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Biotechnology Progress

Sercan Karav, Joshua L Cohen, Daniela Barile, Juliana Maria Leite Nobrega de Moura Bell
Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions...
October 7, 2016: Biotechnology Progress
Claire F Komives, Elda E Sanchez, Anurag S Rathore, Brandon White, Montamas Suntravat, Michael Balderrama, Angela Cifelli, Varsha Joshi
An eleven amino acid ribosomal peptide was shown to completely neutralize Western Diamondback Rattlesnake (Crotalus atrox) venom in mice when a lethal dose of the venom was pre-incubated with the peptide prior to intravenous injection. We have expressed the peptide as a concatenated chain of peptides and cleaved them apart from an immobilized metal affinity column using a protease. After ultrafiltration steps, the mixture was shown to partially neutralize rattlesnake venom in mice. Preliminary experiments are described here that suggest a potential life-saving therapy could be developed...
October 7, 2016: Biotechnology Progress
William Kelly, Jonathan Rubin, Jennifer Scully, Hari Kamaraju, Piotr Wnukowski, Ravinder Bhatia
Within the last decade, fully disposable centrifuge technologies, fluidized-bed centrifuges (FBC), have been introduced to the biologics industry. The FBC has found a niche in cell therapy where it is used to collect, concentrate, and then wash mammalian cell product while continuously discarding centrate. The goal of this research was to determine optimum FBC conditions for recovery of live cells, and to develop a mathematical model that can assist with process scaleup. Cell losses can occur during bed formation via flow channels within the bed...
October 6, 2016: Biotechnology Progress
Marcin Zabadaj, Karolina Chreptowicz, Jolanta Mierzejewska, Patrycja Ciosek
Soft sensors are powerful tools for bioprocess monitoring due to their ability to perform on-line, non-invasive measurement and possibility of detection of multiple components in cultivation media, which in turn can provide tools for the quantification of more than one metabolite/substrate/product in real time. In this work soft sensor based on excitation-emission fluorescence is for the first time applied for the monitoring of biotransformation production of 2-Phenylethanol (2-PE) by yeast strains. Main process parameters, such as optical density, glucose and 2-PE concentrations were determined with high accuracy and precision by fluorescence fingerprinting coupled with Partial Least Squares Regression...
October 3, 2016: Biotechnology Progress
Soumyadeep Chakraborty, T Jagan Mohan Rao, Arun Goyal
Recombinant pectate lyase from family 1 polysaccharide lyase (PL1B) was immobilized on synthesized magnetic nanoparticles (MNPs) after 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) activation. At 70 mg/ml MNPs 100% binding of 1 mg/ml PL1B was achieved. The immobilized PL1B-MNP displayed activity of 20.3 and 18.2 U/mg against polygalacturonic acid and citrus pectin, respectively, which was higher than the activity of free PL1B, on the same substrates of 17.8 and 16.2 U/mg. The immobilized PL1B-MNP showed 32 fold and 14 fold enhanced thermal stability at 80°C and 90°C, respectively as compared with free PL1B at same temperatures...
October 3, 2016: Biotechnology Progress
Ying Wang, Linli Wang, Weili Yu, Dawei Gao, Guoxing You, Penglong Li, Shan Zhang, Jun Zhang, Tao Hu, Lian Zhao, Hong Zhou
Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been used as blood substitutes in surgery medicine and oxygen therapeutics for ischemic stroke. As a potent HBOC, the PEGylated Hb has received much attention for its oxygen delivery and plasma expanding ability. Two PEGylated Hbs, Euro-Hb and MP4 have been developed for clinical trials, using human adult hemoglobin (HbA) as the original substrate. However, HbA was obtained from outdated human blood and its quantity available from this source may not be sufficient for mass production of PEGylated HbA...
October 3, 2016: Biotechnology Progress
Kevin C Sales, Filipa Rosa, Bernardo R Cunha, Pedro N Sampaio, Marta B Lopes, Cecília R C Calado
Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However genetic, physiological and environmental factors influence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rapid, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable...
October 3, 2016: Biotechnology Progress
W N Reichelt, F Haas, P Sagmeister, C Herwig
Microbial bioprocesses need to be designed to be transferable from lab scale to production scale as well as between setups. Although substantial effort is invested to control technological parameters, usually the only true constant parameter is the actual producer of the product: the cell. Hence, instead of solely controlling technological process parameters the focus should be increasingly laid on physiological parameters. This contribution aims at illustrating a workflow of data life cycle management with special focus on physiology...
September 30, 2016: Biotechnology Progress
Rahul Sadavarte, Carlos D M Filipe, Raja Ghosh
Heterologous overexpression of genes in Escherichia coli has made it possible to obtain high titers of recombinant proteins. However, this can result in the formation of aggregated protein particles known as 'inclusion bodies'. Protein sequestered as inclusion body is inactive and needs to be converted back to its functional form by refolding using appropriate techniques. In the current study inclusion bodies of the enzyme aminoglycoside nucleotidyl transferase (or ANT(2")-Ia) were first solubilized in urea and subsequently subjected to thermal-cycling under controlled conditions as part of the refolding strategy...
September 30, 2016: Biotechnology Progress
Li-Kuan Zhu, Dominique T Monteil, Xiao Shen, David L Hacker, Bo-Yan Song, Zhen-Long Wang, Maria De Jesus, Florian M Wurm
Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50-ml tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high-throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three-dimensional simulation model of the unsteady liquid forms (waves) in this vessel...
