Biology subjects

Villiger, T. K.

Publications and source records attributed to Villiger, T. K..

3 recordsLinked to original sources

bioRxiv2026

Integrated Continuous Biomanufacturing of Recombinant Adeno-Associated Virus

Recombinant adeno-associated virus (rAAV) manufacturing remains constrained by low yields, costly purification, and process complexity, limiting cost-effective access to gene therapy. Here, we demonstrate an integrated continuous biomanufacturing platform that transfers proven antibody-manufacturing technology to rAAV production by coupling perfusion to semi-continuous twin-column affinity capture (CaptureSMB). This architecture enables continuous harvest of extracellular rAAV without cell lysis, depth filtration, or endonuclease treatment, sustaining stable operation over five days. Relative to conventional batch processing, perfusion improved capsid or vector genome yield depending on the production process, while affinity capture alone reduced total DNA to batch-comparable levels, with no measurable loss in impurity clearance or potency. By consolidating multiple manual unit operations into one continuous workflow, the platform may reduce manual intervention and process footprint. Its modular design may generalize to other rAAV serotypes, inducible producer cell lines, and viral vector classes, offering a potential route toward more efficient gene therapy manufacturing.

bioengineering↗

bioRxiv2026

Continuous Capture of recombinant AAV Particles Using Twin-Column CaptureSMB

Recombinant adeno-associated viruses (rAAVs) have gained increasing importance in gene therapy due to their safe and precise gene delivery. However, certain indications require substantially higher vector doses, pushing manufacturing capacity and cost of goods (COG) to its limits. In this study, we present for the first time a continuous twin-column capture process (CaptureSMB) enabling direct purification of rAAV5 from unprocessed perfusion harvest without prior concentration or processing. This approach differs fundamentally from conventional batch workflows which typically mandate clarification and concentration before affinity capture and offers a novel process integration in viral vector manufacturing. A single-column batch capture process was developed first and subsequently compared to continuous CaptureSMB configurations. Optimized CaptureSMB operation achieved consistent yields over four cycles, with recoveries exceeding batch operation (+ 14.3%) with concomitant higher productivity (+ 11.4%) and reduced buffer consumption (- 79.2%). Critical quality attribute analysis showed lower host cell protein levels and lower residual DNA in early CaptureSMB cycles, while full capsid ratios, thermal stability and transduction efficiency of rAAV5 particles remained unaltered across cycles and process modes. These findings highlight that continuous twin-column CaptureSMB directly from perfusion harvest can not only improve yield and manufacturing efficiency but also maintain and in some respects enhance product quality. This novel strategy provides a promising route to address manufacturing capacity and cost challenges in rAAV gene therapy production.

bioengineering↗

bioRxiv2017

Glycosylation Flux Analysis Reveals Dynamic Changes Of Intracellular Glycosylation Flux Distribution In Chinese Hamster Ovary Fed-Batch Cultures

N-linked glycosylation of proteins has both functional and structural significance. Importantly, the glycan structure of a therapeutic protein influences its efficacy, pharmacokinetics, pharmacodynamics and immunogenicity. In this work, we developed glycosylation flux analysis (GFA) for predicting intracellular production and consumption rates (fluxes) of glycoforms, and applied this method to CHO fed-batch monoclonal antibody (mAb) production using two different media compositions, with and without additional manganese feeding. The GFA is based on a constraint-based modelling of the glycosylation network, employing a pseudo steady state assumption. While the glycosylation fluxes in the network are balanced at each time point, the GFA allows the fluxes to vary with time by way of two scaling factors: (1) an enzyme-specific factor that captures the temporal changes among glycosylation reactions catalyzed by the same enzyme, and (2) the cell specific productivity factor that accounts for the dynamic changes in the mAb production rate. The GFA of the CHO fed-batch cultivations showed that regardless of the media composition, the fluxes of galactosylation decreased with the cultivation time in comparison to the other glycosylation reactions. Furthermore, the GFA showed that the addition of Mn, a cofactor of galactosyltransferase, has the effect of increasing the galactosylation fluxes but only during the beginning of the cultivation period. The results thus demonstrated the power of the GFA in delineating the dynamic alterations of the glycosylation fluxes by local (enzyme-specific) and global (cell specific productivity) factors.

systems biology↗