Biotechnology Journal最新文献

Adeno-associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome-containing from empty AAV capsids, is usually time-consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow-through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl₂ resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4- to 7.5-fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids’ functional potency and structural integrity. Following the successful establishment of the flow-through polishing approach, it was adapted to a manual syringe-based system. Manual flow-through polishing using the monolith or membrane adsorber achieved 3.6- and 5.4-fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow-through polishing provides a rapid and easy-to-perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies.

Heme is a key ingredient required to mimic the color and flavor of meat in plant-based alternatives. This study aimed to develop a yeast-based microbial cell factory for efficient and sustainable production of heme. To this end, first, Hem12p (uroporphyrinogen decarboxylase) was identified as the rate-limiting enzyme in the heme biosynthetic pathway present in Saccharomyces cerevisiae D452-2. Next, we investigated the effects of disruption of the genes involved in the competition for heme biosynthesis precursors, transcriptional repression, and heme degradation (HMX1) on heme production efficiency. Of the knock-out strains constructed in this study, only the HMX1-deficient strain produced heme at a higher concentration than the background strain without gene disruption. In addition, overexpression of PUG1 encoding a plasma membrane transporter involved in protoporphyrin IX (the precursor to heme biosynthesis) uptake led to a significant increase in intracellular heme concentration. As a result, among the various engineered strains constructed in this study, the ΔHMX1/H3&12 + PUG1 strain, the HMX1-deficient strain overexpressing HEM3, HEM12, and PUG1, produced the highest concentration of heme (4.6 mg/L) in batch fermentation, which was 3.9-fold higher than that produced by the wild-type D452-2 strain. In a glucose-limited fed-batch fermentation, the ΔHMX1/H3&12 + PUG1 strain produced 28 mg/L heme in 66 h.

In light-based 3D-bioprinting, gelatin methacrylate (GelMA) is one of the most widely used materials, as it supports cell attachment, and shows good biocompatibility and degradability in vivo. However, as an animal-derived material, it also causes safety concerns when used in medical applications. Gelatin is a partial hydrolysate of collagen, containing high amounts of hydroxyproline. This causes the material to form a thermally induced gel at ambient temperatures, a behavior also observed in GelMA. This temperature-dependent gelation requires precise temperature control during the bioprinting process to prevent the gelation of the material. To avoid safety concerns associated with animal-derived materials and reduce potential issues caused by thermal gelation, a recombinant human alpha-1 collagen I fragment was expressed in Komagataella phaffii without hydroxylation. The resulting protein was successfully modified with methacryloyl groups and underwent rapid photopolymerization upon ultraviolet light exposure. The developed material exhibited slightly slower polymerization and lower storage modulus compared to GelMA, while it showed higher stretchability. However, unlike the latter, the material did not undergo physical gelation at ambient temperatures, but only when cooled down to below 10°C, a characteristic that has not been described for comparable materials so far. This gelation was not caused by the formation of triple-helical structures, as shown by the absence of the characteristic peak at 220 nm in CD spectra. Moreover, the developed recombinant material facilitated cell adherence with high cell viability after crosslinking via light to a 3D structure. Furthermore, desired geometries could be easily printed on a stereolithographic bioprinter.

Oligoclonal antibodies, which are carefully defined mixtures of monoclonal antibodies, are valuable for the treatment of complex diseases, such as infectionss and cancer. In addition to these areas of medicine, they could be utilized for the treatment of snakebite envenoming, where recombinantly produced monoclonal human antibodies could overcome many of the drawbacks accompanying traditional antivenoms. However, producing multiple individual batches of monoclonal antibodies in an industrial setting is associated with significant costs. Therefore, it is attractive to produce oligoclonal antibodies by mixing multiple antibody-producing cell lines in a single batch to have only one upstream and downstream process. In this study, we selected four antibodies that target different toxins found in the venoms of various elapid snake species, such as mambas and cobras, and generated stable antibody-producing cell lines. Upon co-cultivation, we found the cell line ratios to be stable over 7 days. The purified oligoclonal antibody cocktail contained the anticipated antibody concentrations and bound to the target toxins as expected. These results thus provide a proof of concept for the strategy of mixing multiple cell lines in a single batch to manufacture tailored antivenoms recombinantly, which could be utilized for the treatment of snakebite envenoming and in other fields where oligoclonal antibody mixtures could find utility.