Scalable process for protein purification
AssigneeKuros Biosciences AG
InventorsSusanne Richter, Simon Topell
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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.
Claims19
A process for the purification of a VLP from a recombinant bacterial host expressing said VLP, wherein said VLP is a VLP of RNA bacteriophage Qβ comprising coat protein of SEQ ID NO:3, 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.
A process for the purification of a VLP of RNA bacteriophage Qβ from a recombinant bacterial host expressing said VLP, wherein said VLP comprises coat protein of SEQ ID NO:3, 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;
- (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;
- (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.
Dependencies are inferred from the claim text.