Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1999-02-01
2002-01-29
Minniefield, N. M. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C530S350000, C530S402000, C530S404000, C530S408000, C530S412000, C530S399000, C435S069100, C435S069400, C435S069500, C435S254100, C435S254200
Reexamination Certificate
active
06342585
ABSTRACT:
The present invention concerns a simplified process for the solubilization and renaturation of denatured proteins, in particular of recombinantly produced denatured proteins.
Sparingly soluble inactive protein aggregates (inclusion bodies) are frequently formed when proteins are produced in prokaryotic cells such as
E. coli
. In order to convert these proteins into their active form it is necessary to solubilize and to renature these proteins. Such processes are known and are for example described in EP-A 0 361 475, EP-A 0 114 506, EP-A 0 093 619, EP-A 0 253 823, WO 87/02673, EP-A 0 364 926 and EP-A 0 241 022. An important factor in the activation which limits the yield of renatured protein is the competing reaction between conversion of the renatured protein into the correctly folded intermediate and an aggregation of several protein molecules. For this reason the concentration of renatured protein in the renaturation solution is an important parameter for the yield of the renaturation process. Aggregation is favoured by increasing concentrations of renatured protein and the relative yield of renatured protein with the conformation of the native protein decreases (critical concentration).
In a large-scale production of recombinant proteins the amount of protein to be renatured is usually much higher than the critical concentration. Since the proteins often have a low solubility in the activation buffer used, this therefore results in considerable disadvantages such as low yield, long time requirement and large buffer volumes.
A process is known from WO 87/02673 in which the inactive soluble protein is solubilized with denaturing agents and reducing agents, subsequently the reducing agent is separated and then heterologous mixed disulfides between protein and for example glutathione are prepared from the solubilized proteins. Such mixed disulfides are advantageous for the further purification and renaturation since after modification of the thiol groups the protein is protected against air oxidation and it is thus stable in a larger pH range. The change in the net charge also facilitates the purification since it enables non-modified proteins to be separated by means of ion exchange chromatography.
In order to form the mixed disulfides, the solubilized, dialysed and reduced protein that has been purified of reducing agents is incubated with a solution which contains a denaturing agent and a disulfide component for the derivatization (e.g. GSSG, cystine, cystamine). A renaturation is carried out in the usual manner after separation of the disulfide component. Although this process is efficient, it requires many individual process steps especially for separation of the reducing agent before the derivatization.
A process for folding and purifying insulin-like growth factor I is known from WO 93/19084. According to this the inclusion bodies are dissolved under reducing conditions, subsequently an excess of oxidizing agent is added without separating the reducing agent. The renaturation is initiated by subsequent dilution (without dialysis) and new addition of reducing agent (to construct a redox system). WO 91/08762 describes the preparation of biologically active platelet derived growth factor. In this process a solubilization is firstly carried out at pH 3 without a reducing agent and subsequently a purification under denaturing conditions. Only afterwards is an oxidizing agent added to produce a derivative. According to EP-A 0 450 386 an extract of inclusion bodies (denatured dissolved NGF protein) is prepared by adding solubilizing buffer with subsequent centrifugation. The extract is then treated with a reducing agent, incubated and oxidized by addition of an oxidizing agent without previous dialysis. Subsequently it is diluted and further components are added for the denaturation. Thus in this process a solubilization is firstly carried out as a separate process step without adding redox active substances. None of these processes is suitable for a pulse renaturation according to U.S. Pat. No. 4,933,434.
In addition processes are known which already allow a derivatization during the solubilization. The method of sulfitolysis has been known for a long time (e.g. Bailey, J. L., Cole, R. D., 1959, J. Biol. Chem. 234, 1733-1739; Cole, R. D., 1967, In: Meth. Enzymol. 11, 206-208; EP 0 114 507). In this process disulfide bridges in proteins are treated with salts of sulfurous acid to form a mixture of 50% thio-sulfonated (RS-SO-
−
3
) and 50% free (RS
−
) protein-SH groups as the reaction product. The latter free SH groups are in turn converted into disulfides by reoxidation (e.g. with copper ions, iodosobenzoate or preferably with tetrathionate) which can be almost completely converted into the thiosulfonate by repeated cycles of the process. This process is relatively simple and can be carried out under mild boundary conditions (e.g. neutral pH value). As already set forth in J. Biol. Chem. 234, 1733 a disadvantage is that the thiosulfonate that is formed is chemically labile, it is not possible to check the completion of the conversion and above all the tryptophan residues are partially destroyed by the reoxidation agent. A further disadvantage is that it is very difficult to completely separate by-products containing thiosulfonated protein-SH groups and oxidizing agents such as the said iodosobenzoate in the final product and it is extremely laborious to detect this analytically. However, this is absolutely necessary for proteins which are intended for a therapeutic application in order to exclude possible side effects of a therapeutic agent that has been chemically modified in such an unphysiological manner.
WO 95/30686 also describes such a sulfitolysis to renature neurotrophic factors of the NGF/BDNF family. A similar process is described for the renaturation of human proinsulin by R. Wetzel et al., Gene 16 (1981) 63-71 as well as by W. F. Heath et al., J. Biol. Chem. 267 (1992) 419-425.
A similar method which avoids the use of reoxidation conditions that damage side chains is also known (Thannhauser, T. W., Konishi, Y., Scheraga, H. A., 1984, Analyt. Biochem. 138, 181-188; Thannhauser, T. W., Scheraga, H. A., 1985, Biochemistry 24, 7681-7688): In this case instead of a reoxidation, the cysteine obtained when the disulfide bridge is reduced is directly derivatized by reaction with 2-nitro-5-(sulfothio)-benzoate; 2-nitro-5-thiobenzoate is released in this process which can be measured photometrically and thus enables a quantification of the converted SH groups. A disadvantage of this method is that a complex chemical substance is introduced whose complete separation from the final product is very time-consuming and difficult to check. Furthermore the authors (Biochemistry 24, 7681) observed that the thiosulfonate obtained is only stable when thiol groups are completely absent. In addition a side chain modification is also observed in this case (deamination of asparagine).
The object of the present invention is to simplify and to improve these processes and to provide stable, storable proteins whose SH groups are derivatized and which can be renatured in a high yield.
Surprisingly it was found that the process according to the invention allows the solubilization and derivatization to be carried out in a single step without having to previously reduce. It is particularly surprising that the derivatization can also take place under acidic conditions (pH value less than 7.0, preferably pH 3-6.5) preferably for neurotrophins such as NGF and that this is achieved without essentially affecting the kinetics and the completeness of a reaction compared to a reaction with thiol components in the usual pH range of about 7-10. It is usually assumed that such reactions can only proceed in the presence of the free thiolate anion; this only occurs in effective concentrations at pH values above about 7 due to the high pK value of thiolate anions of about 9.
The invention therefore concerns a process for producing mixed disulfides composed of a protein and a disulfide component w
Fulbright & Jaworski LLP
Minniefield N. M.
Roche Diagnostics GmbH
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