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  • 1
    Language: English
    In: The Journal of Membrane Biology, 2013, Vol.246(11), pp.861-867
    Description: The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli . In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.
    Keywords: Alkaline lysis ; Electroextraction ; Plasmid DNA ; Escherichia coli
    ISSN: 0022-2631
    E-ISSN: 1432-1424
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  • 2
    Language: English
    In: Journal of chromatography, 2013, Vol.1281, pp.87-93
    Description: To exploit different chromatographic modes for efficient plasmid DNA (pDNA) purification a novel monolithic chromatographic support bearing multimodal histamine (HISA) groups was developed and characterized. Electrostatic charge of HISA groups depends on the pH of the mobile phase, being neutral above pH 7 and becoming positively charged below. As a consequence, HISA groups exhibit predominantly ion-exchange character at low pH values, which decreases with titration of the HISA groups resulting in increased hydrophobicity. This feature enabled separation of supercoiled (sc) pDNA from other plasmid isoforms (and other process related impurities) by adjusting salt or pH gradient. The dynamic binding capacity (DBC) for a 5.1kbp large plasmid at pH 5 was 4.0mg/ml under low salt binding conditions, remaining relatively high (3.0mg/ml) even in the presence of 1.0M NaCl due to the multimodal nature of HISA ligand. Only slightly lower DBC (2.7mg/ml) was determined under preferentially hydrophobic conditions in 3.0M (NH₄)₂SO₄, pH 7.4. Open circular and sc pDNA isoforms were baseline separated in descending (NH₄)₂SO₄ gradient. Furthermore, an efficient plasmid DNA separation was possible both on analytical as well as on preparative scale by applying the descending pH gradient at a constant concentration (above 3.0M) of (NH₄)₂SO₄. ; p. 87-93.
    Keywords: Chromatography ; Sodium Chloride ; Titration ; Plasmids ; Ion Exchange ; Hydrophobicity ; Ph ; Histamine ; Binding Capacity
    ISSN: 0021-9673
    Source: AGRIS (Food and Agriculture Organization of the United Nations)
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  • 3
    Language: English
    In: Journal of Chromatography A, 15 March 2013, Vol.1281, pp.87-93
    Description: ► A new chromatographic method for the separation of pDNA isoforms was developed. ► The method employs a multimodal histamine ligand on a monolith. ► Hydrophobic and electrostatic interactions are expressed simultaneously. ► The balance of interaction is determined by the titration state of the histamine. ► Both plasmid isoforms were proven to be selectively eluted on an analytical as well as on preparative scale. To exploit different chromatographic modes for efficient plasmid DNA (pDNA) purification a novel monolithic chromatographic support bearing multimodal histamine (HISA) groups was developed and characterized. Electrostatic charge of HISA groups depends on the pH of the mobile phase, being neutral above pH 7 and becoming positively charged below. As a consequence, HISA groups exhibit predominantly ion-exchange character at low pH values, which decreases with titration of the HISA groups resulting in increased hydrophobicity. This feature enabled separation of supercoiled (sc) pDNA from other plasmid isoforms (and other process related impurities) by adjusting salt or pH gradient. The dynamic binding capacity (DBC) for a 5.1 kbp large plasmid at pH 5 was 4.0 mg/ml under low salt binding conditions, remaining relatively high (3.0 mg/ml) even in the presence of 1.0 M NaCl due to the multimodal nature of HISA ligand. Only slightly lower DBC (2.7 mg/ml) was determined under preferentially hydrophobic conditions in 3.0 M (NH ) SO , pH 7.4. Open circular and sc pDNA isoforms were baseline separated in descending (NH ) SO gradient. Furthermore, an efficient plasmid DNA separation was possible both on analytical as well as on preparative scale by applying the descending pH gradient at a constant concentration (above 3.0 M) of (NH ) SO .
    Keywords: Plasmid DNA ; Chromatography ; Immobilized Histamine ; Multimodal Interactions ; Monoliths ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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  • 4
    Language: English
    In: Journal of Chromatography A, 2011, Vol.1218(17), pp.2432-2437
    Description: Monoliths are chromatographic stationary phases, which were specially designed for efficient purification of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60 × 10 viral particles/mL to at least 1.64 × 10 viral particles/mL, the limit of detection was found to be 2.56 × 10 virus particles/mL and the limit of quantification was 5.12 × 10 virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus.
