Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1992-05-06
1993-10-12
Wityshyn, Michael G.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
530410, A61K 4502, C07K 308, C12N 1526, C12P 2102
Patent
active
052527085
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a preparation process for a protein containing at least one intramolecular disulphide bridge by oxidation, at a pH of less than 5.0, of the corresponding reduced recombinant protein.
Many recombinant proteins for use in therapeutics are expressed in various host systems, amongst which E. Coli, yeast and CHO cells represent those most frequently used.
Among these proteins, heterologous proteins expressed but not secreted for example in yeast or in E. Coli are generally stored in the form of denatured aggregates in an insoluble granular state. The extraction processes require the latter to be put in solution in a denaturating medium using agents such as urea or guanidine. The correct folding of the molecule, which can then be carried out by various known methods and which is necessary for the protein to exhibit its biological activity, is not always obtained selectively and results in variable yields, often low, according to the recombinant protein concerned due to the associated formation of non-active conformers which must be separated by known purification techniques, for example by chromatography.
In particular when the protein contains at least one intramolecular disulphide bridge, and optionally one or more additional cysteines, the non-secreted recombinant protein is generally produced in a reduced state in the granules. Thus when the restoration of one or more specific disulphide bridges, either necessary for the biological activity of the protein or which contribute to an increase in this activity, is desired, the oxidation process must ensure the correct folding of the protein without formation of the optional undesirable isomers corresponding to the random formation of intramolecular or intermolecular bridges.
One development concerning the regioselective reactions permitting the formation of disulphide bridges from cysteines in peptide chains, obtained by chemical synthesis, describes a multiplicity of methods which highlight the difficulty of optimizing the yields in a general manner and the absence of quantitative results (F, Cavelier et al. Bull. Soc. Chim. Franc. 1989 No. 6 788-798).
Controlled oxidation processes for proteins produced by genetic engineering techniques have been described, notably in the domain of recombinant cytokins containing at least 2 cysteines and produced in E. Coli, for example for beta interferon and interleukin 2 (IL2) of which the corresponding natural proteins contain 3 cysteines and a disulphide bridge having a specific position: reduced recombinant proteins illustrated by the application to a reduced mutein of beta interferon which contains 2 cysteines (Example 1), to reduced desAla-IL2 which contains 3 cysteines (Example 3) or to a reduced mutein of desAla-IL2 which contains 2 cysteines (Example 4), using iodosobenzoate at a pH comprised between 5.5 and 9.0, with which, according to the operating conditions, up to 10 to 15% of undesired formed oxidized oligomers are observed. chloride at pH 8.0 in the presence of air at 25.degree. C. (Example 2) or at 37.degree. C. (Example 3) of the above mutein of desAla-IL2 whose application to the above reduced desAla-IL2 results in the formation of about 15% of inactive oxidized products having undesired isomer disulphide bridges. process of the recombinant IL2 expressed in E. Coli in aqueous solution at a concentration of 1 to 2 .mu.g/ml, in the presence of guanidine, by the action of cupric sulphate at a pH equal to 8.5 which then requires a purification by reversed-phase chromatography in order to obtain the purified IL2. Furthermore the authors indicate that the oxidation becomes "extremely slow" when the pH is lower than 6.
Thus in the absence of a selective quantitative oxidation method for reduced recombinant proteins containing at least 2 cysteines, it is necessary after oxidation at a pH of higher than 5.0 to carry out a purification, generally by chromatography, which eliminates the oxidation products corresponding to the incorrect formation of isomer intramolecular bridges as wel
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Creighton, "Methods in Enzymology", Academic Press Inc., (N.Y., U.S.), vol. 107 (1984) p. 314.
Abecassis Pierre Y.
Leclaire Jacques
Riberon Philippe
Roussel Uclaf
Sayala C.
Wityshyn Michael G.
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