Methods and devices for producing biomolecules

Abstract

A scalable process and device for producing a biomolecule, in particular pharmaceutical grade plasmid DNA. The process includes the steps of alkaline lysis and a neutralization. For separating the lysate and the precipitate, the mixture is allowed to gently flow downward through a clarification reactor that is partially filled, in its lower part, with retention material like glass beads, whereby the precipitate is retained on top of and within the retention. In a preferred embodiment of the lysis step, cell suspension and alkaline lysis solution flow through a lysis reactor that is filled with particulate material like glass beads. The process can be run continuously and fully automated.

1 - 39 . (canceled) 
     
     
         40 . A method of purifying a polynucleotide of interest from host cells comprising the polynucleotide of interest using a device, wherein the device comprises a lysis reactor, a neutralization reactor and a clarification reactor fluidly connected to one another, the method comprising:
 (a) providing a cell suspension of the host cells that have been cultivated to produce the polynucleotide of interest, wherein the cell suspension is a fermentation broth within which the host cells were cultivated or a re-suspension of the cultivated host cells that were harvested from the fermentation broth;   (b) introducing a flow of the cell suspension and a flow of a lysis solution into the lysis reactor, the lysis reactor containing filling elements made of glass, plastic, stainless steel or fibrous material, such that the flow of the cell suspension and the flow of the lysis solution through the lysis reactor filling elements provides homogenous mixing of the flows in the absence of shear forces and whereby the cultivated host cells of the flowed suspension are substantially disintegrated by alkaline lysis alone to produce a lysed solution;   (c) neutralizing the cell solution via the neutralization reactor, wherein the lysed cell solution is mixed with a neutralization solution to produce a mixture comprising a lysate and a precipitate comprising cellular debris and impurities, and wherein the lysate contains the polynucleotide of interest;   (d) introducing the mixture comprising the precipitate and the lysate into the clarification reactor and mixing slowly with a stirrer or introducing gas through a distributor from the top or from an inlet in the bottom of the clarification reactor;   (e) purifying the polynucleotide of interest, where the polynucleotide of interest is purified from the lysate that flows out of the clarification reactor, wherein said method is operated on a manufacturing scale.   
     
     
         41 . The method of  claim 40 , wherein in step d), the mixing is carried out by introducing air through a distributor from the top or from an inlet in the bottom of the reactor. 
     
     
         42 . The method of  claim 41 , wherein in step d), the mixing is carried out by introducing air through an inlet in the bottom of the reactor. 
     
     
         43 . The method of  claim 40 , wherein the device is an automated or semi-automated system. 
     
     
         44 . A method of purifying a polynucleotide of interest from host cells comprising the polynucleotide of interest using device, wherein the device comprises a lysis reactor and a clarification reactor fluidly connected to one another, the method comprising:
 (a) providing a cell suspension of the host cells that have been cultivated to produce the polynucleotide of interest, wherein the cell suspension is a fermentation broth within which the host cells were cultivated or a re-suspension of the cultivated host cells that were harvested from the fermentation broth;   (b) introducing a flow of the cell suspension and a flow of a lysis solution into the lysis reactor, the lysis reactor containing filling elements made of glass, plastic, stainless steel or fibrous material, such that the flow of the cell suspension and the flow of the lysis solution through the lysis reactor filling elements provides homogenous mixing of the flows in the absence of shear forces and whereby the cultivated host cells of the flowed suspension are substantially disintegrated by alkaline lysis alone to produce a lysed solution;   (c) introducing the lysed cell solution into the clarification reactor;   (d) neutralizing the cell solution, wherein the lysed cell solution is mixed with a neutralization solution to produce a mixture comprising a lysate and a precipitate comprising cellular debris and impurities, and wherein the lysate contains the polynucleotide of interest;   (e) mixing slowly with a stirrer or introducing gas through a distributor from the top or from an inlet in the bottom of the clarification reactor;   (f) purifying the polynucleotide of interest, where the polynucleotide of interest is purified from the lysate that flows out of the clarification reactor, wherein said method is operated on a manufacturing scale.   
     
     
         45 . The method of  claim 44 , wherein in step e), the mixing is carried out by introducing air through a distributor from the top or from an inlet in the bottom of the reactor. 
     
     
         46 . The method of  claim 44 , wherein in step e), the mixing is carried out by introducing air through an inlet in the bottom of the reactor. 
     
     
         47 . The method of  claim 44 , wherein the device is an automated or semi-automated system. 
     
     
         48 . The method of  claim 44 , wherein the lysis reactor and the neutralization reactor are combined to form a two-step automated or semi-automated system.