Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-11-20
2004-08-31
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S176000, C435S178000, C435S180000, C435S803000, C435S815000, C436S524000, C436S529000, C436S531000, C530S412000, C530S413000, C530S415000, C530S811000, C530S813000, C530S815000, C536S022100
Reexamination Certificate
active
06783962
ABSTRACT:
FIELD OF THE INVENTION
A fluidised/expanded bed adsorption process for purification of bio-macromolecules is described. The method has been developed to be a first capture step in a downstream process in the production of bio-macromolecules such as nucleic acids, e.g. plasmid DNA, chromosomal DNA, RNA and virus DNA, and viruses themselves, and even bacteria.
BACKGROUND OF THE INVENTION
With the growing interest in gene therapy, the need to produce large quantities of gene therapy vectors becomes more pressing. Currently 24% of protocols under trial employ plasmid DNA as the delivery vehicle for gene therapy and vaccine applications. Viral DNA is also used as delivery vehicle in many gene therapy cases. Given the large size of gene vector molecules and the shear sensitivity of the solids produced following cell lysis and neutralisation to extract the gene vectors from microbial cells or viral material, traditional unit operations such as centrifugation, filtration and packed bed chromatography are not especially attractive. Fluidised bed adsorption or expanded bed adsorption, techniques based on fluidisation, offer considerable promise with intractable biological feedstock containing insoluble impurities.
An expanded bed is characterised by low degree of back-mixing of the adsorbent media. This means that each adsorbent particle move within a limited volume of the total bed. An expanded bed is called a stabilised fluidised bed. The stability occur when the adsorbent particles make up a so called classified bed where the larger and/or most dense adsorbent particles are positioned furthest down in the bed and the smaller and/or less dense adsorbent particles are positioned further up in the bed. The adsorbent media used in this invention is designed for use in a fluidised and preferable in an expanded bed. The size distribution and density variation determine the stability of the bed. As used herein, the term “fluidised/expanded” bed refers to that the unit operation at least is a fluidised bed and preferable an expanded bed.
There are at present at least two commercial suppliers of adsorbents for expanded bed chromatography. Pharmacia Biotech AB (Uppsala, Sweden) market Streamline™ (which utilises adsorbents of cross-linked polysaccharides (agarose) with quartz particles incorporated as high density fillers. The adsorbents have a density of about 1.2 g/ml with diameters being in the range of 125-315 &mgr;m. WO92/18237 (Pharmacia LKB Biotechnology AB) describe beads for down stream processing comprising a polymer matrix into which glass or silica particles have been incorporated, and their use in down stream processing, especially stabilised fluidised bed separations. The beads have a diameter of 100-1000 &mgr;m and a density of 1.10-1.50 g/ml of hydrated beads. The core materials are typically glass or silica.
WO 92/00799 (Kem-En-Tec, UpFront Chromatography A/S) describe adsorbent particles having a structure that is characterised by being pellicular or a conglomerate. This publication discloses a large number of adsorbents for use in fluidised/expanded bed chromatography.
WO 97/17132 (Pharmacia Biotech AB (Uppsala, Sweden) describes adsorbents for use in expanded bed chromatography, that is characterised by having a density of more than 1 g/ml and comprising a porous polymer base matrix in which a particulate filler is incorporated. The filler is characterised by having a density ≧3 g/ml, but the size of the core material particles “will always be much smaller than the size of the beads”. Thus, the density of the beads are normally just above 1.0 g/cm
3
.
Also described in the literature (oral presentation by E. Boschetti, BioSepra, on Second International Conference on Expanded Bed Adsorption, Napa Valley; Calif., USA, 21-23 June, 1998, see conference abstract book, page 14) is the use of small, high density particles suitable for fluid bed applications. The adsorbents described are designed to reduce the diffusion distance in a porous media inside the adsorbents. The adsorbents described are favourable to rapid diffusion and therefore compatible with high flow rates.
The adsorbent media commercially available today for fluidised/expanded bed chromatography has mainly been developed for protein purification. A fraction of the media has a high density (>1 g/ml) to ensure the sedimentation properties needed. The high density fraction is ideally inert and non corrosive under the conditions used during the particular purification. The high density fraction is combined with a polymer phase with an open pore structure where the target molecule (usually a protein) of interest can bind to a ligand coupled to the polymer. The target molecule is able to diffuse into the pores.
Thereby, the target molecule is exposed to binding sites in the whole volume of the polymer phase. The target bio-macromolecules described in the method here, e.g. plasmid DNA, chromosomal DNA, RNA, virus DNA and viruses themselves, and even bacteria are not able to diffuse into the pores of the commercially available media designed for expanded bed chromatography. Therefore is the binding likely to occur mainly to the surface of the media. With the media available today this constitute an important limitation of capacity, because of their relatively low available surface ares.
WO 97/29190 describes a technique for production of highly purified plasmid DNA in
E. coli
, which method includes growing plasmid containing cells to a high bio-mass in exponential growth, and lysing the cells by raising the pH of the culture to a carefully controlled pH value in which chromosomal DNA is denatured, but plasmid DNA is reversibly renatured. The plasmid containing feedstock is subsequently processed on a diethylaminoethyl (DEAE) anion exchanger in an expanded bed process.
Journal of Chromatography A, 806 (1998) 31-45 describes preparative purification of super-coiled plasmid DNA using quaternary amino ethyl (QAE) anion exchange chromatography.
BRIEF DESCRIPTION OF THE INVENTION
Thus the problem behind the present invention is to provide improved materials for fluidised bed adsoption or expanded bed adsorption, in particular materials for the purification of bio-macromolecules.
The present invention, thus, provides a method for the use of fluidised bed adsorption or expanded bed adsorption as a first capture step for recovery of bio-macromolecules, e.g. plasmid DNA, chromosomal DNA, RNA, viral DNA and viruses themselves, and even bacteria, from crude biologically feedstock, e.g. recovery of plasmid DNA from an
E. coli
, lysate feedstock. Novel particulate materials are also described.
A first objective is to provide a particulate material (an adsorbent media) that have an improved binding capacity for bio-macromolecules, e.g. plasmid DNA, chromosomal DNA, RNA, virus DNA, viruses as such and bacteria, through a hitherto unrealised combination of particle size, particle density and pendant groups (ligands) thereby offering certain advantages in chromatographic processes such as fluid bed processes. Thus, the present invention also provides a particulate material having a density of at least 2.5 g/ml, where the particles of the particulate material have an average diameter of 5-75 &mgr;m, and the particles of the particulate material are essentially constructed of a polymeric base matrix and a non-porous core material, said core material having a density of at least 3.0 g/ml, said polymeric base matrix including pendant groups which are positively charged such as at pH 4.0 or pH 6.0 or which are affinity ligands for a bio-molecule.
A second objective of the invention is to provide a method that can handle large scale production of bio-macromolecules, e.g. plasmid DNA, chromosomal DNA, RNA, virus DNA and viruses themselves, and even bacteria from crude feedstock using fluidised/expanded bed adsorption as a first capture step. Thus, the present invention provides a method for the isolation or purification of a bio-macromolecule, wherein said bio-macromolecule is adsorbed to a particulate material as defined in any of the c
Hobley Timothy John
Lihme Allan Otto Fog
Olander Morten Aae
Simon Marcos
Theodossiou Irini
Naff David M.
UpFront Chromatography
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