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Biotechnology Progress

Kahina Mellahi, Florian Cambay, Denis Brochu, Michel Gilbert, Michel Perrier, Sven Ansorge, Yves Durocher, Olivier Henry
Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10x106 cells/mL)...
November 10, 2018: Biotechnology Progress
Guoshuai Geng, Guangbin Dai, Dandan Li, Shengling Zhou, Zaoxia Li, Zhongbo Yang, Yuehong Xu, Jiaguang Han, Tianying Chang, Hong-Liang Cui, Huabin Wang
Photoconductive antenna microprobe (PCAM)-based terahertz (THz) near-field imaging technique is promising for biomedical detection due to its excellent biocompatibility and high resolution yet it is limited by its imaging speed and the difficulty in the control of the PCAM tip-sample separation. In this work, we successfully realized imaging of mouse brain tissue slices using an improved home-built PCAM-based THz near-field microscope. In this system, the imaging speed was enhanced by designing and applying a voice coil motor-based delay-line...
November 10, 2018: Biotechnology Progress
Ruth C Rowland-Jones, Colin Jaques
The biopharmaceutical industry is moving towards a more quality by design (QbD) approach that seeks to increase product and process understanding and process control. Miniature bioreactor systems offer a high-throughput method enabling the assessment of numerous process variables in a controlled environment. However, the number of off/atline samples that can be taken is restricted due to the small working volume of each vessel. This limitation may be resolved through the use of Raman spectroscopy due to its ability to obtain multi-analyte data from small sample volumes fast...
October 31, 2018: Biotechnology Progress
Enrique Cubas-Cano, Cristina González-Fernández, Mercedes Ballesteros, Elia Tomás-Pejó
Lactic acid is a versatile chemical that can be produced via fermentation of lignocellulosic materials. The heterolactic strain Lactobacillus pentosus CECT 4023 T, that can consume glucose and xylose, was studied to produce lactic acid from steam exploded wheat straw prehydrolysate. The effect of temperature and pH on bacterial growth was analysed. Besides, the effect of oxygen on lactic acid production was tested and fermentation yields were compared in different scenarios. This strain showed very high tolerance to the inhibitors contained in the wheat straw prehydrolysate...
October 31, 2018: Biotechnology Progress
Juliana Lebeau, Thomas Petit, Patricia Clerc, Laurent Dufossé, Yanis Caro
Filamentous fungi have gained growing interest as sources of diverse pigmented secondary metabolites. Some specific polyketides from Ascomycetous species have demonstrated a wide range of industrial applications in food, cosmetic, textile and in the design of pharmaceutical products. The formulation of recipes containing fungal polyketides has increased over recent years. Fusarium strains were proven useful to mankind in a variety of technologies. Nevertheless, there is still need of new isolates of Fusarium for use in emerging and already existing fields...
October 26, 2018: Biotechnology Progress
Kutay İçöz, Tayyibe Gerçek, Ayşegül Murat, Servet Özcan, Ekrem Ünal
One way to monitor Minimal Residual Disease is to screen cells for multiple surface markers using flow cytometry. In order to develop an alternative microfluidic based method, isolation of B type Acute Lymphoblastic Cells using two types of antibodies should be investigated. The immunomagnetic beads coated with various antibodies are used to capture the B type Acute Lymphoblastic cells. Single beads, two types of beads and surface immobilized antibody were used to measure the capture efficiency. Both micro and nano size immunomagnetic beads can be used to capture B type Acute Lymphoblastic cells with a minimum efficiency of 94% and maximum efficiency of 98%...
October 24, 2018: Biotechnology Progress
Loukas I Goulatis, Rasika Ramanathan, Eric V Shusta
Expressing antibodies as fusions to the non-self cleaving Mxe GyrA intein allows for site-specific chemical functionalization via expressed protein ligation. It is highly desirable to maximize the yield of functionalizable protein; and previously an evolved intein, 202-08, was identified that could increase protein fusion production in yeast. Given that the -1 amino acid residue upstream of inteins can affect cleavage efficiency, we examined the effects of amino acid variability at this position on 202-08 intein cleavage efficiency and secretion yield...
October 20, 2018: Biotechnology Progress
Sara Arana-Peña, Yuliya Lokha, Roberto Fernández-Lafuente
Lipase A from Candida antarctica (CALA, commercialized as Novocor ADL) was immobilized on octyl agarose, which is a very useful support for lipase immobilization, and coated with polyethylenimine (PEI) to improve the stability. The performance was compared to that of the form B of the enzyme (CALB) immobilized on the same support, as both enzymes are among the most popular ones used in biocatalysis. CALA immobilization produced a significant increase in enzyme activity versus p-nitrophenyl butyrate (by a factor of 7) and the coating with PEI did not have a significant effect on enzyme activity...
October 20, 2018: Biotechnology Progress
Haibin Luo, Liu Tie, Mingyan Cao, Alan K Hunter, Timothy M Pabst, Jiali Du, Raymond Field, Yuling Li, William K Wang
We report co-purification of the protease Cathepsin L that results in product fragmentation during purification processing and storage. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in CHO cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies (mAbs), antibody fragments (Fabs), bi-specific antibodies, and fusion proteins...
October 15, 2018: Biotechnology Progress
Clarissa Hamaio Okino-Delgado, Milene Stefani Pereira, João Vitor Inácio da Silva, Daniela Kharfan, Débora Zanoni do Prado, Luciana Francisco Fleuri
Brazil is the world's leading orange supplier for juice production purposes. The production process generates high amount of wastes, which leads to disposal problems. Orange wastes can be used for lipases production, incorporating the biorefinery concept into juice industries. Thus, the aim of the present study was to investigate the wastes of orange production chain as source of lipases based on different varieties (Pera, Hamlin, Valencia and Natal), as well as on different fractions of wastes. The mass balance of the juice/wastes (2007-2016 crops) was evaluated, and lipases from different varieties and fraction were biochemically characterized...
