Mária Tóth

Stable cell lines have recently achieved recombinant adeno-associated virus (rAAV) titers comparable to the standard triple transfection approach, making them a promising alternative to plasmid-based production systems. However, whether integration of the rAAV transgene into the host genome influences packaging efficiency and vector quality remains unclear. In this study, we generated stable HEK293 cell lines carrying the rAAV transgene in their genome. rAAV production was enabled by supplying the rep/cap and helper genes on two plasmids, rendering vector genome generation dependent on the chromosomally integrated transgene. Although the stable cell lines produced a 4.5-fold lower titer of viral genomes (VGs) compared to the standard triple transfection method, VG-normalized potency was four times higher. Detailed particle characterization further revealed 3-fold lower plasmid backbone DNA packaging in rAAVs produced by stable cell lines relative to triple transfection. Consistent results were obtained from mass photometry and ELISA/ddPCR analyses for the double transfection condition, while discrepancies emerged under triple transfection. These findings emphasize the importance of functional and qualitative assessments for evaluating different rAAV production approaches.

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

A unique collection of high-quality late medieval (fifteenth–sixteenth century) glazed and unglazed stove tiles from the northern part of the Carpathian Basin is of great interest to archaeologists and art historians. It is yet to be determined if these products, which are characterised by similar features, were produced in a single workshop, perhaps in Besztercebánya/Banská Bystrica (in present-day Slovakia), or in several workshops throughout the region. The first systematic multi-analytical investigation was carried out on the ceramic body and glaze of one hundred and seventeen tile fragments from six sites (Besztercebánya/Banská Bystrica, Fülek/Fiľakovo, and Csábrág/Čabraď in Slovakia; Salgó, Eger, and Szécsény in Hungary) using polarising microscopy, X-ray diffraction, electron microprobe, and Raman microspectroscopy analyses to determine the raw materials and production techniques used. Based on the petrographic characteristics, phase and chemical composition of the ceramic body, and the chemical composition and colourants of the glazes, the stove tiles can be classified into three primary groups. Tiles from different sites are different to each other, only the tiles from the Hungarian sites and from Fülek/Fiľakovo are similar. Thus, it is probable that the tiles were produced in several (at least three) workshops in the region from where they were then dispersed. The technological knowledge of the master(s) producing the polychrome Csábrág/Čabraď tiles with tin-opacified glazes was higher than that of the master(s) producing the other tiles. However, the exact location of the workshops as well as their existence through time is still in unknown.