Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Insulin; related peptides
Reexamination Certificate
2001-01-23
2003-07-08
Kunz, Gary (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Insulin; related peptides
C530S324000, C530S350000, C435S069100, C435S070100
Reexamination Certificate
active
06590072
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a method for extractive refolding of scrambled and polymerised single-chain polypeptides and proteins into their native conformation directly from a microbial fermentation broth.
BACKGROUND OF THE INVENTION
Many polypeptides or proteins feature one or more intra molecular disulfide bonds serving to lock the tertiary conformation in place. If such polypeptides or proteins are recovered with incorrectly positioned disulfide bonds, they are often less biologically active than the corresponding molecule with correct disulfide bonds. Such scrambled or polymerised products are often found in proteins and polypeptides formed by fermentation of recombinant microorganisms or during the growth of a mammalian cell culture and may accumulate, stabilised by the incorrect disulfide bonds. The incorrect disulfide bonds may be intramolecular, giving rise to a misfolded monomeric product, or intermolecular, giving rise to dimeric or polymeric products.
The desired product and its accompanying, incorrectly formed, by-products may be secreted from the cells and found in soluble form in the broth. The may also be found in the periplasm as incorrectly folded and aggregated products resembling insoluble inclusion bodies. This will typically be the case when yeast is being used as the host organism for the recombinant production.
Alternatively, the desired product and its accompanying, incorrectly formed, by-products may be found either in the periplasm or inside the cells, in the form of insoluble inclusion bodies. This is the case when many polypeptides or proteins such as human proinsulin are expressed in
E. coli
Formation of such scrambled or polymerised by-products may be caused by a number of factors, e.g. that the conditions in the production host, such as pH, temperature, redox potential, concentration and type of chaperones etc. are different from those in the original (natural) site of production. Alternatively, the rate of production may be to high to cope with the time necessary for the molecule to fold into the native conformation which is usually believed to be the state having the lowest free energy. These scrambled and polymerised by-products represent a serious loss in commercial production. Therefore great efforts have been exerted to extract and refold these incorrectly folded proteins.
Thus, Steiner and Clark, Proc. Nat. Acad. Sci. 60, 622-629, 1968, disclose a method for isolation of reduced proinsulin and oxidation of the reduced form to native proinsulin; EP 600372 discloses a method for dissolving and extracting reduced proinsulin from
E. coli
inclusion bodies after cell homogenisation; EP 906918 discloses an improvement of this process by dilution of the reduced and extracted product in water; Frank, Pettee, Zimmerman and Burck, in: Peptides, Synthesis—Structure—Function, Proceedings of the Seventh American Peptide Symposium, Eds.: Rich, Gross, Pierce Chemical Company, Rockford, Ill., pp 729-738, 1981, describe a process in which human proinsulin is recovered from
E. coli
via its purified hexa S-sulfonate; WO 96/32407 discloses an unfolding and refolding process of secreted IGF-1; Markussen, in: Proinsulin, Insulin, C-Peptide, Proceedings of the Symposium on Proinsulin, Insulin and C-Peptide, Eds.: Baba, Kaneko, Yanaihara, Excerpta Medica, Amsterdam-Oxford, pp 50-61, 1979, discloses reduction of a single-chain des(B30) insulin precursor; Markusen, in: Int. J. Peptide Protein Res. 25, 431-434, 1985 compares refolding of a mini-proinsulin B(1-29)-A(1-21) and porcine insulin by air oxidation of the reduced and isolated forms of the molecules; and U.S. Pat. No. 6,003,875 discloses a method for improving the yield of IGF-1 when secreted from a yeast cell. A common feature with the prior art is that rather harsh conditions are used in the unfolding/refolding steps and that chaotropic agents are used requiring additional purification steps.
SUMMARY OF THE INVENTION
The present invention relates to a simple method for extractive refolding of scrambled and/or polymerised single-chain polypeptides or proteins to their native conformation directly from a microbial fermentation broth, in which they may appear in a variety of scrambled, misfolded and polymer forms together with the native, monomolecular form.
In the process according to the present invention, the sulphur chemistry is conducted directly on the crude product in the broth from the fermentation or, in the case the scrambled products are retained inside the cells, after disruption of the cell walls.
Thus, the present inventions is related to a method for extractive refolding scrambled and/or polymerised single-chain polypeptides contained in a microbial culture broth said method comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11;
b) adding a catalyst for refolding of disulfide bonds without adding a chaotropic agent;
c) adjusting pH if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) subjecting the supernatant to oxidation;
f) and isolating single-chain polypeptide material with correctly positioned disulfide bonds by suitable purification steps.
Steps a) to f) may be conducted in a temperature interval from about 4 to about 35° C. or from about 20 to about 25° C. and may be completed within a period from about 30 to about 180 minutes or from about 30 to 120 minutes. The order of one or more of steps a) to e) may be changed or reversed and some of the steps may even be omitted, e.g. step c). Thus in one embodiment, cells and cell debris are removed prior to step b). The catalyst for refolding disulfide bonds is typically a thiol compound, such as cysteine, HCl.
In one embodiment the present invention is related to a process comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11 by addition of diluted alkali hydroxide;
b) adding a thiol to the culture broth;
c) adjusting pH if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) stirring the supernatant under aeration;
f) and isolating single-chain polypeptide material with correctly positioned disulfide bonds by suitable purification steps.
In still another embodiment the present invention is related to a process comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11 by addition of 0.1-8 M sodium hydroxid;
b) adding solid or liquid cysteine, HCl to the culture broth to make 0.5-100 mM;
c) after a short period, e.g. 5 minutes, adjusting pH to about 7.0-11.0 if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) stirring the supernatant under aeration for a period of about 30 to about 180 minutes; and
f) and isolating single-chain polypeptide material with correctly positioned disulfide bonds by suitable purification steps.
In step a) the culture broth is advantageously diluted with water either before, simultaneously or after adjustment of pH. In one embodiment of the present invention, dilution of the culture broth is accomplished by addition of diluted alkali hydroxide. The dilution of the culture broth in step a) may be from about 2 to about 500%, from about 2 to about 300%, from about 2 to about 200%; from about 5 to 200%; from about 50 to 200% or from about 50 to 150%. Very high yields are obtained at a dilution of about 50%.
Thus in a further embodiment, the present invention is related to a method for extractive refolding scrambled and/or polymerised single-chain polypeptides or proteins contained in a microbial culture broth said method comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11;
a1) dilution of the culture broth to from about 2% to 500%;
b) adding a catalyst for refolding of disulfide bonds without adding a chaotropic agent;
c) adjusting pH if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) subjecting the supernatant to oxidation;
f) and isolating single-chain polypeptide material with correctly positi
Began, Esq. Marc A.
Bork, Esq. Richard W.
Green, Esq Reza
Kunz Gary
Li Ruixiang
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