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Biotechnology Progress

Thais M de Borba, Taiele B Machado, Adriano Brandelli, Susana J Kalil
The parameters half-life, z-value, enthalpy, entropy, and free energy were evaluated in the temperature range of 40 to 55°C for a Bacillus sp. protease P45 present in a medium composed of ionic liquid (IL) and organic solvent. The protease was previously treated in IL [Emim][Tf2 N] and increased activity was observed in four out of five organic solvents tested. The reaction medium containing acetone and IL (1:1 v/v ratio) was more stable than the buffer medium, with half-life of 2.4 h at 55°C. Thermodynamic parameters values showed there was probably less rupture of non-covalent bonds that stabilize the protein structure, which promoted the protease P45 stabilization...
July 10, 2018: Biotechnology Progress
Szymon Skoneczny
The paper presents a mathematical model and steady-state characteristics of a continuous-flow two-phase fluidized-bed bioreactor with external aeration. The quantitative description includes biofilm growth on inert carriers, distributions of density and diffusion coefficients in biofilm, decay of microorganisms, gas-liquid and liquid-biofilm mass transfer, biofilm detachment, biomass thickening and recirculation. The method for the determination of steady-states and the assessment of stability based on the orthogonal collocation method was presented...
June 12, 2018: Biotechnology Progress
Simel Bağder Elmacı, Filiz Özçelik
In this study, it was aimed to enhance the enzymatic hydrolysis of recalcitrant yellow pine wood and subsequently to produce ethanol efficiently through the application of 1-ethyl-3-methylimidazolium acetate (EMIMAc) ionic liquid. The effect of biomass particle size (<2.5 mm, 500-850 μm, 363-500 μm and <363 μm) prior to pretreatment and pretreatment time (15, 30 and 45 min) on the structural properties and enzymatic hydrolysis yield of EMIMAc pretreated pine wood were also investigated. In general, EMIMAc functioned more efficiently at larger biomass particle sizes compared to smaller ones...
June 9, 2018: Biotechnology Progress
Jenna Williamson, Jennifer Miller, Joseph McLaughlin, Rodney Combs, Chia Chu
N-linked glycosylation profiles are routinely characterized on mammalian-derived protein therapeutic products and achieving consistency in the product-associated glycan attributes is an important indicator that the manufacturing process is under control. More importantly, meeting target glycan profile is a common criterion for ensuring product efficacy. During laboratory process development and subsequent scale up for pilot demonstration for a monoclonal antibody program, discrepancies in the molecule's terminal galactosylation level at 2-L, 100-L, and 6000-L scales were observed...
June 8, 2018: Biotechnology Progress
Rüdiger Ohs, Martin Leipnitz, Marie Schöpping, Antje C Spiess
Thiamine diphosphate (ThDP)-dependent enzymes catalyze a broad range of reactions with excellent enantioselectivity. Among these reactions, carboligations of aldehydes are of particular interest since the products, chiral hydroxy ketones, are valuable building blocks in the pharmaceutical industry. However, the substrates, e.g. benzaldehyde, inactivate the biocatalysts, e.g. the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL). Since only few mechanistic kinetic models for carboligation and simultaneous inactivation are available today, we quantitatively determined the reaction kinetics and inactivation of the self-carboligation of benzaldehyde yielding the product (R)-benzoin catalyzed by PfBAL directly from progress curves using model-based experimental analysis...
June 8, 2018: Biotechnology Progress
Mahesh D Patil, Ashok S Shinde, Manoj J Dev, Gopal Patel, Kiran D Bhilare, Uttam Chand Banerjee
Disruption of Pseudomonas putida KT2440 by ultrasound treatment in a bath sonicator, in presence of the glass beads, was carried out for the release of arginine deiminase (ADI) and the results were compared with that of by Dyno-mill. The release of ADI depended mainly on the bead size and cellmass concentration being disrupted in bead mill. Nearly 23 U/mL ADI was released when slurry with a cell-mass concentration of 250 g/L was disintegrated for 9 min with 80% bead loading (0.25 mm) in Dyno-mill. Marginally higher amount of ADI (24...
June 8, 2018: Biotechnology Progress
Tharmala Tharmalingam, Hedieh Barkhordarian, Nicole Tejeda, Kristi Daris, Sam Yaghmour Pheng Yam, Fang Lu, Chetan Goudar, Trent Munro, Jennitte Stevens
Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long-term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics...
June 8, 2018: Biotechnology Progress
Alejandro González-Mora, Federico Ruiz-Ruiz, Jorge Benavides, Marco Rito-Palomares
Aqueous two-phase systems (ATPS) have been widely exploited for the recovery and partial purification of biological compounds. Recently our research group characterized the primary recovery and partial purification of bacteriophage M13 using polymer-salt and ionic liquid-salt ATPS. From such study, it was concluded that PEG 400-potassium phosphate ATPS with a volume ratio (VR ) of 1 and 25% w/w TLL were the best suitable for the primary recovery of bacteriophage M13 from a crude extract, achieving a recovery yield of 83...
June 8, 2018: Biotechnology Progress
Natarajan Vijayasankaran, Sharat Varma, Yi Yang, Steve Meier, Robert Kiss
This manuscript summarizes the effect of certain cell culture medium additives on antibody drug substance coloration and acidic charge variants. It has been shown previously that B-vitamins and iron in the cell culture medium could significantly impact color intensity. In this manuscript, we detail the effect of several other cell culture components that have been shown to impact coloration. It is shown that if cystine is used instead of cysteine in the cell culture medium, coloration was reduced. Hydrocortisone has been shown to reduce coloration and boost specific productivity...
June 8, 2018: Biotechnology Progress
Martin Kögler, Andrea Paul, Emmanuel Anane, Mario Birkholz, Alex Bunker, Tapani Viitala, Michael Maiwald, Stefan Junne, Peter Neubauer
The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman) and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples...