    Keywords: Monolithic Column ; Chromatography ; Hplc Analysis ; Influenza ; Haemagglutinin ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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  • 5
    Language: English
    In: Journal of chromatography, 2013, Vol.927, pp.80-89
    Description: Chromatographic monoliths have already penetrated in many different areas of separation sciences. This is due to their properties, especially advantageous for fast separation and purification of large biologic macromolecules, even at low pressure drop. Probably the most outstanding features are flow unaffected binding capacity and resolution, later resulting in very short analysis times. Furthermore, since large biomolecules interact with the matrix via many binding sites, efficient separation can be achieved with the monolithic columns of a very short length, further reducing pressure drop over matrix. In this review brief introduction to the monoliths is given with the emphasize on the theory of separation of large molecules, particularly on a linear gradient elution and estimation of peak broadening. As an outcome of this analysis the most efficient separation is expected when short monolithic column with accordingly adjusted gradient is implemented, especially for macromolecules interacting with the monolith functionalities via over 10 binding sites. This is experimentally demonstrated by several recent examples of short monolithic column applications for analysis of antibodies, viruses, virus like particles (VLPs) and polynucleotides like plasmid DNA (pDNA) and RNA, indicating their potential for process monitoring, control and optimization but also for product final formulation and quality control. ; p. 80-89.
    Keywords: Chromatography ; Rna ; Binding Sites ; Antibodies ; Quality Control ; Viruses ; Plasmids ; Polynucleotides ; Process Monitoring ; Binding Capacity
    ISSN: 1570-0232
    Source: AGRIS (Food and Agriculture Organization of the United Nations)
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  • 6
    Language: English
    In: Genome announcements, 10 November 2016, Vol.4(6)
    Description: Here, we present the whole-genome sequences of bacteriophages PC5 and PC14 specific for Campylobacter jejuni, a leading cause of gastroenteritis in developed countries. Their genomes are syntenic to those of group III Campylobacter bacteriophages and share more than 90% identity at the nucleotide level with members of this group.
    Keywords: Biology;
    ISSN: 2169-8287
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  • 7
    Language: English
    In: Virology journal, 12 November 2012, Vol.9, pp.265
    Description: The NanoSight LM10 with Nanoparticle tracking analysis (NTA) software was evaluated for the quantification of latex particles, adenovirus 5, and influenza virus. The inter-day variability was determined by measuring the same sample over several consecutive days and the method's accuracy was demonstrated by using known concentrations of the subject particles. NTA analysis was also used to quantify chromatographic fractions of adenovirus and influenza virus after purification on a CIM monolithic column. NTA results were compared and evaluated against hemagglutination (HA) and end point dilution assay, determining total and infection virus particle number, respectively. The results demonstrated that nanoparticle tracking analysis is a method for fast estimation of virus concentration in different samples. In addition, it can provide a better insight into the sample status, regarding the level of virus aggregation.
    Keywords: Nanoparticles ; Adenoviridae -- Isolation & Purification ; Orthomyxoviridae -- Isolation & Purification ; Viral Load -- Methods
    E-ISSN: 1743-422X
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 8
    Language: English
    In: Virology Journal, 01 November 2012, Vol.9(1), p.265
    Description: Abstract The NanoSight LM10 with Nanoparticle tracking analysis (NTA) software was evaluated for the quantification of latex particles, adenovirus 5, and influenza virus. The inter-day variability was determined by measuring the same sample over several consecutive days and the method’s accuracy...
    Keywords: Nanosight ; Nta ; Adenovirus 5 ; Influenza ; Latex Particles ; Cim Monolithic Column ; Medicine ; Biology
    ISSN: 1743-422X
    E-ISSN: 1743-422X
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  • 9
    Language: English
    In: Journal of Chromatography B, 15 May 2013, Vol.927, pp.80-89
    Description: ► Application of short monolithic columns for analytics of macromolecules and nanoparticles – recent overview. ► Impact of macromolecule interactions on peak spreading and resolution in IEX. ► Overview of binding sites number for different class of macromolecules and nanoparticles. Chromatographic monoliths have already penetrated in many different areas of separation sciences. This is due to their properties, especially advantageous for fast separation and purification of large biologic macromolecules, even at low pressure drop. Probably the most outstanding features are flow unaffected binding capacity and resolution, later resulting in very short analysis times. Furthermore, since large biomolecules interact with the matrix via many binding sites, efficient separation can be achieved with the monolithic columns of a very short length, further reducing pressure drop over matrix. In this review brief introduction to the monoliths is given with the emphasize on the theory of separation of large molecules, particularly on a linear gradient elution and estimation of peak broadening. As an outcome of this analysis the most efficient separation is expected when short monolithic column with accordingly adjusted gradient is implemented, especially for macromolecules interacting with the monolith functionalities via over 10 binding sites. This is experimentally demonstrated by several recent examples of short monolithic column applications for analysis of antibodies, viruses, virus like particles (VLPs) and polynucleotides like plasmid DNA (pDNA) and RNA, indicating their potential for process monitoring, control and optimization but also for product final formulation and quality control.
    Keywords: Monoliths ; Analytics ; Peak Spreading ; Convective Interaction Media (Cim) ; Short Columns ; Biomolecues ; Anatomy & Physiology
    ISSN: 1570-0232
    E-ISSN: 1873-376X
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  • 10
    Language: English
    In: Journal of Thoracic Oncology, January 2017, Vol.12(1), pp.S1156-S1156
    Keywords: Medicine
    ISSN: 1556-0864
    E-ISSN: 1556-1380
    Source: ScienceDirect Journals (Elsevier)
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