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US 8,940,878 B2Granted
Capture purification processes for proteins expressed in a non-mammalian system
AssigneeAmgen Inc.
InventorsJoseph Edward Shultz, Roger Hart
Life of this patent
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Abstract
Methods of purifying proteins expressed in non-mammalian expression systems in a non-native soluble form directly from cell lysate are disclosed. Methods of purifying proteins expressed in non-mammalian expression systems in a non-native limited solubility form directly from a refold solution are also disclosed. Resin regeneration methods are also provided.
Claims25
A method of purifying a protein expressed in a non-native soluble form in a non-mammalian expression system comprising:
- (a) lysing a non-mammalian cell in which the protein is expressed in a non-native soluble form to generate a cell lysate;
- (b) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
- (c) washing the separation matrix;
- and
- (d) eluting the protein from the separation matrix, wherein the separation matrix is an affinity resin selected from the group consisting of Protein A, Protein G and a synthetic mimetic affinity resin.
2 more elements
A method of purifying a protein expressed in a non-native limited solubility form in a non-mammalian expression system comprising:
- (a) expressing a protein in a non-native limited solubility form in a non-mammalian cell;
- (b) lysing a non-mammalian cell;
- (c) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
- (i) a denaturant;
- (ii) a reductant;
- and
- (iii) a surfactant;
- (d) forming a refold solution comprising the solubilization solution and a refold buffer, the refold buffer comprising one or more of the following:
- (i) a denaturant;
- (ii) an aggregation suppressor;
- (iii) a protein stabilizer;
- and
- (iv) a redox component;
- (e) directly applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the matrix;
- (f) washing the separation matrix;
- and
- (g) eluting the protein from the separation matrix, wherein the separation matrix is a non-affinity resin selected from the group consisting of ion exchange, mixed mode, and a hydrophobic interaction resin.
14 more elements
Dependencies are inferred from the claim text.