Search Results (11)

The production of influenza A virus (IAV) using Madin-Darby Canine Kidney (MDCK) cells is a key strategy for efficient influenza vaccine manufacturing. However, challenges remain in optimizing cell culture processes for higher yield and efficiency. This study aims to evaluate different process intensification strategies on two distinct clonal MDCK suspension cell lines (C59 and C113) for improved IAV production. A semi-perfusion strategy was used to push cells towards high cell density (HCD), achieving up to 17 × 10⁶ C113 cells/mL and 42 × 10⁶ C59 cells/mL, respectively. Next, a Tangential Flow Depth Filtration (TFDF)-based perfusion process with direct harvest during IAV production was established, resulting in high titers and a 10-fold higher space-time yield for C59 and a 4-fold improvement for C113 compared to batch operation. In addition, the suitability of N-1 perfusion was evaluated for batch and intensified fed-batch processes. Cells taken from the N-1 perfusion showed different cell-specific growth rates, but this had no effect on virus titers except for processes started from oxygen-deprived precultures. Finally, comparable virus titers were obtained when the production bioreactor was directly inoculated from an HCD cryovial. Taken together, seed train intensification and TFDF-based perfusion majorly reduced process times and improved IAV production. Full article

Intra-experimental factor setting shifts in intensified design of experiments (iDoE) enhance understanding of bioproduction processes by capturing their dynamics and are thus essential to fulfill quality by design (QbD) ambitions. Determining the influence of process history on the cellular responses, often referred to as memory effect, is fundamental for accurate predictions. However, the current iDoE designs do not explicitly consider nor quantify the influence of process history. Therefore, we propose the one-factor-multiple-columns (OFMC)-format for iDoE planning. This format explicitly describes stage-dependent factor effects and potential memory effects as across-stage interactions (ASIs) during a bioprocess. To illustrate its utility, an OFMC-iDoE that considers the characteristic growth phases during a fed-batch process was planned. Data were analyzed using ordinary least squares (OLS) regression as previously described via stage-wise analysis of the time series and compared to direct modeling of end-of-process outcomes enabled by the OFMC-format. This article aims to provide the reader with a framework on how to plan and model iDoE data and highlights how the OFMC-format simplifies planning, and data acquisition, eases modeling and gives a straightforward quantification of potential memory effects. With the proposed OFMC-format, optimization of bioprocesses can leverage which factor settings are most beneficial in which state of the mammalian culture and thus elevate performance and quality to the next level. Full article

Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder characterized by the insufficient production of the AAT protein. Due to availability limitations, not all AATD patients receive protein therapy treatment. In this study, the technoeconomic analysis of different processes (conventional and intensified) producing 200 kg/year of PEGylated recombinant AAT (PEG-AAT) using a Chinese hamster ovary cell line was investigated. All bioprocesses consist of upstream, downstream, and PEGylation sections. A base-case model (process A) of the conventional fed-batch production bioreactor was developed using SuperPro Designer software (Version 13) to evaluate the economic feasibility of the process. The cost of goods (COG) was estimated to be approximately USD 387.6/g. Furthermore, an intensified process (B) was modeled and evaluated to reduce the COG. Process intensification was implemented in the process (N-1 perfusion bioreactor). The specific operating COG for process B was found to be 10% less than that of process A. Scenario analysis was performed to assess the impact of process capacity (100–1000 kg/year) and cell-specific productivity (30–90 pg/cell/day). With an increase in process capacity, the specific operating COG was reduced for all processes. Increasing cell-specific productivity decreases the specific operating COG at different rates for each process, depending on the titer level. Future investigations into the PEGylation section are required since it has the highest COG of all the sections. Full article

Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism. Full article

Major efforts in the intensification of cell culture-based viral vaccine manufacturing focus on the development of high-cell-density (HCD) processes, often operated in perfusion. While perfusion operations allow for higher viable cell densities and volumetric productivities, the high perfusion rates (PR) normally adopted—typically between 2 and 4 vessel volumes per day (VVD)—dramatically increase media consumption, resulting in a higher burden on the cell retention device and raising challenges for the handling and disposal of high volumes of media. In this study, we explore high inoculum fed-batch (HIFB) and low-PR perfusion operations to intensify a cell culture-based process for influenza virus production while minimizing media consumption. To reduce product retention time in the bioreactor, produced viral particles were continuously harvested using a tangential flow depth filtration (TFDF) system as a cell retention device and harvest unit. The feeding strategies developed—a hybrid fed-batch with continuous harvest and a low-PR perfusion—allowed for infections in the range of 8–10 × 10⁶ cells/mL while maintaining cell-specific productivity comparable to the batch control, resulting in a global increase in the process productivity. Overall, our work demonstrates that feeding strategies that minimize media consumption are suitable for large-scale influenza vaccine production. Full article

Monoclonal antibodies are the workhorse of the pharmaceutical industry due to their potential to treat a variety of different diseases while providing high specificity and efficiency. As a consequence, a variety of production processes have been established within the biomanufacturing industry. However, the rapidly increasing demand for therapeutic molecules amid the recent COVID-19 pandemic demonstrated that there still is a clear need to establish novel, highly productive, and flexible production processes. Within this work, we designed a novel discontinuous process by combining two intensification strategies, thus increasing inoculation density and media exchange via a fluidized bed centrifuge, to fulfill the need for a flexible and highly productive production process for therapeutic molecules. To establish this new process, firstly, a small-scale experiment was conducted to verify synergies between both intensification strategies, followed by a process transfer towards the proof-of-concept scale. The combination of these two-process intensification measures revealed overall synergies resulting in decreased process duration (−37%) and strongly enhanced product formation (+116%) in comparison to the not-intensified standard operation. This led to an impressive threefold increase in space-time yield, while only negligible differences in product quality could be observed. Overall, this novel process not only increases the ways to react to emergency situations thanks to its flexibility and possible short development times, but also represents a possible alternative to the current established processes due to high increases in productivity, in comparison to standard fed-batch operations. Full article

The conventional fed-batch process characterized by a low titer currently challenges pharmaceutical development. Process optimization by applying a perfusion process in the pre-stage and subsequent production phase at a high seeding density (HSD) can meet this challenge. In this study, we employed a simplified approach based on measured experiments, namely segmented modeling, to systematically analyze an HSD fed-batch process compared to a standard process. A comparison indicated that the metabolic phases of HSD processes are not only shifted in time, but metabolite trends show an altered metabolism. In an extended study, we integrated the intracellular fluxes determined by a metabolic flux analysis into the segmented modeling approach. Compared to using only extracellular rates, similar phases are identified, and this highlights the reliability of phase identification modeling using extracellular rates only. Furthermore, the segmented linear regression approach is used to create a model that describes cellular behavior and that can be used to predict potential improvements in the feeding strategy and in harvest viability. Here, overfeeding was eliminated and a significantly higher titer was achieved. This work provides insights into the overall metabolic changes in the HSD process and paves the way towards the optimization of the feeding regime. Full article

White wine fermentations are typically performed in an entirely batchwise manner, with yeast nutrients only added at the beginning of fermentation. This leads to slow (2+ weeks) fermentation cycle times, with large capital expenditures required to increase winery processing capacity. Prior attempts to speed fermentations via increasing temperature have resulted in unpalatable wine, and continuous fermentation processing is uneconomical and impractical in the winery setting. In this work, we measured yeast nutrient consumption as a function of fermentation progression at the 300 mL scale, and from this derived an equation to optimize yeast nutrient concentration as a function of fermentation progression. These findings were applied at the pilot scale in 150 L fermentors, which resulted in a 60% cycle time reduction versus “best practices” control fermentations. The resultant wines were compared via GC-MS as well as by a trained sensory panel. Organoleptic analysis found statistically significant, but overall, small differences in sensory characteristics between the control and process intensified wines. This intensified fermentation process shows great promise for fermented beverage producers wishing to maximize equipment utilization and debottleneck wineries or other beverage fermentation facilities. Full article

