Scalable Process for Protein Purification

Abstract

The invention provides a process for the purification recombinantly expressed, self-assembled VLP from the homogenate of a bacterial host, wherein the process can be scaled up to a commercial production scale in a cost effective manner. The process comprises a first chromatography using an anion exchange matrix, a second chromatography using hydroxyapatite and, optionally, a size exclusion chromatography. VLP preparations obtained by the process of the invention are essentially free of endotoxin contaminations.

1 . A process for the purification of a VLP from a recombinant bacterial host expressing said VLP, the process comprising the steps of:
 (a) homogenizing said bacterial host;   (b) clarifying the homogenate obtained by said homogenizing;   (c) purifying said VLP from the clarified homogenate obtained by said clarifying in a first chromatography comprising the steps of:
 (i) binding said VLP to a first chromatography matrix; 
 (ii) washing said first chromatography matrix; and 
 (iii) eluting said VLP from said first chromatography matrix; and 
   (d) further purifying said VLP from the eluate obtained by said first chromatography in a second chromatography, wherein said second chromatography is performed on a second chromatography matrix, wherein said second chromatography matrix is a hydroxyapatite matrix;   wherein said steps are performed in the given order.   
     
     
         2 . The process of  claim 1 , wherein said second chromatography comprises the steps of:
 (i) binding said VLP to said second chromatography matrix, wherein said second chromatography matrix is a hydroxyapatite matrix;   (ii) washing said second chromatography matrix; and   (iii) eluting said VLP from said second chromatography matrix;   
       wherein said steps are performed in the given order. 
     
     
         3 . The process of  claim 1 , said process additionally comprising the step of finally purifying said VLP obtained by said second chromatography by at least one third chromatography, wherein said at least one third chromatography is selected from:
 (a) hydrophobic interaction chromatography (HIC);   (b) immobilized metal ions affinity chromatography (IMAC); and   (c) size exclusion chromatography.   
     
     
         4 . The process of  claim 1 , wherein said VLP comprises capsid protein of a virus selected from the group consisting of:
 (a) RNA bacteriophage;   (b) bacteriophage;   (c) Hepatitis B virus;   (d) measles virus;   (e) Sindbis virus;   (f) rotavirus;   (g) foot-and-mouth-disease virus;   (h) Norwalk virus;   (i) Alpha Virus;   (j) retrovirus;   (k) retrotransposon Ty;   (l) human Papilloma virus;   (m) Polyoma virus;   (n) Tobacco mosaic virus; and   (o) Flock House Virus.   
     
     
         5 . The process of  claim 1 , wherein said clarifying of said homogenate is performed by a method selected from the group consisting of:
 (a) centrifugation;   (c) tangential flow filtration, preferably using a filter having a membrane comprising a pore size of about 0.45 μm; and   (c) a combination of (a) and (c).   
     
     
         6 . The process of  claim 1 , wherein said first chromatography matrix is an anion exchange matrix, preferably an anion exchange matrix comprising TMAE groups. 
     
     
         7 . The process of  claim 1 , wherein said first chromatography matrix is a tentacle anion exchange matrix comprising (i) resin particles of cross-linked methacrylate polymer or cross-linked vinyl polymer (ii) acrylamide tentacles, wherein said acrylamide tentacles are attached to the surface of said resin particles, and wherein said acrylamide tentacles are substituted with TMAE (Trimethylaminoethyl-) groups. 
     
     
         8 . The process of  claim 1 , wherein said first chromatography matrix is selected from the group consisting of
 (a) Fractogel® EMD TMAE (M), preferably having a particle size of 40-90 μm;   (b) Fractogel® EMD TMAE Hicap (M), preferably having a particle size of 40-90 μm;   (c) Fractoprep® DEAE, preferably having a particle size of 30-150 μm;   (d) Macro-Prep® CHT Ceramic Hydroxyapatite Type I, preferably having a particle size of about 80 μm;   (e) Macro-Prep® CHT Ceramic Hydroxyapatite Type II, preferably having a particle size of about 80 μm;   (f) Matrex® Granular Silica PEI-300 Å, preferably having a particle size of 35-70 μm;   (g) Matrex® Granular Silica PEI-1000 Å, preferably having a particle size of 35-70 μm;   (h) Poros 50 HQ;   (i) CIM-QA (quarternary amino group, BIA Separations Cat. No. 210.5113); and   (j) CIM-DEAE.   
     
