Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Lysis of micro-organism
Reexamination Certificate
1999-06-02
2004-03-02
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Lysis of micro-organism
C435S283100, C435S298100, C435S298200, C435S306100
Reexamination Certificate
active
06699706
ABSTRACT:
This invention relates to a process for treating suspensions of microbiological cells, for example for performing cell lysis, and for treating the fluid produced as a result of cell lysis.
A range of complex organic molecules can be synthesised within cells, such as bacterial cells, in a fermentation process. The desired product molecules must then be recovered and purified from the cells. In many cases, for example the synthesis of genetic material in the form of plasmid DNA, this is achieved using an initial cell lysis step followed by subsequent processing to remove contaminants through techniques such as precipitation. Cell lysis may be brought about by contacting a suspension of the cells with a lysis reagent such as sodium hydroxide solution. The rate at which homogenous mixing can be achieved is crucial to optimal performance of the lysis reaction. If the rate of mixing is too slow then lysis will occur in discrete regions within the fluids held within the mixer. This will reduce the rate at which lysis agent is able to contact the cells suspended within the fluid, as local increases in viscosity will impede the efficiency of the mixing process. This may result in damage to the desired product as a result of prolonged exposure to shear forces induced by the mixer, and loss of product due to incomplete lysis of the cells, and it increases the possibility of fouling due to the formation of by-products from the lysis reagent cross reacting with other cell components released subsequent to lysis.
According to the present invention there is provided a method for processing cells wherein an aqueous suspension of cells is intimately mixed with a lysis reagent by passage through a fluidic vortex mixer, the dimensions of the mixer and the flow rates being such that the residence time of the cell suspension in the mixer is less than the time for lysis to be completed.
A fluidic vortex mixer comprises a substantially cylindrical chamber with an axial outlet duct at the centre of an end wall of the chamber, and with at least one substantially tangential inlet near the periphery of the chamber to create a spiralling flow in the chamber. A second liquid may be supplied through a second tangential inlet, or through a radial inlet. The chamber contains no vanes or baffles. Such a mixer achieves a very intimate mixing of the two liquids in a very short time, yet it does not subject the liquids to high shear. It is also much less prone to being fouled, for example by proteinaceous deposits, than other types of mixer.
The residence time means the time taken by the cell suspension to pass through the mixer. This is preferably much less than the time taken for lysis to be completed, and is preferably less than 0.1 seconds, more preferably less than 0.01 seconds, so that the emerging liquid mixture is of viscosity similar to that of the in-flowing cell suspension. In this specification ‘completion of lysis’ refers to both the breaking down of the cell walls and the resulting increase in viscosity as cellular proteins and chromosomal DNA released from the cells interact with the lysis reagent. Preferably the viscosity increases during passage through this mixer by a factor of no more than ten times, more preferably no more than five times, for example less than twice. The low shear stress to which the cell suspension is subjected minimizes loss of product through denaturation or fragmentation of the product, and indeed of contaminants. Furthermore the rapid, intimate mixing ensures that all the cells are subjected to optimal conditions for lysis to occur.
The cell processing procedure usually involves subsequent treatment of the lysed cells with a precipitation agent. The invention also envisages a mixture of cell suspension and lysis reagent being mixed with the precipitation agent by passage through a fluidic vortex mixer. This may be a second such vortex mixer. Alternatively the precipitation agent may be mixed with the mixture of cell suspension and lysis reagent during its passage through the first vortex mixer, by supplying the precipitation agent to a third inlet duct into the first vortex mixer; this third inlet duct preferably communicates with the outlet duct from the chamber, and the open end of this third inlet duct may be adjustable in its position along the axis of the outlet duct.
REFERENCES:
patent: 4652517 (1987-03-01), Scholl
patent: 4775622 (1988-10-01), Hitzeman
patent: 4880746 (1989-11-01), Wohlleben
patent: 5047345 (1991-09-01), DeBonville
patent: 5096818 (1992-03-01), DeBonville
patent: 5437985 (1995-08-01), Quintana
patent: 5837529 (1998-11-01), Wan
patent: 0271667 (1988-06-01), None
patent: 0341215 (1989-11-01), None
patent: 0626456 (1994-11-01), None
patent: 1576299 (1969-07-01), None
patent: 2241796 (1991-09-01), None
patent: WO 91/01367 (1991-02-01), None
patent: WO 96/12956 (1996-05-01), None
Michael Mutsakis and Robert Rader, “Static Mixers Bring Benefits to Water/Wastewater Operations,” Water/Engineering & Management, Nov., 1986, pp. 30-31.
Accentus PLC
Holt William H.
Naff David M.
Ware Deborah K.
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