin process alternatives, revealing performance differences in productivity and cost of goods attributed to variations in dynamic binding capacity, media geometry, and process residence time. From the set of optimal process configurations, we selected one membrane and one resin platform alternative yielding the highest net present values to undergo sensitivity analyses involving variations in batch cadence and product selling price. For the scenarios considered in this work, membrane continuous platforms showed benefits in the cost of goods and process mass intensity. Their shorter residence time compared to resins positions them as a viable alternative for single-use capture chromatography. Moreover, this low residence time makes membrane platforms more flexible to changes in throughput, an essential feature for integrating capture into fully continuous processes.
A novel multiparameter sensor for shake flask cultivations: Online biomass, dissolved oxygen, and fluorescence monitoring for comprehensive bioprocess characterization.
Strehl L, Kuhn AL, Hoffmann K … +2 more , Mann M, Magnus JB
Biotechnol Prog · 2025 · PMID 40265675 · Full text
Shake flasks are one of the most widely used cultivation vessels in biotechnological process development. To improve the process understanding, new technologies have been reported for online monitoring of different param...
Shake flasks are one of the most widely used cultivation vessels in biotechnological process development. To improve the process understanding, new technologies have been reported for online monitoring of different parameters like oxygen, pH, or biomass in the last couple of years. However, most reports address the monitoring of a single parameter per shake flask. This work evaluates the ability to measure dissolved oxygen (DO), biomass, and fluorescence in parallel with a new Multiparameter Sensor (MPS). Therefore, abiotic tests for reproducibility, sensitivity, and accuracy were performed. In biological tests, different microbial systems were used to evaluate if a wide range of applications is feasible. This work demonstrates that three different parameters: DO, biomass, and fluorescence can be monitored online, in parallel, for various biological systems. The online data obtained provide crucial process knowledge, such as the start of intracellular product formation. Abiotic and biological tests showed good reproducibility, resolution, and sensitivity to changing environmental conditions. Compared to other existing measurement systems for DO or oxygen transfer rate, similar or in the former case, more data points can be recorded, allowing a detailed overview and a better understanding of the process.
Modulation of the cell cycle and inhibition of histone deacetylases by small molecules increase recombinant adeno-associated virus productivity across different HEK293 cell lines.
Krämer N, Teschner K, Buve A … +4 more , Scheller L, Brinkert P, Ortseifen V, Klausing S
Biotechnol Prog · 2025 · PMID 40259814 · Publisher ↗
Recombinant adeno-associated viruses (rAAV) are one of the most popular gene therapy vectors. To date, low-product yields are limiting a broader clinical application. To identify targets for improving productivity, two h...
Recombinant adeno-associated viruses (rAAV) are one of the most popular gene therapy vectors. To date, low-product yields are limiting a broader clinical application. To identify targets for improving productivity, two human embryonic kidney cell lines (HEK293) with varying productive profiles were transiently transfected for rAAV2 production and transcriptomes were compared at 18 h after transfection. As expected, high-producing cell lines exhibited elevated levels of plasmid-derived viral gene expression. Gene set enrichment analysis indicated that these cells demonstrated increased transcriptional activity and upregulation of mRNA-processing mechanisms. Furthermore, transcriptomic analysis suggested increased transcription of histone-coding genes and a modulated cell cycle under the influence of viral gene expression, with differences being more prominent in the high-producer cell line. Aiming to increase rAAV yield, cyclin-dependent kinases and histone deacetylases were targeted by treatment with the small molecule inhibitors Flavopiridol and M344, respectively. Without compromising biological activity, Flavopiridol increased rAAV titer by 2-fold, and M344 increased it up to 8-fold in a cell line-independent manner, while also enhancing the percentage of filled capsids. A DoE-based approach also revealed the potential for combining both molecules to enhance rAAV production, exhibiting an additive effect across three different HEK293 derivatives. Consequently, novel functions of M344 and Flavopiridol as enhancers of rAAV production were unraveled, which can be employed to enhance the accessibility of in vivo gene therapy applications.
Improving trans-cinnamic acid production in a model cyanobacterium.