September 30, 2016: Biotechnology Progress
Michael Sokolov, Jonathan Ritscher, Nicola MacKinnon, Jean-Marc Bielser, David Brühlmann, Dominik Rothenhäusler, Gian Thanei, Miroslav Soos, Matthieu Stettler, Jonathan Souquet, Hervé Broly, Massimo Morbidelli, Alessandro Butté
This work presents a multivariate methodology combining Principal Component Analysis, the Mahalanobis distance and Decision Trees for the selection of process factors and their levels in early process development of generic molecules. It is applied to a high throughput study testing more than 200 conditions for the production of a biosimilar monoclonal antibody at microliter scale. The methodology provides the most important selection criteria for the process design in order to improve product quality towards the quality attributes of the originator molecule...
September 30, 2016: Biotechnology Progress
María García-Torreiro, María López-Abelairas, Thelmo A Lu-Chau, Juan M Lema
In this study, a flow cytometry (FC) protocol was implemented to measure poly(3-hydroxybutyrate) (PHB) content in a halophilic bacterium, in order to have a faster and easier control of the process. The halophilic bacterium Halomonas boliviensis was stained with BODIPY 493/503 and analyzed using FC. Bacterial polymer accumulation induced by two different nutrient limitations during the operation of a 2 L bioreactor was studied using traditional gas chromatography (GC) analysis and FC. The application of this rapid and straightforward method is useful to obtain complex and precise information about PHB accumulation that could be used for the monitoring, control and optimization of the production of PHB...
September 30, 2016: Biotechnology Progress
Marc Feary, Andrew J Racher, Robert J Young, C Mark Smales
In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent...
September 30, 2016: Biotechnology Progress
Ashima Dua, Swati Joshi, T Satyanarayana
Chitinase from the thermophilic mould Myceliopthora thermophila BJA (MtChit) is an acid tolerant, thermostable and organic solvent stable biocatalyst which does not require any metal ions for its activity. In order to produce high enzyme titres, reduce fermentation time and overcome the need for induction, this enzyme has been heterologously expressed under GAP promoter in the GRAS yeast, Pichia pastoris. The production medium supplemented with the permeabilizing agent Tween-20 supported 2-fold higher rMtChit production (5...
September 30, 2016: Biotechnology Progress
Mahmoud Kamal Ahmadi, Blaine A Pfeifer
Biosynthesis of complex natural products like polyketides and nonribosomal peptides using Escherichia coli as a heterologous host provides an opportunity to access these molecules. The value in doing so stems from the fact that many compounds hold some therapeutic or other beneficial property and their original production hosts are intractable for a variety of reasons. In this work, metabolic engineering and induction variable optimization were used to increase production of the polyketide-nonribosomal peptide compound yersiniabactin, a siderophore that has been utilized to selectively remove metals from various solid and aqueous samples...
September 28, 2016: Biotechnology Progress
Jyoti Batra, Anurag S Rathore
Protein based therapeutics have started to dominate the pharmaceutical landscape primarily due to the high efficacy that they have demonstrated against complex diseases. Despite the significant success, issues with regards to safety, efficacy and quality of biotherapeutics have been a point of considerable debate and concern among the various stakeholders. The correlation between the glycoform profile and the safety and efficacy of a drug, in particular, has garnered significant attention of researchers worldwide...
September 27, 2016: Biotechnology Progress
Daniel Eugenio Gaytán Luna, Ana Erika Ochoa Alfaro, Alejandro Rocha-Uribe, Ana Silvia Pérez-Martínez, Ángel Gabriel Alpuche-Solís, Ruth Elena Soria-Guerra
Microalgae have the potential to accumulate triacylglycerols (TAGs) under different light spectra. In this work Chlamydomonas reinhardtii was grown under white (400-700 nm), red (650 nm) and green (550 nm) lights. According to our results, red light (650nm) has a positive effect in the microalgae growth and chlorophyll concentration. Regarding the lipid content, the control culture (white light-illuminated) reached a 4.4% of dry cell weight (dcw) while the culture grown at 550nm showed an increase in the lipids accumulation of 1...
September 27, 2016: Biotechnology Progress
Mahsa Hadidi, John J Buckley, Andrew L Zydney
Charged polysaccharides are used in the food industry, as cosmetics, and as vaccines. The viscosity, thermodynamics, and hydrodynamic properties of these charged polysaccharides are known to be strongly dependent on the solution ionic strength because of both inter- and intramolecular electrostatic interactions. The goal of this work was to quantitatively describe the effect of these electrostatic interactions on the ultrafiltration behavior of several charged capsular polysaccharides obtained from Streptococcus pneumoniae and used in the production of Pneumococcus vaccines...
September 27, 2016: Biotechnology Progress
André Kiesewetter, Peter Menstell, Lars H Peeck, Andreas Stein
Rapid development of chromatographic processes relies on effective high-throughput screening (HTS) methods. This article describes the development of pseudo-linear gradient elution for resin selectivity screening using RoboColumns(®) . It gives guidelines for the implementation of this HTS method on a Tecan Freedom EVO(®) robotic platform, addressing fundamental aspects of scale down and liquid handling. The creation of a flexible script for buffer preparation and column operation plus efficient data processing provided the basis for this work...
September 7, 2016: Biotechnology Progress
Maryam Ahmadi, Fereidoun Mahboudi, Mohammad Reza Akbari Eidgahi, Reza Nasr, Fatemeh Nemat Pour, Samira Ahmadi, Saeedeh Ebadat, Mojtaba Aghaee Poor, Fatemeh Davami
Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells...
September 7, 2016: Biotechnology Progress
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