Methods for processing inclusion bodies
AssigneeMedImmune, LLC
InventorEllen T. O'Connor
Life of this patent
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Abstract
The present application relates to methods for purifying recombinant proteins, including antibodies and antibody fragments. Suitably, the methods utilize depth filtration to clarify the desired proteins from a solubilized mixture, and provide refolding methods and refolding buffers to allow for refolding of the recombinant proteins into functional and active proteins. Exemplary antibody fragments include anti-CD22 antibody fragments that comprise VH and VL chains refolded into a functional and active fragment.
Claims32
A method for purifying a recombinant protein from a mixture comprising the recombinant protein and inclusion bodies, the method comprising:
- a) solubilizing the mixture comprising the recombinant protein and inclusion bodies with a solubilization buffer;
- b) clarifying the recombinant protein from the solubilized mixture with one or more depth filters;
- and
- c) recovering the clarified recombinant protein,
- wherein the method does not include centrifuging the solubilized mixture of recombinant protein and inclusion bodies prior to the clarifying.
A protein refolding buffer for refolding a solubilized recombinant protein comprising:
- about 20 mM to about 70 mM ethanolamine;
- about 500 mM to about 2 M arginine;
- about 0.5 mM to about 3 mM EDTA;
- and about 0.5 mM to about 1.5 mM GSSG,
- wherein the refolding buffer has a pH of about 9 to about 10.
A method of refolding a solubilized recombinant protein, comprising:
- a) concentrating the solubilized recombinant protein;
- b) diluting the concentrated recombinant protein in a refolding buffer comprising:
- about 20 mM to about 70 mM ethanolamine;
- about 500 mM to about 2 M arginine;
- about 0.5 mM to about 3 mM EDTA;
- and about 0.5 mM to about 1.5 mM GSSG;
- c) incubating the diluted recombinant protein at a pH of about 9 to about 10 and at a temperature of about 10° C. to about 15° C., (2-15° C.)for about 48 hours to about 96 hours;
- and
- d) recovering the refolded recombinant protein.
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A method of producing a recombinant antibody fragment comprising a VH antibody fragment and a VL antibody fragment, the method comprising:
- a) expressing a polynucleotide encoding a VH antibody fragment in a first bacterial cell;
- b) expressing a polynucleotide encoding a VL antibody fragment in a second bacterial cell;
- c) mixing the VH antibody fragment and the VL antibody fragment to generate a mixture, wherein the mixture further comprises inclusion bodies;
- d) solubilizing the mixture comprising the VH antibody fragment, the VL antibody fragment and the inclusion bodies with a solubilization buffer;
- e) clarifying the VH antibody fragment and the VL antibody fragment from the solubilized mixture with one or more depth filters;
- f) recovering the clarified VH antibody fragment and the clarified VL antibody fragment,
- wherein the method does not include centrifuging the solubilized mixture of VH antibody fragment, VL antibody fragment and inclusion bodies;
- g) concentrating the clarified VH antibody fragment and the clarified VL antibody fragment;
- h) diluting the concentrated clarified VH antibody fragment and the concentrated clarified VL antibody fragment with a refolding buffer comprising:
- about 20 mM to about 70 mM ethanolamine;
- about 0.5 M to about 2 M arginine;
- about 0.5 mM to about 3 mM EDTA;
- and about 0.5 mM to about 1.5 mM GSSG;
- i) incubating the diluted clarified VH antibody fragment and the diluted clarified VL antibody fragment at a pH of about 9 to about 10 and at a temperature of about 10° C. to about 15° C., (2-15° C.)for about 48 hours to about 96 hours;
- and
- j) recovering the recombinant antibody fragment.
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Dependencies are inferred from the claim text.