     
         9 . The process of  claim 1 , wherein said first chromatography comprises the steps of:
 (i) equilibrating said first chromatography matrix with a first equilibration buffer;   (ii) binding said VLP to a first chromatography matrix;   (iii) washing said first chromatography matrix with a first washing buffer; and   (iv) eluting said VLP from said first chromatography matrix with a first elution puffer;   wherein said first equilibration buffer, said first washing buffer and said first elution buffer comprise an inorganic salt, preferably an alkaline metal halogenide, more preferably potassium chloride or sodium chloride, most preferably sodium chloride.   
     
     
         10 . The process of  claim 9 , wherein said first equilibration buffer comprises at most about 200 mM sodium chloride, said first washing buffer comprises about 425 mM sodium chloride, and said first elution buffer comprises least about 500 mM sodium chloride or a gradient of sodium chloride, wherein preferably said gradient is from at most about 400 to at least about 650 mM sodium chloride, preferably from 425 to 650 mM sodium chloride. 
     
     
         11 . The process of  claim 9 , wherein said first equilibration buffer, said first washing buffer and said first elution buffer comprise a pH of about 7.2, wherein preferably said pH is stabilized by a phosphate buffer, more preferably by about 20 mM phosphate buffer, most preferably by about 20 mM sodium phosphate buffer. 
     
     
         12 . The process of  claim 1 , wherein said hydroxyapatite matrix is a ceramic hydroxyapatite matrix, wherein preferably said ceramic hydroxyapatite matrix comprises a particle size of about 80 μm and a pore size of the particles of about 800-1000 Å, wherein further preferably said ceramic hydroxyapatite matrix is Macro-Prep® CHT Ceramic Hydroxyapatite Type II. 
     
     
         13 . The process of  claim 2 , wherein said second chromatography comprises the steps of:
 (i) equilibrating said second chromatography matrix with a second equilibration buffer;   (ii) binding said VLP to said second chromatography matrix, wherein said second chromatography matrix is a hydroxyapatite matrix;   (iii) washing said second chromatography matrix with a second washing buffer; and   (iv) eluting said VLP from said second chromatography matrix;   wherein said second equilibration buffer, said second washing buffer and said second elution buffer, comprise an inorganic salt, preferably an alkaline metal halogenide, more preferably potassium chloride or sodium chloride, most preferably sodium chloride.   
     
     
         14 . The process of  claim 13 , wherein said second equilibration buffer comprises about 100 to 400 mM sodium chloride, said second washing buffer comprises about 150 mM sodium chloride, and said second elution buffer comprises 900 mM sodium chloride and about 200 mM sodium phosphate buffer. 
     
     
         15 . The process of  claim 13 , wherein said second equilibration buffer, said second washing buffer and said second elution buffer comprise a pH of about 7.2, wherein preferably said pH is stabilized by a phosphate buffer, preferably by a sodium phosphate buffer. 
     
     
         16 . The process of  claim 1 , wherein said clarifying further comprises the step of exposing said VLP to oxidative conditions. 
     
     
         17 . The process of  claim 1 , wherein said at least one third chromatography is at least one, preferably exactly one, size exclusion chromatography, wherein said size exclusion chromatography is preferably performed using a gel filtration matrix selected from the group consisting of:
 (a) Sephadex G-25;   (b) Sepharose CL-4B; and   (c) Sephacryl-S400.   
     
     
         18 . A process for the purification of a VLP of RNA bacteriophage Qβ from a recombinant bacterial host expressing said VLP, the process comprising the steps of:
 (a) homogenizing said bacterial host; 
 (b) clarifying the homogenate obtained by said homogenizing, wherein said clarifying further comprises the step of exposing said VLP to oxidative conditions; 
 (c) purifying said VLP from the clarified homogenate obtained by said clarifying in a first chromatography comprising the steps of:
 (i) equilibrating a tentacle anion exchange matrix, wherein said equilibrating is performed with a first equilibration buffer, wherein said first equilibration buffer comprises about 150 mM sodium chloride and a pH of 702; 
 (ii) binding said VLP to said tentacle anion exchange matrix; 
 (iii) washing said tentacle anion exchange matrix, wherein said washing is performed with a first washing buffer comprising about 425 mM sodium chloride and a pH of 72; and 
 (iv) eluting said VLP from said tentacle anion exchange matrix, wherein said eluting is performed with a first elution buffer comprising a gradient of 425 to 650 mM sodium chloride and a pH of 71; 
 wherein preferably said tentacle anion exchange matrix is a tentacle anion exchange matrix as defined in  claim 7 , most preferably Fractogel® EMD TMAE (M); 
 
