Compositions comprising high concentration of biologically active molecules and processes for preparing the same

Abstract

Large scale processes for producing high purity samples of biologically active molecules of interest from bacterial cells are disclosed. The methods comprise the steps of producing a lysate solution by contacting a cell suspension of said plurality of cells with lysis solution; neutralizing said lysate solution with a neutralizing solution to produce a dispersion that comprises neutralized lysate solution and debris; filtering the dispersion through at least one filter; performing ion exchange separation on said neutralized lysate solution to produce an ion exchange eluate; and performing hydrophobic interaction separation on said ion exchange eluate to produce a hydrophobic interaction solution. Further, provided are compositions comprising large scale amounts of plasmid DNA produced by the disclosed large scale processes.

1 . A large scale process for producing high purity samples of at least one biologically active molecule of interest from bacterial cells, comprising the steps of:
 a) producing a lysate solution by contacting a cell suspension of said plurality of cells with lysis solution;   b) neutralizing said lysate solution with a neutralizing solution to produce a dispersion that comprises neutralized lysate solution and debris;   c) filtering the dispersion through at least one filter;   d) performing ion exchange separation on said neutralized lysate solution to produce an ion exchange eluate; and   e) performing hydrophobic interaction separation on said ion exchange eluate to produce a hydrophobic interaction solution.   
     
     
         2 . The method of  claim 1 , wherein step a) comprises mixing said cell suspension with lysis solution in a high shear, in-line mixer. 
     
     
         3 . The method of  claim 1 , wherein step b) comprises mixing said lysate solution with said neutralizing solution in a bubble mixer. 
     
     
         4 . The method of claim any one of  claims 1 , wherein step e) comprises performing hydrophobic interaction separation using a hydrophobic interaction column or a hydrophobic interaction membrane to form a hydrophobic interaction solution. 
     
     
         5 . The method of any of  claims 1 , further comprising the step:
 f) preparing a solution of at least one biologically active molecule by ultrafiltration of said hydrophobic interaction solution.   
     
     
         6 . The method of  claims 5 , further comprising the step:
 g) preparing a sterile solution of at least one biologically active molecule by sterile filtration of said solution of biologically active molecules.   
     
     
         7 . The method of  claim 1 , further comprising holding the dispersion for a period of time to separate the neutralized lysate solution from the debris and filtering the neutralized lysate solution through at least one filter. 
     
     
         8 . The method of  claim 1 , wherein the producing step comprises contacting the cell suspension with the lysis solution in a mixer over a duration of from about 1 minutes to about 20 minutes. 
     
     
         9 . The method of  claim 8 , wherein the duration is from about 4 minutes to about 8 minutes. 
     
     
         10 . The method of  claim 8 , wherein the duration is about 5 minutes. 
     
     
         11 . The method of  claim 1 , wherein said biologically active molecule is a plasmid. 
     
     
         12 . The method of  claim 1  wherein said ion exchange is an anion exchange membrane. 
     
     
         13 . The method of  claim 1 , wherein step e) comprises performing hydrophobic interaction separation comprises butyl hydrophobic interaction chromatography in order to produce a hydrophobic interaction solution that is a butyl hydrophobic interaction chromatography solution eluate. 
     
     
         14 . The method of  claim 1 , wherein the method comprises transitioning from one step to a subsequent step substantially continuously and comprises separating the neutralized lysate solution from the debris in the dispersion by collecting the lysate in a container and passing the dispersion through a primary filter to produce an initial crude neutralized lysate solution. 
     
     
         15 . The method of  claim 14 , further comprising passing the first crude neutralized lysate solution through a secondary filter to produce a subsequent crude neutralized lysate solution. 
     
     
         16 . A large scale process for producing high purity samples of at least one biologically active molecule of interest from bacterial cells, comprising the steps of:
 contacting the bacterial cells in a dispersion of cells with lysis solution to form a lysate solution;   neutralizing the lysate solution by mixing a neutralization solution into the lysate solution with a bubble column mixer to form a neutralized mixture;   filtering the neutralized mixture through a primary filter and a secondary filter to form a filtered solution;   passing the filtered solution through an ion-exchange column to form a ion-exchange solution;   passing the ion-exchange solution through a hydrophobic interaction column or a hydrophobic interaction membrane to form a hydrophobic interaction solution; and   ultrafiltration of the hydrophobic interaction solution to form a high purity sample of at least one biologically active molecules of interest;   wherein each transition from one step to a subsequent step in the large scale process from the contacting step to the passing the filtered solution step occur substantially continuously.   
     
     
         17 . A composition comprising of at least one biologically active molecule of interest prepared by the method of  claims 1 , wherein at least one biologically active molecule of interest is a DNA plasmid. 
     
     
         18 . The composition of  claim 17 , where the composition comprises DNA plasmids at a quantity of about 10 mg or more in solution, wherein the high purity of said plasmids is the plasmids being present at greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. 
     
     
         19 . The composition of  claim 18 , wherein the purity of said plasmid is greater than about 99%. 
     
     
         20 . The composition of  claim 18 , wherein concentration of plasmid in the solution is about 5, 6, 7, 8, 9, 10, 11, 12, or 13 mg/mL. 
     
     
         21 . The composition of  claim 18 , wherein said plasmid comprises greater than about 50% supercoiled plasmid. 
     
     
         22 . The composition of  claim 18 , wherein said plasmid comprises greater than about 80% supercoiled plasmid. 
     
     
         23 . The composition of  claim 18 , wherein said solution contains less than or equal to about 10 EU endotoxin per mg of plasmid. 
     
     
         24 . The composition of  claim 18 , wherein said solution contains less than or equal to about 1 EU endotoxin per mg of plasmid. 
     
     
         25 . The composition of  claim 18 , wherein said solution contains less than or equal to about 0.1 EU endotoxin per mg of plasmid. 
     
     
         26 . The composition of  claim 18 , wherein said solution contains less than or equal to about 1.0% RNA. 
     
     
         27 . The composition of  claim 18 , wherein said solution contains less than or equal to about 0.4% RNA. 
     
     
         28 . The composition of  claim 18 , wherein said solution contains less than or equal to about 1.0% protein. 
     
     
         29 . The composition of  claim 18 , wherein said solution contains less than or equal to about 0.20% protein. 
     
     
         30 . The composition of  claim 18 , wherein said solution contains less than or equal to about 1% genomic DNA. 
     
     
         31 . The composition of  claim 18 , wherein said solution contains less than or equal to about 0.01% genomic DNA.