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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 Herrmann Mobayed, Ruan da Silva Rafael, Rafael C Rodrigues, Raul Antonio Sperotto, Giandra Volpato, Claucia Fernanda 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
Jakeline Kathiele Poppe, Carla Roberta Matte, Vitória Olave de Freitas, Roberto Fernandez-Lafuente, Rafael C Rodrigues, Marco Antônio Záchia Ayub
This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed-bed continuous reactor, using mixtures of immobilized lipases (combi-lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the use of different solvents, and flow rate on reaction conditions were studied. All experiments were conducted using the best combination of lipases according to the fatty acid composition of the waste oil (combi-lipase composition: 40% of TLL, 35% of CALB, and 25% of RML), and soybean oil (combi-lipase composition: 22...
April 30, 2018: Biotechnology Progress
Rajesh Kumar Kante, Sandeep Vemula, Maheswara Reddy Mallu, Srinivasa Reddy Ronda
Recombinant proteins are revolutionizing present day therapeutics. They are generally expressed as insoluble inclusion bodies in the E. coli and mis-folding, loss of protein, high cost of down streaming are the hurdles in their recovery. For the first time, we are reporting the refolding with simultaneous purification of rhASP in E. coli using a single step utilizing Protein Folding - strong anion exchange chromatography (PF-SAX). The purification method is also standardized for optimal concentration of solution additives, pH and mobile phase composition...
April 30, 2018: Biotechnology Progress
Matthew R Brown, Michael S Burnham, Scott C Lute, Sarah A Johnson, Alison A Walsh, Kurt A Brorson, David J Roush
A multi-tiered approach to determine the binding mechanism of viral clearance utilizing a multi-modal anion exchange resin was applied to a panel of four viral species that are typically used in validating viral clearance studies (i.e., X-MuLV, MVM, REO3, and PrV). First, virus spiked buffer-only experiments were conducted to evaluate the virus's affinity for single mode and multi-modal chromatography resins under different buffer conditions in a chromatography column setting. From these results we hypothesize that the mechanisms of binding of the viruses involve binding to both the hydrophobic and anionic functional groups...
April 30, 2018: Biotechnology Progress
Frank DeVilbiss, Aravinda Mandli, Doraiswami Ramkrishna
Diauxic growth of E. coli is driven by a host of internal, complex regulatory actions. In this classic scenario of cellular control, the cell employs a rational algorithm to modulate its metabolism in a competitive fashion. Cybernetic models of metabolism, whose development now spans three decades, were first formulated to describe regulation of cells in complex, multi-substrate environments. They modeled this scenario using the hypothesis that the formation of the enzymatic machinery is regulated to maximize a return on investment...
April 30, 2018: Biotechnology Progress
Joseana R Monte, Debora F Laurito-Friend, André Ferraz, Adriane M F Milagres
Sugarcane bagasses from three experimental sugarcane hybrids and a mill-reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and β-glucosidases (20 UI/g bagasse)...
April 26, 2018: Biotechnology Progress
Yinjun Zhang, Yicheng Fan, Wei Zhang, Guanzhong Wu, Jinghong Wang, Feng Cheng, Jianyong Zheng, Zhao Wang
Methyl (R)-N-(2,6-dimethylphenyl)alaninate ((R)-DMPM) is a key chiral intermediate for the production of (R)-metalaxyl, which is one of the best-selling fungicides. A new strain, Pseudochrobactrum asaccharolyticum WZZ003, was identified as a biocatalyst for the enantioselective hydrolysis of (R,S)-DMPM. The key parameters including pH, temperature, rotation speed and substrate concentrations were optimized in the enantioselective hydrolysis of (R,S)-DMPM. After the 48 h hydrolysis of 256 mM (R,S)-DMPM under the optimized reaction conditions, the enantiomeric excess of product (e...
April 25, 2018: Biotechnology Progress
J P Trasatti, J Woo, A Ladiwala, S Cramer, P Karande
Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use...
