Process for high efficiency refolding of recombinant proteins
AssigneeBiogenomics Limited
InventorsArchana KRISHNAN, Sanjay SONAR, NIKHIL GHADE, FAIZA SHAIKH
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Abstract
A process of refolding a recombinant protein from inclusion bodies (IBs) formed inside a host cell, is provided. The process includes homogenising a wet cells slurry to obtain a cell lysate; incubating the cell lysate with a reducing agent to obtain a reduced cell lysate; isolating reduced IBs from the reduced cell lysate to obtain isolated IBs; solubilising the isolated IBs with a denaturing agent to obtain solubilised IBs; and subjecting the solubilised IBs to a unit process of refolding to obtain a refolded recombinant protein.
Claims15
A process of refolding a recombinant protein isolated from inclusion bodies (IBs), formed inside host cells, comprising:
- (a) homogenising a wet cells slurry to obtain a cell lysate;
- wherein said cell lysate comprises IBs;
- (b) incubating said cell lysate with a reducing buffer to obtain a reduced cell lysate;
- said reduced cell lysate comprising reduced IBs;
- (c) isolating said reduced IBs from said reduced cell lysate to obtain isolated reduced IBs;
- (d) solubilising said isolated reduced IBs with a denaturing agent to obtain reduced solubilised IBs;
- and
- (e) subjecting said reduced solubilised IBs to a unit process of refolding to obtain a refolded recombinant protein.
A process of refolding a recombinant protein from inclusion bodies (IBs) formed inside host cells, comprising:
- (a) homogenising a wet cells slurry to obtain a cell lysate;
- wherein said cell lysate comprises IBs;
- (b) incubating said cell lysate with a reducing agent, at a pH ranging between 7.0 and 9.0, and preferably at 8.0, to obtain a reduced cell lysate;
- said reduced cell lysate comprising reduced IBs;
- (c) diluting said reduced cell lysate with at least 20 volumes buffer at pH ranging between 7.3 and 7.5, to obtain a diluted cell lysate;
- (d) isolating reduced IBs from said diluted cell lysate to obtain isolated IBs at pH ranging between 7.3 and 7.5;
- (e) reducing pH of said isolated IBs to 3.0;
- (f) solubilising said isolated IBs at pH 3.0 with a denaturing agent at pH ranging between 2.5 and 4.0, to obtain acidic pH solubilised IBs;
- and
- (g) diluting said acidic pH solubilised IBs with a continuously flowing refolding buffer at pH ranging between 7.5 and 11.5 to obtain a refolded biologically active recombinant protein, such that when said solubilised IBs are introduced into said continuous flow of refolding buffer, concentration of said denaturing agent is reduced to preferably lesser than 0.3M but not more than 3M, and concentration of said recombinant protein in said solubilised IBs is between 0.1 g/L and 1 g/L in said refolding mixture.
Dependencies are inferred from the claim text.