ABSTRACT Adeno‐associated viruses (AAVs) are the leading vectors for in vivo gene therapy, fueling a growing demand for scalable and robust biomanufacturing processes. Yet, the impact of cell lysis conditions on the performance of midstream and downstream unit operations across serotypes remains poorly characterized. This study presents a serotype‐resolved investigation of cell lysis and its cascading effects on AAV2, AAV5, AAV8, and AAV9 processing. A panel of lysis methods including inorganic agents, non‐ionic and ionic detergents, and mechanical disruption was evaluated using an analytical framework of at‐ and off‐line assays to quantify AAV yield, purity, and activity at each stage. Our results reveal pronounced serotype‐specific differences: cetrimonium bromide (CTAB) delivered the highest transduction output of any condition evaluated for AAV8 (2.25 × 10 9 TU/mL) at 55.0% capture step yield, while sodium deoxycholate achieved ~ 98% host cell protein (HCP) reduction for AAV9 (HCP log reduction value 1.60) and the highest functional enrichment for AAV5 (6.73‐fold) and AAV8 (5.80‐fold). AAV2 proved uniquely challenging, with CTAB enabling the highest tangential flow filtration (TFF) yield. Acid precipitation paired with high salt addition reduced aggregation and improved permeate flux for AAV5 and AAV9. TFF performance was governed by the detergents' physicochemical properties: micelle‐forming agents generated multicomponent complexes that fouled membranes in condition‐ and serotype‐specific patterns. Rheological characterization revealed that all clarified lysates converged to the viscosity of the media after filtration, supporting transferable TFF parameters. Mixed‐mode chromatography using a peptide‐functionalized resin amplified lysis‐imprinted differences, decoupling step yield from functional enrichment. Bayesian optimization of the chromatographic step improved purification outcomes: for AAV8 residual HCPs in the eluate decreased > 300‐fold, from 0.91 mg/mL to 3 μg/mL, while for AAV9 the yield rose to ~95% at residual HCP as low as 14 μg/mL, demonstrating that purity and recovery are co‐optimizable when lysis, midstream, and downstream conditions are rationally paired.
ABSTRACT Mechanistic modeling is important in biopharmaceutical manufacturing by enabling in‐silico experimentation. However, developing a robust mechanistic model for mammalian cell culture remains challenging due to the complexity of the underlying bioprocesses. Existing data‐driven models often suffer from limited extrapolation capability and require extensive datasets. Not all modeling approaches can accurately represent differing metabolic scenarios such as lactate consumption and production. To address these limitations, this study proposes a hybrid mechanistic model for mammalian cell culture to improve predictive accuracy and process understanding. Our model integrates high‐frequency pH data with low‐frequency measurements of viable cell density (VCD), glucose, lactate, monoclonal antibody (mAb) concentrations from 10 runs (7 L). Novel kinetic equations for cell growth and death, and lactate metabolism considering both scenarios were developed. Additionally, our model enables automatic lactate switch detection based on time‐series pH and feeding data. It demonstrated high predictive capability with a mean R 2 of 0.88 for VCD, mAb, glucose, and lactate concentrations across training runs. Real‐time fine‐tuning further improved accuracy, reducing the root mean squared error (RMSE) for glucose by ~50% and for lactate by ~30%. The results indicate that our model provides accurate real‐time predictions of VCD, mAb, glucose, and lactate concentrations under variable bioprocessing conditions.
ABSTRACT Plant virus‐like particles (VLPs) are promising nanotechnology platforms, but their translation requires purification strategies suitable for large‐scale production. Here, we developed a nuclease‐free downstream process for Physalis mottle virus (PhMV) VLPs expressed in Escherichia coli , combining ammonium sulfate precipitation, low‐pH extraction, ultrafiltration/diafiltration (UF/DF), and flow‐through anion‐exchange (AEX) chromatography. Ammonium sulfate precipitation separated the VLPs from bulk host nucleic acids, whereas extraction at low pH removed most host cell proteins (HCPs) while retaining the VLPs in solution. Subsequent UF/DF was used to concentrate VLPs and simultaneously achieve buffer exchange for downstream AEX chromatography, which removed remaining HCPs while the PhMV VLPs were collected in the flow‐through fraction. This combined workflow produced PhMV VLPs with > 95% purity and a recovery of ~68%. Compared with conventional workflows based on ultracentrifugation and size‐exclusion chromatography, the new process reduced the purification time from ~3 days to 1 day and hands‐on processing time from ~18 h to 8 h, also increasing the average yield from ~80 to ~130 mg PhMV VLPs per liter of culture. The resulting particles were intact and monodisperse, with a lower content of encapsulated host nucleic acid than VLPs purified by ultracentrifugation. The VLPs also had increased chemical addressability of lysine and cysteine residues. These results establish a scale‐up compatible, nuclease‐free purification strategy for PhMV VLPs expressed in bacteria and provide a framework for the downstream processing of plant VLP platforms produced in microbial systems.
ABSTRACT Inclusion body (IB) processes yielding the recombinant product only as IBs are long‐established in the biopharmaceutical industry. Yet, for recombinant proteins that accumulate both IBs and soluble fraction depending on the selected upstream processing (USP) strategy, the influence of process parameters on the partitioning between both forms remains poorly characterized. In this study, l ‐lactate dehydrogenase (LDH) was selected as a model protein as it yields IBs and soluble protein concurrently. We investigated how induction temperature and specific substrate uptake rate shift the relative amount of soluble product. A response surface model revealed a sharp switch between exclusively soluble production at 25°C, exclusive IB formation at 35°C and a mixed production regime at 30°C. Further, we investigated whether these trends hold for the structurally related proteins xylose isomerase (Xiso) and pyranose‐2‐oxidase (P2O) using a reduced diagonal design and a projection into the LDH latent space by principal component analysis (PCA). Results revealed that the shared structural features were not sufficient to predict the expression form. Still, the LDH model served as a reference framework for identifying the extent to which other mixed producers follow established process‐response trends.
ABSTRACT Wound healing is a complex and dynamic biological process that occurs in a series of coordinated steps, including hemostasis, inflammation, proliferation, and tissue remodeling. Traditional wound dressings, such as gauze and bandages, are not suitable for healing because they do not maintain moisture levels and cannot prevent infections. So, hydrogels have been recognized as advanced wound‐healing materials because of their high‐water content and their ability to mimic the natural ECM. Additionally, amyloid‐based hydrogels have been recognized for their unique physicochemical and functional properties, which are constructed by the self‐assembly of peptides into highly ordered fibrillar structures. The nanoscale fibrillar structure provides the hydrogels with high mechanical stability and viscoelasticity. The porous structure allows the movement of oxygen and nutrients, as well as the removal of waste products. They can also self‐heal and be formulated in injectable forms for application to wounds with irregular surfaces. Their unique structural and functional properties make them highly effective at accelerating wound healing. This review highlights in vitro and in vivo studies demonstrating the efficacy of amyloid‐based hydrogels in accelerating wound healing, making them highly promising materials for advanced wound healing.
