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Arcalis E.,University of Natural Resources and Life Sciences, Vienna | Stadlmann J.,University of Natural Resources and Life Sciences, Vienna | Rademacher T.,Fraunhofer Institute for Molecular Biology and Applied Ecology | Marcel S.,Caliber Biotherapeutics | And 3 more authors.
Plant Molecular Biology | Year: 2013

Many plant-based systems have been developed as bioreactors to produce recombinant proteins. The choice of system for large-scale production depends on its intrinsic expression efficiency and its propensity for scale-up, post-harvest storage and downstream processing. Factors that must be considered include the anticipated production scale, the value and intended use of the product, the geographical production area, the proximity of processing facilities, intellectual property, safety and economics. It is also necessary to consider whether different species and organs affect the subcellular trafficking, structure and qualitative properties of recombinant proteins. In this article we discuss the subcellular localization and N-glycosylation of two commercially-relevant recombinant glycoproteins (Aspergillus niger phytase and anti-HIV antibody 2G12) produced in different plant species and organs. We augment existing data with novel results based on the expression of the same recombinant proteins in Arabidopsis and tobacco seeds, focusing on similarities and subtle differences in N-glycosylation that often reflect the subcellular trafficking route and final destination, as well as differences generated by unique enzyme activities in different species and tissues. We discuss the potential consequences of such modifications on the stability and activity of the recombinant glycoproteins. © 2013 Springer Science+Business Media Dordrecht.

Iyama T.,U.S. National Institute on Aging | Lee S.Y.,Weatherall Institute of Molecular Medicine | Berquist B.R.,Caliber Biotherapeutics | Gileadi O.,University of Oxford | And 4 more authors.
Nucleic Acids Research | Year: 2015

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5′-3′ exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS. © 2015 Published by Oxford University Press on behalf of Nucleic Acids Research.

Holtz B.R.,Caliber Biotherapeutics | Berquist B.R.,Caliber Biotherapeutics | Bennett L.D.,Caliber Biotherapeutics | Kommineni V.J.M.,Caliber Biotherapeutics | And 6 more authors.
Plant Biotechnology Journal | Year: 2015

Rapid, large-scale manufacture of medical countermeasures can be uniquely met by the plant-made-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plant-based manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

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