October 13, 2018: Biotechnology Progress
Kristin Robin Ko, Meng-Chiao Tsai, John P Frampton
Extracellular matrix-based hydrogels such as Matrigel are easy-to-use, commercially available, and offer environments for three-dimensional (3D) cell culture that mimic native tissue. However, manipulating small volumes of these materials to produce thin-layer 3D culture systems suitable for analysis is difficult due to air-liquid-substrate interfacial tension effects and evaporation. Here, we demonstrate two simple techniques that use standard liquid-handling tools and non-treated 96-well plates to produce uniform, thin-layer constructs for 3D culture of cells in Matrigel...
October 13, 2018: Biotechnology Progress
Yamini Satyawali, David Fernandes Del Pozo, Pieter Vandezande, Ingmar Nopens, Winnie Dejonghe
Hydrophobic pervaporation (PV), allowing for the separation of an organic component from an aqueous stream, was investigated for in situ acetone removal from a transamination reaction. A poly(dimethylsiloxane) membrane was applied in a coupled enzymatic process at 5 L scale. Among the four components, there was no loss of donor and product amines through PV which was highly desirable. However, in addition to removal of acetone, there was also an unwanted loss of acetophenone (substrate ketone) because of PV...
October 13, 2018: Biotechnology Progress
Lalita K Shekhawat, Avinash Godara, Vijesh Kumar, Anurag S Rathore
Development of a chromatographic step in a time and resource efficient manner remains a serious bottleneck in protein purification. Chromatographic performance typically depends on raw material attributes, feed material attributes, process factors, and their interactions. Design of Experiments (DOE) based process development is often chosen for this purpose. A challenge is, however, in performing a DOE with such a large number of process factors. In this paper, we propose a split DOE approach based on process knowledge in order to reduce the number of experiments...
October 13, 2018: Biotechnology Progress
Carol N Flores-Fernández, Max Cárdenas-Fernández, Dragana Dobrijevic, Kosma Jurlewicz, Amparo I Zavaleta, John M Ward, Gary J Lye
Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified...
October 10, 2018: Biotechnology Progress
Xiaotian Zhong, Weijun Ma, Caryl L Meade, Amy S Tam, Eliza Llewellyn, Richard Cornell, Kaffa Cote, John J Scarcelli, Jeffrey K Marshall, Boriana Tzvetkova, Bruno Figueroa, Dana DiNino, Annette Sievers, Christopher Lee, Jane Guo, Evan Mahan, Christopher Francis, Khetemenee Lam, Aaron M D'Antona, Richard Zollner, Hongli L Zhu, Ron Kriz, Will Somers, Laura Lin
Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i...
October 9, 2018: Biotechnology Progress
Norfariza Jamaluddin, Arbakariya B Ariff, Fadzlie Wong Faizal Wong
Aqueous micellar two-phase system (AMTPS) is an extractive technique of biomolecule, where it is based on the differential partitioning behaviour of biomolecule between a micelle-rich and a micelle-poor phase. In this study, an AMTPS composed of a non-ionic surfactant, Triton X-100 (TX-100) was used for purifying a bacteriocin-like inhibitory substance (BLIS) derived from Pediococcus acidilactici Kp10. The influences of the surfactant concentration and the effect of additives on the partitioning behaviour and activity yield of the BLIS were investigated...
October 9, 2018: Biotechnology Progress
Binglin Li, Jiao Wang, Huanyu Li, Xiaoli Zhang, Dandan Duan, Wenyu Yu, Binxia Zhao
The synthesis of nonnatural phospholipid, phosphatidylhydroxybutyrate (PB), was firstly introduced by phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) with sodium γ-hydroxybutyrate (NaGHB) in the aqueous-solid system. Nanoscale silicon dioxide (NSD) was employed as a carrier to provide an "artificial interphase" between PC and PLD. Special attention has been paid to the effect of the PC coverage on the surface area of hybrids of NSD-PC, the PC loading and the yield of PB...
October 9, 2018: Biotechnology Progress
Gyeongho Seon, Hyun Woo Joo, Yong Jae Kim, Juyi Park, Yong Keun Chang
Microalgal biomass was hydrolyzed by using a solid acid catalyst with the aid of liquid acid. The use of solid acid as the main catalyst instead of liquid acid was to omit subsequent neutralization and/or desalination steps, which are commonly required in using the resulting hydrolysates for microbial fermentation. The hydrolysis of 10 g/L of lipid-extracted Chlorella vulgaris containing 12.2% carbohydrates by using 7.6 g/L Amberlyst 36 and 0.0075 N nitric acid at 15 °C resulted in 1.08 g/L of mono-sugars with a yield of 88...
October 9, 2018: Biotechnology Progress
Shengchun Guo, Ayswarya Ravi, Stephen Mayfield, Zivko L Nikolov
Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed-modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel)...
October 9, 2018: Biotechnology Progress
Matthew Gagnon, Shashikant Nagre, Wenge Wang, Gregory W Hiller
In order to address the increasing demand for biologics, cell culture intensification using perfusion offers significantly higher productivities while also reducing manufacturing costs, especially when part of an integrated, continuous bioprocess. An initial study of a long-duration perfusion process using a cell-bleed to maintain a target cell density observed a 2.1-fold higher cell-specific productivity and a gradual decline in the culture growth rate when perfused at an overall lower rate. Subsequent studies sought an alternative process that largely reduced the overall volume of media needed by first perfusing at a high cell-specific perfusion rate (CSPR) to support a high cell density followed by continued perfusion at a low CSPR to promote a more productive stationary phase...
October 9, 2018: Biotechnology Progress
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