The Kluyveromyces marxianus yeast recently has gained considerable attention due to its applicability in high-value-added product manufacturing. In order to intensify the biosynthesis rate of a target product, reaching high biomass concentrations in the reaction medium is mandatory. Fed-batch processes are an attractive and efficient way how to achieve high cell densities. However, depending on the physiology of the particular microbial strain, an optimal media composition should be used to avoid by-product synthesis and, subsequently, a decrease in overall process effi-ciency. Thus, the aim of the present study was to optimise the synthetic growth medium and feeding solution compositions (in terms of carbon, nitrogen, phosphorous, magnesium, and calcium concentrations) for high cell density K. marxianus fed‑batch cultivations. Additionally, the biomass yields from the vitamin mixture and other macro/microelements were identified. A model predictive control algorithm was successfully applied for a fed-batch cultivation control. Biomass growth and substrate consumption kinetics were compared with the mathematical model predictions. Finally, 2‑phenylethanol biosynthesis was induced and its productivity was estimated. The determined optimal macronutrient ratio for K. marxianus biomass growth was identified as C:N:P = 1:0.07:0.011. The maximal attained yeast biomass concentration was close to 70 g·L^-1 and the 2-PE biosynthesis rate was 0.372 g·L⁻¹·h⁻¹, with a yield of 74% from 2-phenylalanine. Full article

Increased aquaculture production is associated with a growing interest in improving fish welfare. For this reason, the search for strategies to mitigate stress has intensified, one of these strategies being food supplementation with amino acids. The objective of this study was to evaluate the effects of dietary phenylalanine (Phe) and Tyrosine (Tyr) on the stress response and metabolism of juvenile gilthead seabreams (Sparus aurata) and meagres (Argyrosomus regius). Fish batches were fed a control diet and two diets supplemented with 5% Phe or Tyr for seven days. At the end of the experiment fish were stressed by air exposure for 3 min and then sacrificed for the extraction of blood and brain. Classical plasma stress markers were analyzed (glucose, lactate, proteins, cortisol), as well as hormones derived from those amino acids (adrenaline, noradrenaline and dopamine). Despite interspecific differences, fish fed the diets supplemented with Phe or Tyr showed a reduction on several stress markers. However, interspecific differences were detected for many indicators. Concretely, hormonal stress markers were significantly attenuated in meagres fed the enriched diets. Moreover, the stress condition favored a mobilization of amino acids towards the brain, especially in supplemented diets, hence this amino acid excess could be used as an energy substrate to cope with stress. Full article

Intensified and accelerated development processes are being demanded by the market, as innovative biopharmaceuticals such as virus-like particles, exosomes, cell and gene therapy, as well as recombinant proteins and peptides will possess no available platform approach. Therefore, methods that are able to accelerate this development are preferred. Especially, physicochemical rigorous process models, based on all relevant effects of fluid dynamics, phase equilibrium, and mass transfer, can be predictive, if the model is verified and distinctly quantitatively validated. In this approach, a macroscopic kinetic model based on Monod kinetics for mammalian cell cultivation is developed and verified according to a general valid model validation workflow. The macroscopic model is verified and validated on the basis of four decision criteria (plausibility, sensitivity, accuracy and precision as well as equality). The process model workflow is subjected to a case study, comprising a Chinese hamster ovary fed-batch cultivation for the production of a monoclonal antibody. By performing the workflow, it was found that, based on design of experiments and Monte Carlo simulation, the maximum growth rate µ_max exhibited the greatest influence on model variables such as viable cell concentration X_V and product concentration. In addition, partial least squares regressions statistically evaluate the correlations between a higher µ_max and a higher cell and product concentration, as well as a higher substrate consumption. Full article