 (d) further purifying said VLP from the eluate obtained by said first chromatography in a second chromatography comprising the steps of:
 (i) equilibrating a hydroxyapatite matrix wherein said equilibrating is performed with a second equilibration buffer comprising about 150 mM sodium chloride and a pH of 7.2; 
 (ii) binding said VLP to hydroxyapatite matrix, preferably in the presence of about 250 mM sodium chloride; 
 (iii) washing said hydroxyapatite matrix, wherein said washing is performed with a second washing buffer comprising about 150 mM sodium chloride and a pH of 7.2; 
 (iv) eluting said VLP from said hydroxyapatite matrix, wherein said eluting is performed with a second elution buffer comprising about 900 mM sodium chloride, about 200 mM sodium phosphate buffer and a pH of 7.2; 
 wherein preferably said hydroxyapatite matrix is a hydroxyapatite matrix as defined in  claim 12 , most preferably a Macro-Prep® CHT Ceramic Hydroxyapatite Type II matrix; 
 
 (e) finally purifying said VLP contained in the eluate of said second chromatography by exactly one size exclusion chromatography, wherein said size exclusion chromatography is performed in the presence of about 150 mM sodium chloride, and wherein further said size exclusion chromatography is performed using a Sepharose CL-4B gel filtration matrix; 
 wherein said steps are performed in the given order. 
 
     
     
         19 . A process for the purification of a VLP of RNA bacteriophage AP205 from a recombinant bacterial host expressing said VLP, the process comprising the steps of:
 (a) homogenizing said bacterial host;   (b) clarifying the homogenate obtained by said homogenizing, wherein said clarifying further comprises the step of exposing said VLP to oxidative conditions;   (c) purifying said VLP from the clarified homogenate obtained by said clarifying in a first chromatography comprising the steps of:
 (i) equilibrating a tentacle anion exchange matrix, wherein said equilibrating is performed with a first equilibration buffer, wherein said first equilibration buffer comprises about 150 mM sodium chloride and a pH of 7.2; 
 (ii) binding said tentacle anion exchange matrix; 
 (iii) washing said tentacle anion exchange matrix, wherein said washing is performed with a first washing buffer comprising about 425 mM sodium chloride and a pH of 7.2; and 
 (iv) eluting said VLP from said tentacle anion exchange matrix, wherein said eluting is performed with a first elution buffer comprising about 550 mM sodium chloride and a pH of 7.2; 
 wherein preferably said tentacle anion exchange matrix is a tentacle anion exchange matrix as defined in  claim 7 , most preferably Fractogel EMD TMAE (M); 
   (d) further purifying said VLP from the eluate obtained by said first chromatography in a second chromatography comprising the steps of:
 (i) equilibrating a hydroxyapatite matrix, wherein said equilibrating is performed with a second equilibration buffer comprising, about 100 mM sodium chloride and about 5 mM sodium phosphate buffer and a pH of 7.2; 
 (ii) binding said VLP to said hydroxyapatite matrix, preferably in the presence of about 100 mM sodium chloride and about 5 mM sodium phosphate buffer; 
 (iii) washing said hydroxyapatite matrix wherein said washing is performed with a second washing buffer comprising about 100 mM sodium chloride, about 5 mM sodium phosphate buffer and a pH of 7.2; 
 (iv) eluting said VLP from said hydroxyapatite matrix, wherein said eluting is performed with a second elution buffer comprising about 250 mM sodium chloride, about 50 mM sodium phosphate buffer and a pH of 7.2; 
 wherein preferably said hydroxyapatite matrix is a hydroxyapatite matrix as defined in  claim 12 , most preferably a Macro-Prep® CHT Ceramic Hydroxyapatite Type II matrix; 
   (e) finally purifying said VLP contained in the eluate of said second chromatography by exactly one size exclusion chromatography, wherein said size exclusion Chromatography is performed in the presence of about 150 easel sodium chloride, and wherein further said size exclusion chromatography is performed using a Sepharose CL-4B gel filtration matrix;   
       wherein said steps are performed in the given order.