Preferential exclusion chromatography as a capture step for extracellular AAV harvest from adherent and suspension productions.

Preferential exclusion chromatography (PXC) sometimes described as hydrophobic interaction chromatography is a well-known, but not widely used technique for purification of Adeno-associated viruses. It employs high molarity of preferentially excluded cosolvent (salt in our case). The downside of this method is that high molarity of salt can lead to aggregation and precipitation of different compounds from the sample. In the case of viruses that are excreted to medium, the concentration of impurities is much lower compared to cell lysates, and PXC can be used as a first chromatographic, serotype independent step to concentrate and purify adeno-associated virus (AAV). Here, we explored PXC for adherent and suspension harvests using monolithic chromatographic columns (CIMmultus). Suspension extracellular adeno-associated virus, serotype 9 (AAV9) harvest had more impurities compared to adherent harvest, therefore it required higher input regarding method development. Final conditions for suspension harvest included higher molarity of binding salt and using more open channel format of chromatographic column (6 µm channel size). Vector genome analysis with droplet digital polymerase chain reaction (ddPCR) revealed 84% and 97% recovery for suspension and adherent AAV9 harvest, respectively. After PXC capture step, adherent AAV9 was purified by already described ion exchange techniques. Overall process vector genome recovery, from clarified harvest to anion exchange elution fraction, was 54% measured by ddPCR. Residual host cell DNA was measured at 40 ng per 1E13 vector genome, and empty AAV was below 5% in final anion exchange chromatography fraction.

Optimization of rAAV capture step purification using SO3 monolith chromatography.

Adeno-associated virus (AAV) vectors are crucial tools for gene therapy applications. As AAVs are administered in vivo, stringent purity requirements must be met, necessitating the development of various downstream processing strategies in accordance with regulatory guidelines. In this context, we focus on the non-affinity serotype-independent recombinant AAV (rAAV) capture step, which involves the use of Convective Interaction Media (CIM) cation-exchange SO3 monoliths. We analyzed differentially pretreated viral samples obtained from the Sf9 cell line and applied these samples to the capture SO3 chromatography step. We conducted screening experiments using CIM SO3 0.05 mL monolithic 96-well plates with buffers of varying pH, sodium chloride concentrations, and the inclusion of poloxamer 188, aiming to select the optimal binding mobile phase. Dynamic binding capacity was defined for different pretreatments and the optimal conditions were subsequently retested using the industrial purification CIMmultus line. The results demonstrated a high overall vector recovery (51%) and a significant reduction in impurities (99.98% for protein reduction and 99.25% for DNA reduction) using the selected capture step parameters, thereby confirming the successful optimization of the rAAV capture step in the downstream process using monoliths.

Snezna jama (Slovenia): Interdisciplinary dating of cave sediments and implication for landscape evolution.

Caves are important markers of surface evolution, since they are, as a general rule, linked with ancient valley bottoms by their springs. However, caves can only be dated indirectly by means of the sediments they contain. If the sediment is older than common dating methods, one has to use multiple dating approaches in order to get meaningful results. U/Th dating, palaeomagnetic analysis of flowstone and sediment profiles, cosmogenic dating of quartz pebbles, and mammalian dating allowed a robust estimate of speleogenesis, sediment deposition, climatic change at the surface, and uplift history on the Periadriatic fault line during the Plio-Pleistocene. Our dates indicate that Snezna jama was formed in the (Upper) Miocene, received its sedimentary deposits during the Pliocene in a rather low-lying, hilly landscape, and became inactive due to uplift along the Periadriatic and Sava faults and climatic changes at the beginning of the Quaternary. Although it is only a single cave, the information contained within it makes it an important site of the Southern Alps.

Microbiota entrapped in recently-formed ice: Paradana Ice Cave, Slovenia.

Paradana is one of the biggest ice caves in Slovenia, with an estimated ice volume of 8,000 m3. Reflecting climatological conditions, the cave ice undergoes repeated freeze-thaw cycles and regular yearly deposition of fresh ice. Three distinct ice block samples, collected from the frozen lake in May 2016, were analysed to obtain data on ice physicochemical properties and the composition of associated microbiota. Isotopic composition of the ice samples (18O, 2H) and a local meteoric water line (LMWL) constructed for monthly precipitation at Postojna were used to estimate the isotopic composition of the water that formed the ice, which had high values of deuterium excess and low concentrations of chloride, sulphate and nitrate. The values of total organic carbon (1.93-3.95 mg/l) within the ice blocks fall within the range of those measured in karst streams. Total cell count in the ice was high and the proportion of cell viability increased along the depth gradient and ranged from 4.67 × 104 to 1.52 × 105 cells/ml and from 51.0 to 85.4%, respectively. Proteobacteria represented the core of the cave-ice microbiome (55.9-79.1%), and probably play an essential role in this ecosystem. Actinobacteria was the second most abundant phylum (12.0-31.4%), followed in abundance by Bacteroidetes (2.8-4.3%). Ice phylotypes recorded amounted to 442 genera, but only 43 genera had abundances greater than 0.5%. Most abundant were Pseudomonas, a well-known ice dweller, and Lysobacter, which previously was not reported in this context. Finally, two xanthophytes, Chloridella glacialis and Ellipsoidion perminimum, known from polar environments, were cultured from the ice. This indicates that the abundance and ecological role of phototrophs in such environments might be greater than previously deduced.