Methods for Expression and Purification of Recombinant Human Growth Hormone
First Claim
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1. A process, comprising:
- a) culturing recombinant host cells capable of producing hGH comprising a non-naturally encoded amino acid in a liquid nutrient medium containing the non-naturally encoded amino acid under conditions which favor growth;
b) inducing production of hGH comprising the non-naturally encoded amino acid by said cells; and
c) purifying said hGH comprising a non-naturally encoded amino acid from said cells or culture medium.
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Abstract
The present invention relates generally to the production, purification, and isolation of human growth hormone (hGH). More particularly, the invention relates to the production, purification, and isolation of substantially purified hGH from recombinant host cells or culture medium including, for example, yeast, insect, mammalian and bacterial host cells. The process of the present invention is also useful for purification of hGH linked to polymers or other molecules.
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Citations
12 Claims
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1. A process, comprising:
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a) culturing recombinant host cells capable of producing hGH comprising a non-naturally encoded amino acid in a liquid nutrient medium containing the non-naturally encoded amino acid under conditions which favor growth; b) inducing production of hGH comprising the non-naturally encoded amino acid by said cells; and c) purifying said hGH comprising a non-naturally encoded amino acid from said cells or culture medium. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method comprising the steps:
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(a) reacting hGH comprising a non-naturally encoded amino acid from with a derivatized polyethylene glycol (PEG) under conditions sufficient to form hGH-PEG conjugates; and (b) isolating and purifying said hGH-PEG conjugates by contacting said hGH-PEG conjugate with an anion exchange matrix under conditions that allow binding of the hGH to the matrix followed by elution and collection of the hGH from the anion exchange chromatography matrix to provide substantially purified hGH-PEG conjugate.
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10. A method for isolating substantially purified hGH-PEG conjugates comprising the steps of:
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a) culturing recombinant host cells capable of producing hGH comprising a non-naturally encoded amino acid in a liquid nutrient medium containing the non-naturally encoded amino acid under conditions which favor growth; b) inducing production of hGH comprising the non-naturally encoded amino acid by said cells; c) contacting hGH comprising a non-naturally encoded amino acid from step b) with an anion exchange chromatography matrix under conditions that allow binding of the hGH to the matrix followed by eluting and collecting the hGH from the matrix; d) contacting the hGH eluted from the anion exchange chromatography of step c) with a hydroxyapatite chromatography matrix under conditions that allow binding of the hGH to the matrix followed by eluting and collecting the hGH from the hydroxyapatite chromatography matrix; e) contacting the hGH from step d) with a hydrophobic interaction chromatography (HIC) matrix under conditions that allow binding of the hGH to the matrix followed by eluting and collecting the hGH from the HIC matrix to provide hGH comprising a non-naturally encoded amino acid; f) reacting hGH comprising a non-naturally encoded amino acid from step e) with a derivatized polyethylene glycol (PEG) under conditions sufficient to form hGH-PEG conjugates; and g) isolating and purifying said hGH-PEG conjugates by contacting said hGH-PEG conjugate with an anion exchange matrix under conditions that allow binding of the hGH to the matrix followed by elution and collection of the hGH from the anion exchange chromatography matrix to provide substantially purified hGH-PEG conjugate.
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11. A method for isolating substantially purified hGH-PEG conjugates comprising the steps of:
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a) culturing recombinant host cells capable of producing hGH comprising a non-naturally encoded amino acid in a liquid nutrient medium containing the non-naturally encoded amino acid under conditions which favor growth; b) inducing production of hGH comprising the non-naturally encoded amino acid by said cells; c) contacting hGH comprising a non-naturally encoded amino acid from step b) with an anion exchange chromatography matrix under conditions that allow binding of the hGH to the matrix followed by eluting and collecting the hGH from the matrix; d) contacting the hGH from step d) with a hydrophobic interaction chromatography (HIC) matrix under conditions that allow binding of the hGH to the matrix followed by eluting and collecting the hGH from the HIC matrix to provide hGH comprising a non-naturally encoded amino acid; e) reacting hGH comprising a non-naturally encoded amino acid from step d) with a derivatized polyethylene glycol (PEG) under conditions sufficient to form hGH-PEG conjugates; and f) isolating and purifying said hGH-PEG conjugates by contacting said hGH-PEG conjugate with an anion exchange matrix under conditions that allow binding of the hGH to the matrix followed by elution and collection of the hGH from the anion exchange chromatography matrix to provide substantially purified hGH-PEG conjugate.
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12. A process, comprising:
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a) culturing recombinant host cells capable of producing hGH comprising a non-naturally encoded amino acid in a liquid nutrient medium containing the non-naturally encoded amino acid under conditions which favor growth; b) inducing production of hGH comprising the non-naturally encoded amino acid by said cells; c) reacting hGH comprising a non-naturally encoded amino acid from step b) with a derivatized polyethylene glycol (PEG) under conditions sufficient to form hGH-PEG conjugates and d) purifying said hGH-PEG conjugate.
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Specification