The paper proposes a modular-based approach to constraint handling in process optimization and control. This is partly motivated by the recent interest in learning-based methods, e.g., within bioproduction, for which constraint handling under uncertainty is a challenge. The proposed constraint handler, called predictive filter, is combined with an adaptive constraint margin and a constraint violation cost monitor to minimize the cost of violating soft constraints due to model uncertainty and disturbances. The module can be combined with any controller and is based on minimally modifying the controller output, in a least squares sense, such that constraints are satisfied within the considered horizon. The proposed method is computationally efficient and suitable for real-time applications. The effectiveness of the method is illustrated through a realistic case study of glycosylation constraint satisfaction in continuous monoclonal antibody biosimilar production using Chinese hamster ovary cells, employing a metabolic network model consisting of 23 extracellular metabolites and 126 reactions. In the case study, the average constraint-violation cost is reduced by more than 60% compared to the case without the proposed constraint-handling method.

Gene therapies are transforming modern medicine, with adeno-associated virus (AAV) being the most used vector for delivery. High manufacturing costs however continue to limit drug discovery, clinical development, and patient access. Plasmid DNA (pDNA) has been identified as a critical bottleneck in AAV development and small-scale production. In this study, we compared conventional manual plasmid purification kits with a scalable, automated purification system across Maxi, Mega, and Giga scales. Time savings increased with purification scale, e.g., when purifying 4 plasmid samples at Giga scale (10 mg column plasmid capacity), 8 h of active time was saved using the automated system and total time was cut from almost 10 h to 5 h, although with generally lower yields (3–67% lower) and higher endotoxin levels. Both purification methods produced comparable AAV9 titres using standard PEI-based transfection of HEK293F cells. Surprisingly the AAV-MAX production system, plasmids purified by the automated method generated 14-fold higher titres than manually purified plasmids (3.2 ×10 ¹¹ vg/ml vs. 2.3 ×10 ¹⁰ vg/ml, respectively). Further analyses, including single and triple plasmid transfections and dynamic light scattering, revealed that the elution buffer used in the automated system formed larger pDNA transfection complexes in the AAV-MAX system and resulted in increased transfection efficiency. These findings provide insights that extend beyond AAV manufacturing, highlighting opportunities to optimise the transient production of other biologics and further improvement of plasmid preparation methods.

Human Mesenchymal Stromal Cells (hMSCs) are a safe option for allogeneic cell therapy across various diseases, but their manufacturing process requires improvement to broaden accessibility. In this study, a state-of-the-art planar multi-vessel process was transferred to a stirred tank bioreactor using microcarriers to support the growth of adherent cells. The frequent medium exchange strategy from planar culture guided the design of the bioreactor process. However, complete medium changes in the bioreactor resulted in limited cell expansion and higher glucose consumption compared to planar culture. To enhance expansion, a 0.1 L perfusion bioreactor was tested, enabling continuous medium exchange. Three perfusion approaches were evaluated: (1) maintaining a target cell-specific glucose consumption rate, (2) varying the perfusion rate, and (3) applying a cell-specific perfusion rate. Implementing targeted glucose feeding (TAFE) reduced lactate production, while increasing perfusion rates improved cell density. The highest expansion was achieved using a cell-specific perfusion rate of 5 nL cell- 1 day- 1 combined with a target glucose consumption rate (qglc) of 15 pmol cell- 1 day- 1, resulting in a 5.4-fold higher expansion factor than daily medium changes in stirred tank bioreactors. This optimized process represents a key advancement toward producing clinically relevant quantities of hMSCs.

Continuous centrifugation is a well-established method for clarifying mammalian cell cultures, but traditional batch-based approaches often fall short of modern biomanufacturing's scalability and flexibility demands. Increasing variability in product volumes and manufacturing setups calls for adaptable, scalable solutions. To address this, we developed a scaled-down continuous centrifuge ("Mini") based on a commercial disc-stack centrifuge, facilitating efficient early-stage development and improving technology transfer and scale-up. This study demonstrates the Mini's potential to bridge the gap between small-scale optimization and industrial-scale centrifugation. Proof-of-concept experiments with Chinese hamster ovary cell culture confirmed its separation efficiency, achieving low turbidity, high product recovery (up to 98.5%), and minimal cell stress. Lactate dehydrogenase activity remained low, with a maximum increase in host cell proteins of 11.9% across various operating conditions. Validation experiments against the pilot-scale Culture OneTMPrimo showed comparable or superior turbidity reduction and lower lactate dehydrogenase activity, highlighting the Mini's gentle cell handling. The Mini enables continuous small-scale centrifugation while replicating key performance parameters of the pilot-scale system, ensuring accurate performance predictions and reliable scale-up. It provides a scalable, flexible solution that meets the evolving needs of modern biomanufacturing for efficient and adaptable clarification processes.

