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| 2. |
- Napoleone, Antonino, et al.
(författare)
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Fed-batch production assessment of a tetravalent bispecific antibody : A case study on piggyBac stably transfected HEK293 cells
- 2021
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 65, s. 9-19
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Tidskriftsartikel (refereegranskat)abstract
- The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 x 106 viable cells/mL with over 90 % viability 12 days postinoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 s, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody.
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| 3. |
- Robinson, Kathryn M., 1975-, et al.
(författare)
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Variation in non-target traits in genetically modified hybrid aspens does not exceed natural variation
- 2021
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 64, s. 27-36
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Tidskriftsartikel (refereegranskat)abstract
- Genetically modified hybrid aspens (Populus tremula L. x P. tremuloides Michx.), selected for increased growth under controlled conditions, have been grown in highly replicated field trials to evaluate how the target trait (growth) translated to natural conditions. Moreover, the variation was compared among genotypes of ecologically important non-target traits: number of shoots, bud set, pathogen infection, amount of insect herbivory, composition of the insect herbivore community and flower bud induction. This variation was compared with the variation in a population of randomly selected natural accessions of P. tremula grown in common garden trials, to estimate how the “unintended variation” present in transgenic trees, which in the future may be commercialized, compares with natural variation. The natural variation in the traits was found to be typically significantly greater. The data suggest that when authorities evaluate the potential risks associated with a field experiment or commercial introduction of transgenic trees, risk evaluation should focus on target traits and that unintentional variation in non-target traits is of less concern.
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| 4. |
- Tegel, Hanna, et al.
(författare)
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High throughput generation of a resource of the human secretome in mammalian cells
- 2020
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 58, s. 45-54
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic beta-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.
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| 5. |
- Yan, Ruoyu, et al.
(författare)
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Structural characterization of the family GH115 α-glucuronidase from Amphibacillus xylanus yields insight into its coordinated action with α-arabinofuranosidases
- 2021
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 62, s. 49-56
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Tidskriftsartikel (refereegranskat)abstract
- The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.
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| 6. |
- Zimny, Tomasz
(författare)
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Legal and practical challenges to authorization of gene edited plants in the EU
- 2021
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 60, s. 183-188
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Tidskriftsartikel (refereegranskat)abstract
- According to a predominant interpretation of the C-528/16 judgment of the Court of Justice of the European Union, mutants resulting from gene editing, even those featuring only single nucleotide variants, should be subject to the authorization procedures designed for organisms developed through genetic modification (i.e. insertion of large DNA fragments). In this article, we illustrate practical problems with the authorization of products of gene editing in the EU. On the basis of these problems, we analyze the influence of the current interpretation of EU legislation and judgment on the practical ability to authorize and detect such products on the EU market. We show that the predominant interpretation of the judgment leads to legally unacceptable consequences, in particular to the violation of the principle of proportionality with regard to individuals who wish to develop and market products of gene editing. As a result of our considerations, we show that the C-528/16 judgment did not need to be interpreted in the dominant way.
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