June 8, 2018: Biotechnology Progress
Jaromír Vašíček, Medhat Shehata, Susanne Schnabl, Martin Hilgarth, Rainer Hubmann, Ulrich Jäger, Miroslav Bauer, Peter Chrenek
Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581 and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3)...
June 8, 2018: Biotechnology Progress
Sedigheh Kianpour, Alireza Ebrahiminezhad, Monica Negahdaripour, Milad Mohkam, Fatemeh Mohammadi, SeyyedVahid Niknezhad, Younes Ghasemi
A new technological approach to nanoparticle synthesis is using microorganisms, such as bacteria, which have the ability to synthesize nontoxic nanoparticles with high biocompatibility. In addition, bacteria have strict control over size, structure, shape, and dimension of produced nanoparticles. In the present work, Fe (III)-binding exopolysaccharide (Fe-EPS) nanoparticles were biosynthesized by Ralstonia pickettii sp. SK03, a bacterium isolated from a mineral spring. 16S rRNA gene sequencing and biochemical tests were done for identification of the isolated bacterium...
June 8, 2018: Biotechnology Progress
Lara E Krebs, Daniel M Bowden, Christopher M Bray, Margaret M Shaw, Christopher C Frye
Mammalian cell line development is critical to bioproduct manufacturing. Success requires selecting a line with desirable performance characteristics, including consistent expression throughout the proposed manufacturing window. Given the genetic and phenotypic flux inherent to immortalized lines such as Chinese hamster ovary (CHO) cells, clonally-derived cell line (CDCL) characterization is vital. We describe here the development and implementation of a novel addition to our characterization approach to ensure production cell line suitability: automated intracellular staining with statistical modeling...
June 8, 2018: Biotechnology Progress
Anabella Origone, Grisel Bersi, Andrés Illanes, Héctor Sturniolo, Constanza Liggieri, Fanny Guzmán, Sonia Barberis
In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Tyr-Gln-Gln (Z-YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris - HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr-Gln-Gln (YQQ) produced by solid-phase chemical synthesis. The in vitro anticoagulant activity of the above mentioned peptides was determined using Wiener Lab Test (Wiener, Argentina)...
June 8, 2018: Biotechnology Progress
Jared K Raynes, Laura J Domigan, F Grant Pearce, Juliet A Gerrard
Tobacco etch virus (TEV) protease is widely used for the removal of poly-histidine affinity tags from proteins. In solution, it is a one-time use enzyme for tag cleavage that has low stability, and is therefore a good candidate for immobilisation. Amyloid fibrils can act as a versatile nanoscaffold by providing a large surface area for biomolecule immobilisation. Immobilisation of TEV protease to amyloid fibrils grown from the surface of a small glass bead, using physisorption, successfully immobilised active TEV protease...
June 7, 2018: Biotechnology Progress
Sholeh Hamzehzadeh, Samira Touri
The potential of {polyethylene glycol 400 + potassium citrate} aqueous biphasic system (ABS) with ionic liquid (IL) 1-butyl-3-methylimidazolium bromide ([C4 C1 im]Br) as an adjuvant is examined for the extraction of L-phenylalanine (Phe), as a model biomolecule, at different temperatures and system compositions. The binodal curves and liquid-liquid equilibrium data were determined by the addition of 5 wt% IL to investigate its effect on phase diagrams and Phe partition coefficients. The results indicate that binodal curves of systems with and without IL are more deviated from each other with decreasing temperature...
June 7, 2018: Biotechnology Progress
Caryn L Heldt, Ashish Saksule, Pratik U Joshi, Majid Ghafarian
Vaccine manufacturing has conventionally been performed by the developed world using traditional unit operations like filtration and chromatography. There is currently a shift in the manufacturing of vaccines to the less developed world, requiring unit operations that reduce costs, increase recovery, and are amenable to continuous manufacturing. This work demonstrates that mannitol can be used as a flocculant for an enveloped and non-enveloped virus and can purify the virus from protein contaminants after microfiltration...
May 1, 2018: Biotechnology Progress
Adriano Gennari, Francielle H Mobayed, Ruan da Silva Rafael, Rafael C Rodrigues, Raul A Sperotto, Giandra Volpato, Claucia F Volken de Souza
We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead-Glu) or carboxyl groups through acid solution (Immobead-Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β-galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead-Epx) and modified supports...
May 1, 2018: Biotechnology Progress
Neha A Patel, Colin R Anderson, Sarah E Terkildsen, Ray C Davis, Laura D Pack, Swapnil Bhargava, Howard R G Clarke
Routine CHO cell line development practices involve a lengthy process of iteratively screening clonally-derived cell lines to identify a single line suitable for IND filing and clinical manufacture. Paramount in this process is development of a stable production cell line having consistent growth, productivity and product quality for the entire generational length of the manufacturing process. Scale-down stability models used to screen clones for consistency are time consuming and often a rate-limiting step in clone selection...
May 1, 2018: Biotechnology Progress
Marina Arredondo-Santoyo, Ma Soledad Vázquez-Garcidueñas, Gerardo Vázquez-Marrufo
The isolation and characterization of fungal strains from poorly described taxa allows undercover attributes of their basic biology useful for biotechnology. Here, a wild fungal strain (CMU-196) from recently described Paraconiothyrium genus was analyzed. CMU-196 was identified as Paraconiothyrium brasiliense by phylogenetic analysis of the rDNA internal transcribed spacer region (ITS). CMU-196 metabolized 57 out of 95 substrates of the Biolog FF microplates. Efficient assimilation of dextrins and glycogen indicates that CMU-196 is a good producer of amylolytic enzymes...
April 30, 2018: Biotechnology Progress
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