Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1999-07-07
2003-04-22
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S041000, C435S069100, C435S069400, C530S350000
Reexamination Certificate
active
06551801
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a process for the production of a purified dimeric bone morphogenetic factor. More particularly, it is concerned with a process for the production of a dimeric bone morphogenetic factor, characterizing in acquiring a purified dimeric bone morphogenetic factor from an inclusion body of a bone morphogenetic factor produced by means of a genetic engineering technology.
BACKGROUND OF THE INVENTION
A proteinaceous bone morphogenetic factor was discovered to be present in the bone matrix (Science 150, pp.893-899, 1965) and was named as “bone morphogenetic protein” (hereinafter abbreviated as BMP). Recently, cloning of plural BMP-related genes has been attempted and it has been found that all of them belong to the transforming growth factor-&bgr; (hereinafter abbreviated as TGF-&bgr;) superfamily. Recombinants of some of these factors have been produced by means of a genetic engineering technology and they have been confirmed to have a bone morphogenetic activity, from which their application to the treatment of bone diseases is expected.
Of these factors, the human GDF-5 (MP52) recently discovered and belonging to the human BMP family (Biochem. Biophys. Res. Commun., 204, pp. 646-652, 1994) has been confirmed by animal tests to be effective as a bone morphogenetic factor, while it has been technically reviewed to carry forward the large-scale production thereof by expression using recombinant
Escherichia coli
(
E. coli
). However, when expressed in a large scale in
E. coli
and others, for instance, when the protein is produced at an amount of several grams per liter of cultured broth, the desired protein generally tends to form an inactive and insoluble inclusion body. This inclusion body is monomeric and, in order to obtain a dimer which is active as a bone morphogenetic factor, the inclusion body must be solubilized, renatured to a dimer of an original structure (the procedure generally called “refolding”), separated and purified to obtain the desired protein.
The active form of MP52 has the following or the like problems;
1) because of its low solubility in an aqueous solution, it should be handled in the presence of a denaturing agent or under acidic conditions,
2) the protein used for separation tends to nonspecifically adsorb onto a resin for liquid chromatography, and
3) the surfactant essential for refolding tends to disturb separation,
and thus it has been very difficult to establish a process for the purification thereof.
The purification process recently developed for solving the above-mentioned problems (WO 96/33215) successful in obtaining a single active form of MP52 comprises the following steps;
1. solubilizing an inclusion body by a denaturing agent,
2. separation by ion exchange chromatography,
3. sulfonation,
4. separation by gel filtration chromatography,
5. refolding,
6. recovery by isoelectric precipitation, and
7. separation by reverse-phase chromatography.
However, the above process if scaled up industrially has encountered the following and the like problems;
1) a large amount of a denaturing agent is used in order to solubilize the MP52 inclusion body, whereby modification of the protein (for example, carbamylation reaction in the case of urea) may be induced,
2) an expensive resin for chromatography, especially, for gel filtration chromatography, such as Sephacryl S-200HR or Superdex 200 pg (all available from Pharmacia Biotech) is used in a large amount,
3) a reagent used in refolding, inter alia, CHAPS and oxidized glutathione essential for dimerization reaction is extremely expensive, and
4) when isoelectric precipitation is carried out, the dilution is necessary to decrease the concentration of detergent, thus the volume of the solution is increased.
DISCLOSURE OF THE INVENTION
An object of this invention is to solve the above-mentioned problems, i.e.,
1) to use a denaturing agent in an amount as low as possible;
2) to use a chromatography resin in an amount as low as possible;
3) to replace the reagent used for refolding by other inexpensive ones and to simplify concomitant procedures with refolding,
4) to decrease the volume of the solution by removing a detergent selectively; that is, to considerably shorten the process time.
The present inventors have made feasible a simplification of the purification steps by solubilizing an inclusion body extracted from
E. coli
in the presence of a denaturing agent, conducting a direct refolding according to a dilution procedure and then subjecting an ultrafiltration substititing the refolding solution. This procedure appears to be similar to the first step of a process for the production of human insulin from
E. coli
(EP 600372A1). However, since a bone morphogenetic factor is different in properties from a soluble protein such as human insulin, it was difficult to apply the process for the production of insulin as depicted above in case of a bone morphogenetic factor. MP52 (active form) dimerized as depicted above has a low solubility and tends to adsorb onto a chromatographic resin, thus in the large-scale production, the ion exchange chromatography or hydrophobic chromatography used for human insulin or the gel filtration chromatography used in the above-mentioned WO 96/33215 could not be applied. When an ion exchanger (SP Sepharose FF, Pharmacia Biotech) is used, for example, MP52 is not completely eluted because of its strong adsorption onto the resin, even if a denaturing agent and a maximum salt concentration is used. When gel filtration (Sephacryl S-200HR, Pharmacia Biotech) is used, a strong adsorption of the protein onto a resin occurs even if a denaturing agent is used, causing an excessively broadened fractionation range and thus a very poor separation. Further, properties of the resin is altered by influence with of a surfactant such as CHAPS, which leads to loss of reproducibility. This is also applicable to the elution with an acidic solution in which MP52 is soluble. In conclusion, it is not feasible to make use of the original properties of the resin.
As explained above, it has become apparent that the purification of the desired protein in large-scale production can not be accomplished according to a general chromatographic means using aqueous system. Reverse-phase chromatography using organic solvent is the only means that could be utilized. In view of this, it was necessary to develop a purification means wherein many columns are not used. As purification means other than using columns, a fractionating method by ammonium sulfate seemed promising. However, since it had low purification efficiency and led to unnecessarily low yield, its use was cast aside. In addition, isoelectric precipitation procedure by pH adjustment was adopted, but prior to the actual procedure, an ultrafiltration procedure to remove a surfactant, CHAPS, was carried out which enabled the performance of isoelectric precipitation without increasing the volume of the solution. Conventionally, when the solution contained CHAPS, the protein solubility was high and no precipitation occurred. Therefore, a dilution was necessary to decrease the concentration of CHAPS, but a resultant extensive increase in solution volume has been a problem in process development.
This invention is directed to a process for the production of a purified dimeric bone morphogenetic factor, characterizing in subjecting an inclusion body of a bone morphogenetic factor produced by means of a genetic engineering technology to the following steps a)-e) in order, thereby producing a dimeric bone morphogenetic factor;
a) treating an inclusion body of a bone morphogenetic factor with a denaturing agent to obtain a solubilized monomer,
b) treating the solubilized monomer with a refolding solution to obtain a dimeric bone morphogenetic factor,
c) treating the dimeric bone morphogenetic factor by ultrafiltration and substitution of solvent,
d) subjecting the dimeric bone morphogenetic factor in solvent thus substituted to isoelectric precipitation, and
e) subjecting the dimeric bone morphogenetic factor
Andou Hidetoshi
Honda Jun
Sugimoto Sjunjiro
Andres Janet L.
Biopharm Gesellschaft zur Biotechnologischen Entwicklung von Pha
Eyler Yvonne
Muserlian Lucas and Mercanti
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