Direct visualisation of plasmid DNA in individual chromatography adsorbent particles by confocal scanning laser microscopy

Ljunglöf, Anders; Bergvall, Peder; Bhikhabhai, Ramagauri; Hjorth, Rolf

doi:10.1016/s0021-9673(99)00386-6

Cited by 91 publications

(53 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With a brush like configuration all molecules arrange in the same distance along with reduced electrostatic repulsion and lowest possible energy. Higher experimental values for q max of Source 30 Q and Q Sepharose HP can be explained by a partial penetration into the pores, which had already been illustrated by Ljunglof et al [4]. PL SAX 4000 30 lm cannot take full advantage of the internal surface area since the calculated binding capacity for binding in upright position cannot be reached experimentally.…”

Section: Modelling Of Theoretic Capacitymentioning

confidence: 84%

“…D values being a magnitude of 10 smaller than proteins and Mw in the range of 10 6 Da make conventional media suffer from low capacity and slow mass transfer [3]. Ljunglof et al [4] showed by confocal microscopy that media designed primarily for protein purification act as nonporous beads for pDNA, i. e. molecules are too big to diffuse into the pores and hence bind as outer layer only. Gustavsson et al [5] on the other hand took advantage of this drawback and introduced a flow-through purification protocol for pDNA on anion exchange chromatography (AEC) with a noncharged outer surface.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

View full text Add to dashboard Cite

Original PaperAdsorption of plasmid DNA on anion exchange chromatography media Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 30 lm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of selfassembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.

show abstract

Section: Modelling Of Theoretic Capacitymentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

View full text Add to dashboard Cite

show abstract

“…Such structures can be advantageous in the purification of newly emerging biomolecules such as plasmids and viruses that are large in size (up to 300 nm, potentially biggerlarger) [12]. Current purification of such entities using conventional adsorbents designed and optimized for proteins, such as Sepharose 4B, results in adsorption almost exclusively on the outer surface of the adsorbent as has been observed by poor chromatographic performance [27] and confirmed by confocal microscopy [28,29]. Although larger pore size due to extensive interfiber aggregation indicates reduction in internal surface area, an adsorbent with a very wide structure would provide larger apparent surface area for adsorption by allowing such large molecules to adsorb in the interior of the particles, thus increasing adsorption capacity and separation efficiency.…”

Section: Comparison With Commercial Sepharose 4b and Sepharose CL -4bmentioning

confidence: 97%

Manufacturing of agarose-based chromatographic adsorbents – Effect of ionic strength and cooling conditions on particle structure and mechanical strength

Ioannidis

Bowen

Pacek

et al. 2012

Journal of Colloid and Interface Science

View full text Add to dashboard Cite

The effect of ionic strength of agarose solution and quenching temperature of the emulsion on the structure and mechanical strength of agarose-based chromatographic adsorbents was investigated. Solutions of agarose containing different amounts of NaCl were emulsified at elevated temperature in mineral oil using a high-shear mixer. The hot emulsion was quenched at different temperatures leading to the gelation of agarose and formation of soft particles. Analysis of Atomic Force Microscopy (AFM) images of particle surfaces shows that pore size of particles increases with ionic strength and/or high quenching temperature. Additionally it has been found that the compressive strength of particles measured by micromanipulation also increases with ionic strength of the emulsion and/or high quenching temperature but these two parameters have practically no significant effect on the resulting particle size and /particle size distribution. Results from both characterization methods were compared with Sepharose 4B, a commercial agarose-based adsorbent. This is the first report examining the effect of ionic strength and cooling conditions during manufacturing, on the microstructure of micron-sized agarose beads for bioseparation.Keywords: agarose beads; microstructure; pore size; mechanical properties; AFM; micromanipulation 2 IntroductionAgarose is one of the two constituents of agar (the other being agaropectin) which is extracted from red algae [1]. It is a linear polysaccharide consisting of 1, 3-linked β-D-galactopyranose and 1, 4-linked 3, 6-anhydro-α-L-galactopyranose, while agaropectin is a more complicated polysaccharide containing sulphuric and uronic acid residues [2]. The gelation of agarose involves a change from a random coil in solution to a double helix in the initial stages of gelation and then to bundles of double helices in the final stage [3]. The gel is formed when an infinite extensive three-dimensional network of agarose fibres (consisting of helices) develops [4]. At this state the material is characterized by a co-continuous macroporous morphology with polymer-rich regions (fibres) and solvent-rich regions (pores holding water), and therefore the gelation can be interpreted as a phase separation [5].Because of its physical and chemical stability, neutral charge, hydrophilic character, open structure [6, 7] and biocompatibility, agarose has been extensively used in chromatographic separation of biomolecules [8], cell microencapsulation [9] and food additives [10]. Following functionalization with the appropriate chemistry a selective porous adsorbent can be produced for the purification of biomolecules both in packed beds and, after inclusion of densifiers, in fluidized beds [11]. The efficiency of purification depends both on macro-and microscopic properties of adsorbent. Macroscopic properties such as particle size/size distribution, shape and mechanical strength have a critical effect on throughput, pressure drop and mechanical stability of adsorbent [12]. Particles must also be sufficientl...

show abstract

“…Plasmids are as large as or larger than the pores of most beaded chromatography materials and thus any binding that occurs is strictly limited to the exterior surface [16,18,60]. Two 10 architectural features of Hyper media are likely to be responsible for elevated pDNA and sDNA binding [18].…”

Section: Selection Of Q Hyperz For Plasma Modificationmentioning

confidence: 99%