April 25, 2018: Biotechnology Progress
Martin Vanderlaan, Judith Zhu-Shimoni, Sansan Lin, Feny Gunawan, Thomas Waerner, Kevin van Cott
In the forty-year history of biopharmaceuticals there have been a few cases where the final products contained residual host cell protein (HCP) impurities at levels high enough to be of concern. This review article summarizes the industry experience in these cases where HCP impurities have been presented in public forums and/or published. Regulatory guidance on HCP impurities is limited to advising that products be as pure as practical, with no specified numerical limit because the risk associated with HCP exposure often depends on the clinical setting (route of administration, dose, indication, patient population) as well as the particular impurity...
April 25, 2018: Biotechnology Progress
Bohye Ahn, So Eun Park, Byung-Keun Oh, Young-Kee Kim
Magnetic cobalt ferrite/silica nanoparticles (MSNs) and methyl functionalized MSNs (methyl-MSNs) were used to enhance lipid production in Chlorella vulgaris culture through enhancement of gas-water mass transfer and increased dissolved concentration of CO2 . Methyl-MSNs enhanced CO2 -water mass transfer rate better than MSNs, and 0.3 wt% methyl-MSNs are more effective than 0.1 wt% MSNs. In the cultivation experiment, 0.3 wt% methyl-MSNs yielded the highest dry cell weight and subsequently, the highest mass transfer rate...
April 25, 2018: Biotechnology Progress
Nasib Qureshi, K Thomas Klasson, Badal C Saha, Siqing Liu
In these studies we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H2 SO4 (2 g·L-1 ) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250-300 gL-1 . This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 gL-1 sugars, 7.60 gL-1 acetic acid, 0.33 gL-1 furfural, and 0.07 gL-1 HMF were released...
April 25, 2018: Biotechnology Progress
Brooke E Tam, Yining Hao, Hadley D Sikes
Gene-specific promoter methylation is involved in gene silencing and is an important cancer biomarker. Cancer-specific methylation patterns have been observed and clinically validated for numerous gene promoters, but the knowledge gleaned from this large body of work is currently under-utilized in the clinic. Methylation-specific PCR is currently the gold standard method for clinical methylation assessment, but several research groups have proposed hybridization-based techniques which could be simpler to implement and provide more accurate results...
April 25, 2018: Biotechnology Progress
Nasib Qureshi, K Thomas Klasson, Badal C Saha, Siqing Liu
In these studies liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed-batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 gL-1 SSB hydrolysis, a fed-batch reactor with in-situ butanol recovery was devised...
April 25, 2018: Biotechnology Progress
Silvia M Pirrung, D Parruca da Cruz, A T Hanke, C Berends, Ruud F W C Van Beckhoven, Michel H M Eppink, Marcel Ottens
The application of mechanistic models for chromatography requires accurate model parameters. Especially for complex feedstocks such as a clarified cell harvest, this can still be an obstacle limiting the use of mechanistic models. Another commonly encountered obstacle is a limited amount of sample material and time to determine all needed parameters. Therefore, this study aimed at implementing an approach on a robotic liquid handling system that starts directly with a complex feedstock containing a monoclonal antibody...
April 25, 2018: Biotechnology Progress
Soledad L Saavedra, María C Martínez Ceron, Silvana L Giudicessi, Guillermina Forno, María Belén Bosco, Mariela M Marani, Rosa Erra-Balsells, Fernando Albericio, Osvaldo Cascone, Silvia A Camperi
Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied its ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4...
April 25, 2018: Biotechnology Progress
John J Scarcelli, Megan Hone, Kathryn Beal, Alejaida Ortega, Bruno Figueroa, Jason A Starkey, Karin Anderson
During development of a cell line intended to support production of an IgG2 monoclonal antibody, a sequence variant caused by a genetic mutation was identified in the bulk drug substance. Gene copy number analysis together with the level of the observed variant in genomic DNA indicated that the master cell bank was a mixed population of cells; some harboring the variant copy and some mutation free. Since the cell bank had been single-cell cloned, this variant could be used as a biomarker to demonstrate either that the bank was not derived from a single cell, or that the variant was a result of a post-cloning genetic event, leading to a mixed population of cells...
April 25, 2018: Biotechnology Progress
Margarita D Ramos, Letícia P Miranda, Raquel L C Giordano, Roberto Fernandez-Lafuente, William Kopp, Paulo W Tardioli
The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL-CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA' observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately 5-fold higher...
April 25, 2018: Biotechnology Progress
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