Hunstiger D, Ma H, Paton AJ … +1 more , Peebles CAM
Biotechnol Prog · 2025 · PMID 40235106 · Full text
trans-Cinnamic acid (tCA) is a precursor in the synthesis of many high-value compounds with bio-active qualities useful in applications like medicine, polymers, and cosmetics. Currently tCA is produced by industrial chem...
trans-Cinnamic acid (tCA) is a precursor in the synthesis of many high-value compounds with bio-active qualities useful in applications like medicine, polymers, and cosmetics. Currently tCA is produced by industrial chemical synthesis from fossil fuels or cost-prohibitive isolation from terrestrial plants. Cyanobacteria, a type of photosynthetic bacteria, can be readily engineered to convert sunlight and carbon dioxide into metabolites of interest at relatively high amounts compared to terrestrial plants. The purpose of this study is to advance the industrial and commercial value of cyanobacteria as a biological factory for renewable production of tCA. Production of tCA has previously been demonstrated in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) via expression of non-native phenylalanine ammonia lyase (PAL) from various organisms. This project focuses on developing and characterizing a new high-titer strain of S. 6803 expressing a plant PAL gene controlled by an inducible promoter. We assessed production in shake flasks under constant light, a 12 h:12 h light:dark cycle, and environmental photobioreactors (ePBRs) with a sinusoidal, rapidly fluctuating light environment. Our strain demonstrates a four-fold increase in tCA production to ~500 mg L by 14 days compared to previously reported titers in S. 6803 under shake flask cultivation and a 30-50% improved average tCA production per culture density (60 mg·L·OD ) in ePBRs over comparable previously reported culture methods. Our study progresses S. 6803 tCA bioproduction into higher culture volumes, up to 500 mL, while further validating the strength of an inducible system for tCA production in S. 6803.
Continuous purification of a parvovirus using two aqueous two-phase extraction steps.
Nold NM, Waldack S, James G … +9 more , Colling T, Manchester L, Sarvari T, Bekkala A, Kriz SA, Baldwin M, Agustin-Mazariegos E, Betenbaugh MJ, Heldt CL
Biotechnol Prog · 2025 · PMID 40231435 · Full text
Aqueous two-phase systems (ATPS) are a liquid-liquid extraction method that offers low-cost, continuous-adaptable virus purification. A two-step ATPS using polyethylene glycol (PEG) and sodium citrate that recovered 66%...
Aqueous two-phase systems (ATPS) are a liquid-liquid extraction method that offers low-cost, continuous-adaptable virus purification. A two-step ATPS using polyethylene glycol (PEG) and sodium citrate that recovered 66% of infectious porcine parvovirus with 2.0 logs of protein removal and 1.0 logs of DNA removal in batch has now been run continuously. The continuous system output of <10 ng/mL DNA regardless of starting DNA titer agreed with batch studies. However, the continuous system had a five-fold higher contaminating protein titer than batch studies, likely because of incomplete mixing or settling. Turbidity was tested as a measure of mixing and settling efficiency. Monitoring in-line absorbance at 880 nm directly after mixing and before collection in the settling reservoir could track both mixing and settling during operation. Settling time was reduced by changing the settling line material from PVC to PTFE, which is more hydrophobic. A flow-through AEX filter tested to make impurity removal more robust recovered 90% of PPV and removed an additional 87% of host cell DNA. The filter did not add any additional protein removal. In the future, in-line absorbance sensors could be implemented along with conductivity sensors to measure salt concentration, refractive index sensors to track the PEG-citrate interface, and scales to track mixer and reservoir volumes to enable automated, continuous ATPS. Our vision is to integrate continuous ATPS into a fully continuous end-to-end production for viral vectors.
Scalable process development for rAAV transient transfection production using computational fluid dynamics modeling.
Ou J, Tang Y, Williams A … +7 more , Huang Y, Shimansky R, Salinas G, Keil G, Lee J, Borys MC, Khetan A
Biotechnol Prog · 2025 · PMID 40183256 · Publisher ↗
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 s...
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/m 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/m 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/m 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.
N-1 semi-continuous transient perfusion in shake flask for ultra-high density seeding of CHO cell cultures in benchtop bioreactors.
