Scalable process for protein purification

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 7.2;
• (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 7.2;
• 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 7.2;
• wherein said tentacle anion exchange matrix comprises (i) resin particles of cross-linked methacrylate polymer or cross-linked vinyl polymer, and (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;
• (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;
• (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 said hydroxyapatite matrix is a ceramic hydroxyapatite 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;
• wherein said steps are performed in the given order.

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 said tentacle anion exchange matrix comprises (i) resin particles of cross-linked methacrylate polymer or cross-linked vinyl polymer, and (ii) acrylamide said 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;
• (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;
• (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 said hydroxyapatite matrix is a ceramic hydroxyapatite 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;
• wherein said steps are performed in the given order.