PROCESS FOR PURIFICATION OF IMMUNOGLOBULINS USING A PSEUDOBIOAFFINITY ADSORBENT
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Abstract
The present invention relates to the development of pseudobioaffinity adsorbent and process for purification of immunoglobulin G from immunoglobulin containing solutions such as but not limited to plasma, serum, cell culture supernatant, ascites fluids. The adsorbent consists of a solid support and ligand. The ligand may be attached to the matrix or be part of matrix. The ligand is selected from a group of hydrophobic amino acids such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and tyrosine and the support material being preferably a synthetic hydrophilic polymer of methacrylate or acrylate species, or any of its derivatives. The adsorbent is cheap and stable to harsh conditions such as 1.0 M NaOH used during regeneration of adsorbents. Moreover there is no problem of toxic leachables typically associated with biological ligands. The nature of the adsorbent also allows high flow rate operations at relatively low pressures. The process includes adjusting the pH and conductivity of the feed solution and the use of ionic salts and additives such as polyols or alcohols for eluting the bound IgG.
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Citations
27 Claims
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1-13. -13. (canceled)
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14. A process for immunoglobulin purification/separation using pseudobioaffinity adsorbent comprising:
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a. providing a rigid, porous pseudobioaffinity adsorbent comprising an amalgamation of a hydrophilic polymer base/support and attached/immobilized affinity ligand/s including (i) aliphatic and/or aromatic propanoic acid derivative/s and/or (ii) aliphatic or aromatic hydrophobic amino acids, or (iii) any suitable combinations thereof with average ligand density between 0.1 to 0.2 mol/lit (i.e. 100 to 200 μ
mol/ml) with or without a spacer arm and leading to ionic, hydrophobic and/or mixed mode interaction/s between the said pseudobioaffinity adsorbent and an immunoglobulin;b. contacting the said adsorbent with an immunoglobulin containing solution directly, or after adjusting the pH and conductivity of the immunoglobulin containing solution to adsorb the immunoglobulins on the said adsorbent with high specificity and high selectivity with a high immunoglobulin adsorption capacity between 25 to 120 mg immunoglobulins/ml (i.e. 33 to 160 g/kg) of adsorbent; c. optionally washing the said adsorbent with a washing solution; d. contacting the said adsorbent with desorbing solution in order to elute the bound immunoglobulin and fragments thereof with recovery and purity of 80% to 100% and 90 to 100% respectively; and e. optionally, regenerating and equilibrating the said adsorbent for reuse.
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15. A pseudobioaffinity adsorbent comprising an amalgamation of a rigid porous hydrophilic polymer base/support material of methacrylate or acrylate species, or any of its derivatives and attached/immobilized affinity ligand/s which is selected from a group consisting of (i) aliphatic and/or aromatic propanoic acid derivative/s and/or (ii) aliphatic or aromatic hydrophobic amino acids, and (iii) any suitable combinations thereof with average ligand density between 0.1 to 0.2 mol/lit (i.e. 100 to 200 μ
- mol/ml) with or without a spacer arm;
said adsorbent having high specificity and high selectivity with a high immunoglobulin adsorption capacity between 25 to 120 mg immunoglobulins/ml (i.e. 33 to 160 g/kg) of adsorbent. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23)
- mol/ml) with or without a spacer arm;
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24. A process of separation/purification of immunoglobulin/s, said process comprising:
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a) packing pre-equilibrated pseudobioaffinity adsorbent in a chromatographic column; b) contacting immunoglobulin containing solutions with said pre-equilibrated pseudobioaffinity adsorbent for selective binding of immunoglobulin to the pseudobioaffinity adsorbent under the conditions of pH in the range of 2.5 to 9.0, and salt content in terms of conductivity in the range 0.5 mS/cm to 50 mS/cm, where contaminating proteins remain in the unadsorbed fraction; c) washing the pseudobioaffinity adsorbent with a buffer, having the same or near properties of pH and salt strength as in step (b) above, to remove unadsorbed, or weakly adsorbed, compounds; d) eluting the bound immunoglobulin in 80% to 100% recovery and 90% to 100% purity by washing the pseudobioaffinity adsorbent with desorbing buffer having pH in the range of 2.5 to 9.0, and containing ionic salts so that conductivity is in the range of 20 mS/cm to 140 mS/cm and also containing additives selected from a group consisting of alcohol, polyols, ethanol, sugars, polyethylene glycol, ethylene glycol and glycerol; and e) regenerating and re-equilibrating the pseudobioaffinity adsorbent for reuse. - View Dependent Claims (25, 26, 27)
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Specification