Lemire L, Reyes SJ, Durocher Y … +3 more , Voyer R, Henry O, Pham PL
Biotechnol Prog · 2025 · PMID 40176554 · Full text
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 10 viable cells/mL...
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 10 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.
Continuous glucose feedback control using Raman spectroscopy and deep learning models for biopharmaceutical processes.
Rashedi M, Demers M, Khodabandehlou H … +3 more , Wang T, Garvin C, Rianna S
Biotechnol Prog · 2025 · PMID 40172019 · Publisher ↗
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 convolutio...
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.
Cryopreservation practices in clinical and preclinical iPSC-based cell therapies: Current challenges and future directions.
Dobruskin M, Toner G, Kander R
Biotechnol Prog · 2025 · PMID 40171754 · Full text
Induced pluripotent stem cells (iPSCs) offer significant therapeutic potential, but cryopreservation challenges, particularly the reliance on cytotoxic Dimethyl Sulfoxide (MeSO), hinder their clinical application. This r...
Induced pluripotent stem cells (iPSCs) offer significant therapeutic potential, but cryopreservation challenges, particularly the reliance on cytotoxic Dimethyl Sulfoxide (MeSO), hinder their clinical application. This review examines current cryopreservation practices in clinical and preclinical iPSC-based therapies, highlighting the consistent use of MeSO and the logistical challenges of post-thaw processing. The findings underscore the urgent need for alternative cryopreservation techniques to ensure the safety and efficacy of off-the-shelf iPSC therapies.
N-acetyl-D-mannosamine, a novel additive, effectively reducing high mannose glycosylation of monoclonal antibody without affecting other quality attributes.
Chai M, Shu H, Wang Q … +5 more , Tian C, Wang L, Fan Y, Sun R, Zhou H
Biotechnol Prog · 2025 · PMID 40171753 · Publisher ↗
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 approa...
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.
Enhancement of D-mannitol production by fine-tuned expression of mannitol-1-phosphate dehydrogenase in Synechocystis sp. PCC6803.
Wu W, Du W, Hellingwerf KJ … +1 more , Dos Branco Dos Santos F
Biotechnol Prog · 2025 · PMID 40171712 · Full text
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 promote...
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 P. However, only 5.54 mg L 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 (P, P and P) 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, P, can support the expression of an unmutated mtlD in Synechocystis, which leads to 18.2 mg L 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 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.
Triton CG-110 as an alternative for cell lysis in manufacturing of adeno-associated virus-based gene therapy.
Ming Y, Zhou T, Lu B … +4 more , Ojeda-Lassalle Y, Valerio P, Wang L, Jin M
Biotechnol Prog · 2025 · PMID 40135817 · Publisher ↗
Triton X-100 is one of the most widely used detergents for cell lysis in gene therapy product manufacturing. However, due to the aquatic toxicity of the degradation product, Triton X-100 is regulated by the European Chem...
Triton X-100 is one of the most widely used detergents for cell lysis in gene therapy product manufacturing. However, due to the aquatic toxicity of the degradation product, Triton X-100 is regulated by the European Chemical Agency as a substance of very high concern. In this study, we aim to identify eco-friendly detergent alternatives to replace Triton X-100. Tween 20, Tween 80, n-Dodecyl-β-D-maltoside (DDM), and Triton CG-110 were tested and compared with Triton X-100. Triton CG-110 demonstrated similar performance during the harvest and subsequent purification process for multiple AAV vectors at different scales. Therefore, Triton CG-110 represents an effective and environmentally safe detergent alternative to Triton X-100.
Effect of nucleophilic additives on phosphoric acid pretreatment of lignocelluloses.
Tan X, Wu X, Wang W … +6 more , An J, Zhang Q, Tang S, He B, Lai C, Sheng Y
Biotechnol Prog · 2025 · PMID 40130451 · Publisher ↗
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 en...
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.
Iterative hybrid model based optimization of rAAV production.
Müller C, Siegwart G, Heider S … +5 more , Sokolov M, Botros A, Umprecht A, von Stosch M, Cruz Bournazou MN
Biotechnol Prog · 2025 · PMID 40129076 · Full text
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-b...
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.
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