Virus‐like particles (VLPs) have shown great potential as biopharmaceuticals in the market and in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled and reassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At the industrial scale, cross‐flow filtration (CFF) is the method of choice for performing reassembly by diafiltration. Here, we developed an experimental CFF setup with an on‐line measurement loop for the implementation of process analytical technology (PAT). The measurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as a light scattering photometer. These sensors allowed for monitoring protein concentration, protein tertiary structure, and protein quaternary structure. The experimental setup was tested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, three reassembly processes were performed at different transmembrane pressures (TMPs). While light scattering provided information on the assembly progress, UV/Vis allowed for monitoring the protein concentration and the rate of VLP assembly based on the microenvironment of Tyrosine‐132. VLP formation was verified by off‐line dynamic light scattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experimental results provided evidence of aggregate‐related assembly inhibition and showed that off‐line size‐exclusion chromatography does not provide a complete picture of the particle content. Finally, a Partial‐Least Squares (PLS) model was calibrated to predict VLP concentrations in the process solution. Q 2 values of 0.947–0.984 were reached for the three HBcAg variants. In summary, the proposed experimental setup provides a powerful platform for developing and monitoring VLP reassembly steps by CFF.

Virus-like particles (VLPs) are emerging nanoscale protein assemblies applied as prophylactic vaccines and in development as therapeutic vaccines or cargo delivery systems. Downstream processing (DSP) of VLPs comes both with challenges and opportunities, depending on the complexity and size of the structures. Filtration, precipitation/re-dissolution and size-exclusion chromatography (SEC) are potent technologies exploiting the size difference between product and impurities. In this study, we therefore investigated the integration of these technologies within a single unit operation, resulting in three different processes, one of which integrates all three technologies. VLPs, contained in clarified lysate from Escherichia coli, were precipitated by ammonium sulfate, washed, and re-dissolved in a commercial cross-flow filtration (CFF) unit. Processes were analyzed for yield, purity, as well as productivity and were found to be largely superior to a reference centrifugation process. Productivity was increased 2.6-fold by transfer of the wash and re-dissolution process to the CFF unit. Installation of a multimodal SEC column in the permeate line increased purity to 96% while maintaining a high productivity and high yield of 86%. In addition to these advantages, CFF-based capture and purification allows for scalable and disposable DSP. In summary, the developed setup resulted in high yields and purities, bearing the potential to be applied as an integrated process step for capture and purification of in vivo-assembled VLPs and other protein nanoparticles.

Virus‐like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, named capsomeres, composed of recombinantly expressed viral structural proteins. During downstream processing, in vivo‐assembled VLPs are typically dis‐ and reassembled to remove encapsulated impurities and to improve particle morphology. Disassembly is achieved in a high‐pH solution and by the addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material‐consuming disassembly experiments. To this end, we developed a low‐volume and high‐resolution disassembly screening that provides time‐resolved insight into the VLP disassembly progress. In this study, two variants of C‐terminally truncated hepatitis B core antigen were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. Based on the high‐throughput screening results, a diafiltration‐based disassembly process step was developed. Compared with mixing‐based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration‐based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high‐molecular‐weight species.

BACKGROUND 3D printing and bioprinting in particular are emerging technologies in the field of biotechnology. The developments of bioprinters and applications lie mostly in the highly observed working fields of tissue engineering and regenerative medicine. Until now only little attention has been paid to the application of 3D bioprinting for the investigation of hydrogel–liquid phase interactions in biotechnological applications. This can mostly be attributed to the need for complex and expensive equipment. RESULTS In this work, an entry‐level bioprinter on the base of a commercially available Fused‐Filament‐Fabrication 3D printer and an easy to handle user interface was designed. This newly developed bioprinter allowed the structuring of bioinks and hydrogels in microwell plates and even complex models were printed. The applicability of the presented printer setup in the field of biotechnology was shown by the encapsulation of β‐galactosidase (EC 3.2.1.23) in poly(ethylene glycol) diacrylate based hydrogels. Subsequently, an automated screening of the biocatalytic conversion of the substrate ONPG by the encapsulated enzyme was executed on a liquid handling station. Under varied pH conditions in the surrounding liquid phase highest substrate turnover rates were detected at pH 3 and pH 5 which is in good accordance with previously reported pH optima of β‐galactosidase. CONCLUSION This approach shows an easy access to 3D bioprinting in the field of biotechnology and the implementation of 3D printed hydrogels in high‐throughput experimentation. © 2017 Society of Chemical Industry

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