Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography

A new approach for the removal of protein impurities from purified biologicals using combinatorial solid‐phase ligand libraries

“…Therefore, several strategies have been described whose efficiency depends on the nature of the impurities to be removed. When polishing is intended to remove DNA-related molecules and endotoxins, anion exchangers are most generally used [21,22]. The success of this operation is contingent upon the net charge of the target protein; if the protein has an acidic pI then the separation process may be much less efficient in terms of overall yield.…”

Surface modification of chromatography adsorbents by low temperature low pressure plasma

“…The present study concerns the simplest multi-layered multi-functional support design one can envisage, 30 namely one featuring just two differently functionalised layers -an inert outer size excluding layer and inner ion exchange functionalised core. The benefits of bi-layered size exclusion chromatography -ion exchange chromatography (SEC-IEC) beaded support designs have been clearly demonstrated in the context of 'nanoplex' purification [7,8], fluidised bed separation of organic acids [9,10] and expanded bed adsorption of proteins [11,12]. The important findings from these studies, inherent flaws in the methods employed thus far to manufacture bi-layered SEC-IEC hybrids, and identification of a simple and effective solution 5…”

“…Recently, it has been demonstrated that much greater productivity could be realised if the SEC and AEC operations are 'passively' combined in a single chromatographic operation, employing a new type of multi-functional chromatography material (known as the lid bead) 25 [7,8]. Starting from an SEC matrix with a nucleic acid exclusion limit of 1000 bp, Gustavsson and co-workers [7] made a bi-functional restricted access matrix possessing a positively charged core (to adsorb large amounts of RNA and protein) and an inert outer layer to exclude pDNA from accessing the functionalised bead interior.…”

In situ modification of chromatography adsorbents using cold atmospheric pressure plasmas

“…[8][9][10][11] New types of beaded chromatography adsorbents featuring two or more distinct functional regions spatially separated from one another within the same support bead have been the subject of a handful of research reports since 2002; 5,[12][13][14][15][16][17] the most promising of these describing the manufacture and testing of bi-layered bi-functional supports comprising ion exchange (IEC) functionalized cores surmounted by outer size excluding (SEC) layers. 5,[13][14][15] The main attraction of the bi-layered SEC-IEC support architecture ( Fig. 1(b)) is that it enables efficient separation of certain nanoplex bio-products (e.g., plasmid DNA) from smaller, but chemically very similar "problem" contaminants (protein, RNA) in a "one column-one bead" chromatography process combining size exclusion and ion exchange principles.…”

“…1(b)) is that it enables efficient separation of certain nanoplex bio-products (e.g., plasmid DNA) from smaller, but chemically very similar "problem" contaminants (protein, RNA) in a "one column-one bead" chromatography process combining size exclusion and ion exchange principles. 5,13,14 The ideal bi-layered SEC-IEC support should possess inert mechanically reliable "non-stick" exteriors or barriers, that are freely accessible to smaller components (proteins, RNA), but not larger entities, such as long chain nucleic acids, cell debris fragments, nanoplexes, etc., and in order not to compromise mass transport and sorption properties, they must also be very thin, i.e., ideally <1 lm. 5,18 To date, these criteria have not been met.…”