Natural killer (NK) cell therapies hold great promise for cancer treatment; however, donor-to-donor heterogeneity in the ex vivo expansion process remains a critical bottleneck in their supply. This study aimed to identify factors influencing donor variability in a 2-week-long ex vivo NK cell expansion from peripheral blood mononuclear cells, analyzed across three donors. Single-cell transcriptomics was applied to investigate the distribution of cell types and phenotypes, as well as trajectory inference and differential gene expression. Our results identified that several factors were associated with the variability in the final NK cell fraction and expansion, and that their influence was prevalent between culture days 3 and 8. Compared to a high final NK cell expansion, a culture with a low final NK cell expansion exhibited an upregulation of some stress and inflammatory genes and an increase in one specific subcluster of NK cells already on culture day 3. It showed a low score of CD56^Bright CD16^– phenotype and a high score of CD56^Dim CD16⁺ phenotype. It also had a decreased presence of cytotoxic CD8⁺ Tm cells. Among the observed subclusters of CD8⁺ Tm cells, it exhibited a higher presence of a subcluster associated with a less differentiated and less cytotoxic phenotype, as well as a lower prevalence of a subcluster associated with chemokine and cytotoxic genes. Finally, it had a major expansion of one of the CD8⁺ Tm cells subclusters annotated as NK-like T cell and characterized by a high CCR5 mRNA expression, while the levels of CCL3, CCL4 and CCL5 mRNA were downregulated. The present findings point toward a potential link between CCL signaling and improved NK cell expansion performance, including possible markers for further investigations, and suggest future strategies to increase the final NK cell fraction and expansion based on donor-specific markers.

Background & Aim

rAAV vectors are widely used for many gene therapies. The fast-growing worldwide demand for these therapies presents a major challenge for the rAAV viral vector manufacturing capacity. A prevailing manufacturing method is based on co-transfection of three plasmids pAAV-RC (viral replication and capsid genes for AAV), pHelper (adenovirus gene products required for the production of infective AAV), and pAAV (AAV inverted terminal repeats with the gene of interest) into HEK293 cells with harvest 48-72 hours post transfection (hpT). The present process intensification approach is a combination of high cell density (HCD) transfection at ≥ 20 million cells per mL (MVC/mL) and prolonged production phase of rAAV thanks to repeated transfections in perfusion stirred-tank bioreactors.

Methodology

n this study, repeated triple plasmids co-transfections were performed in HCD (≥ 20 MVC/mL) perfusion cultures of suspension HEK293F-derived cells in stirred tank bioreactors at 250 mL scale for the production of GFP. The rAAV serotype 9 production process was monitored by the transfection efficiency and the quantification of the produced viral capsids (ELISA) as well as the viral genomes (qPCR).

Results

The application of repeated HCD transfection in a perfusion culture ≥20 MVC/mL evidently prolonged the production phase of rAAV beyond 72 hpT with a sustained rAAV productivity per cell.

Conclusion

The successful repeated HCD transfections in bioreactors demonstrated a cost-effective and efficient approach compared to existing rAAV manufacturing processes and paved the way to continuous biomanufacturing of rAAV.

Current monoclonal antibody (mAb) biomanufacturing typically involves expression in mammalian cells during upstream processing (USP), followed by purification during downstream processing (DSP). DSP is a major bottleneck, primarily caused by the prevalent Protein A affinity chromatography capture step. Key drawbacks include the extensive clarification required to prevent clogging of the chromatography column, limited productivity due to diffusional mass transport, and the risk of mAb denaturation under acidic elution conditions. To address these challenges, we introduce the novel concept of calcium-dependent magnetic separation using iron oxide nanoparticles with covalent immobilization of an engineered calcium-dependent affinity ligand (ZCa). The ZCa ligand is employed for mild elution conditions, while the dispersed particle adsorbent promises fast mAb interaction and processing of non-clarified feeds. Using Trastuzumab as a model mAb, we confirmed the intended rapid capture directly from Chinese hamster ovary (CHO) culture and achieved high mAb purities, with reductions in DNA and host cell protein (HCP) comparable to state-of-the-art Protein A chromatography. The demonstrated potential of integrating the clarification and the capture steps is promising for improving productivity and simultaneously reducing costs. Additionally, we developed a mild and sustainable elution protocol based on citrate/NaCl buffers, reaching recoveries above 90% at pH 6.0. The functionality of Trastuzumab after the novel separation approach was proven by high physical integrity and binding functionality to the human epidermal growth factor receptor 2 (HER2). Our findings underline the high potential of calcium-dependent magnetic separation for integrated, gentle, and sustainable bioseparation of mAbs.

The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successfully demonstrated in a process where single cells were first expanded, then cell aggregates were formed by dilution into a different medium 1 day before triple plasmid transfection was conducted. An alternative approach for the STR inoculation using a seed taken from a high cell density perfusion (HCDP) culture was also investigated. It was, however, found that the spent medium of the HCDP inhibited the transfection of HEK293T cell aggregates, which was confirmed when testing with single-cell suspension culture. The formation of aggregates in shaken multi-well plates was also investigated to develop a screening system using the average power input as a scale-down criterion, which revealed that cell aggregates could be generated in 12-well plates, however with a larger size than in a STR. Taking into account the reported higher rAAV production of adherent cells in comparison with single cells for triple-plasmid transfection, HEK293T cell aggregates can possibly surpass single-cell suspension in space–time rAAV yield. The formation of HEK293T cell aggregates in a STR system offers a promising approach for scaling up and intensifying rAAV production by triple-plasmid transfection, in comparison with traditional 2D scale-up methods.