Biotechnology Progress最新文献

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.

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.

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.

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.

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.

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.

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.

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.

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.

Induced pluripotent stem cells (iPSCs) offer significant therapeutic potential, but cryopreservation challenges, particularly the reliance on cytotoxic Dimethyl Sulfoxide (Me₂SO), hinder their clinical application. This review examines current cryopreservation practices in clinical and preclinical iPSC-based therapies, highlighting the consistent use of Me₂SO 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.