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Sunday, 19 November 2017
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Biotechnology and Bioengineering
Wiley Online Library : Biotechnology and Bioengineering

  • cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV?1 envelope glycoprotein vaccine candidate
    We describe the properties of BG505 SOSIP.664 HIV?1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native?like trimers that are the basis for many structure?guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV?1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum?free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size?exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log10. The final cGMP production run yielded 3.52 grams (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native?like as judged by negative?stain electron microscopy, and were stable over a multi?month period at room temperature or below and for at least one week at 50°C. Their antigenicity, disulfide bond patterns and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native?like Env glycoprotein trimers of various designs and genotypes. This article is protected by copyright. All rights reserved

  • Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA?free CRISPR?Cas9 RNP?mediated mutagenesis
    Lutein and zeaxanthin are dietary carotenoids reported to be protective against age?related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC?4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock?out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA?free CRISPR?Cas9 ribonucleoproteins in the CC?4349 strain had a significantly higher zeaxanthin content (56?fold) and productivity (47?fold) than the wild type without the reduction in lutein level. Furthermore, we produced eggs fortified with lutein (2?fold) and zeaxanthin (2.2?fold) by feeding hens a diet containing the mutant. Our results clearly demonstrate the possibility of cost?effective commercial use of microalgal mutants induced by DNA?free CRISPR?Cas9 ribonucleoproteins in algal biotechnology for the production of high?value products. This article is protected by copyright. All rights reserved

  • Modulation of IgG1 Immunoeffector Function by Glycoengineering of the GDP?Fucose Biosynthesis Pathway
    Cross?linking of the Fc? receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody?dependent cell mediated cytotoxicity (ADCC). A conserved N?glycan positioned at the N?terminal region of the IgG CH2 domain is critical in maintaining the quaternary structure of the molecule for Fc? receptor engagement. The removal of a single core fucose residue from the N?glycan results in a considerable increase in affinity for Fc?RIIIa leading to an enhanced receptor?mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti?CD20, IgG1 molecule, we selectively targeted the de novo GDP?fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced Fc?RIIIa binding (13?fold) and in vitro ADCC cell?based activity (11?fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP?fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N?glycan fucosylation level to modulate IgG immune effector function. This article is protected by copyright. All rights reserved

  • Design of a Novel Continuous Flow Reactor for Low pH Viral Inactivation
    Insufficient mixing in laminar flow reactors due to diffusion?dominated flow limits their use in applications where narrow residence time distribution (RTD) is required. The aim of this study was to design and characterize a laminar flow (Re 187.7?375.5) tubular reactor for low pH viral inactivation with enhanced radial mixing via the incorporation of curvature and flow inversions. Towards this aim, the reactor described here, Jig in a Box (JIB), was designed with a flow path consisting of alternating 270° degree turns. The design was optimized by considering the strength of secondary flows characterized by the Dean No., the corresponding secondary flow development length, and the reactor turn lengths. Comprehensive CFD analysis of the reactor centerline velocity profile, cross?sectional velocity, and secondary flow streamlines confirmed enhanced radial mixing due to secondary flows and changes in flow direction. For initial CFD and experimental studies the reactor was limited to a 16.43 m length. Pulse tracer studies for the reactor were computationally simulated and experimentally generated to determine the RTD, RTD variance, and minimum residence time for the tracer fluid elements leaving the reactor, as well as to validate the computational model. The reactor was scaled length wise to increase incubation time and it was observed that as the reactor length increases the RTD variance increases linearly and the dimensionless RTD profile becomes more symmetrical and tighter about the mean residence time. This article is protected by copyright. All rights reserved

  • Production of 1,2?propanediol in photoautotrophic Synechocystis is linked to glycogen turn?over
    We utilized a photoautotrophic organism to synthesize 1,2?propanediol from carbon dioxide and water fueled by light. A synthetic pathway comprising mgsA (methylglyoxal synthase), yqhD (aldehyde reductase), and adh (alcohol dehydrogenase) was inserted into Synechocystis sp. PCC6803 to convert dihydroxyacetone phosphate to methylglyoxal, which is subsequently reduced to acetol and then to 1,2?propanediol. 1,2?propanediol could be successfully produced by Synechocystis, at an approximate rate of 55?µmol h?1 gCDW?1. Surprisingly, maximal productivity was observed in the stationary phase. The production of 1,2?propanediol was clearly coupled to the turn?over of intracellular glycogen. Upon depletion of the glycogen pool, product formation stopped. Reducing the carbon flux to glycogen significantly decreased final product titers. Optimization of cultivation conditions allowed final product titers of almost 1?g?L?1 (12?mM), which belongs to the highest values published so far for photoautotrophic production of this compound. This article is protected by copyright. All rights reserved

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