Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Patent
1983-04-11
1986-07-22
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
435 68, 435 69, C12P 2100, C12P 2106, C12P 2102
Patent
active
046019790
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a novel process for the preparation of human insulin or a B-30 ester thereof.
BACKGROUND ART
For many years various insulins have been used in the treatment of insulin-dependent Diabetes. It would be natural to treat human beings with human insulin, which is not possible, however, in view of the existing demand. Therefore, for practical reasons bovine and porcine insulin is used. However, to a larger or smaller extent, these insulins give rise to the formation of antibodies in the human body, which i.a. involves a reduced effect of the further insulin treatment.
This disadvantage is supposed to be caused partly by "impurities" in the bovine/porcine insulin partly by the alien nature. The latter manifests itself therein that the human insulin molecule differs from other animal insulin molecules in a few differences in the composition of amino acid components.
Great improvements have been obtained as regards the insulin preparations after the introduction of the newest purification methods, but the formation of antibodies in the human body can still occur. It is believed that this can be remedied by using human insulin instead of other animal insulin.
It is known to prepare human insulin chemically, vide U.S. Pat. No. 3,903,068 and Hoppe-Seyler's Z. Physiol. Chem. 357, 759-767 (1976).
These processes comprise condensing a desoctapeptide-(B23-30) porcine insulin with a synthetic octapeptide corresponding to the positions B23-30 in human insulin. However, in the first process an alkaline hydrolysis is carried out, which is accompanied by unfavourable side reactions. The second process comprises a non-specific reaction giving rise to many side reactions and demanding complicated purification procedures. Consequently, these processes are not suitable for use on an industrial scale.
Moreover, U.S. Pat. No. 3,276,961 discloses a process for the preparation of human insulin from other animal insulins by the action of an enzyme, e.g. carboxypeptidase A or trypsin, in the presence of threonine. However, it has not proved to be possible to prepare human insulin to any appreciable extent by this known process. This is probably due to the fact that trypsin and carboxypeptidase A hydrolyze not only the lysyl-alanine peptide bond (B29--B30), but also other positions in insulin under the working conditions. Trypsin preferably hydrolyzes the arginyl-glycine peptide bond (B22--B23) rather than the lysyl-alanine bond (B29--B30). However, carboxypeptidase A cannot exclusively split off the alanine at the C-terminal of the B-chain without also splitting off asparagine at the C-terminal of the A-chain. It has later been shown that a specific condition, i.e. reaction in an ammonium bicarbonate buffer solution, is necessary in order to hinder the asparagine release, cfr. Hoppe-Seyler's Z. Physiol. Chem., 359, 799-802 (1978). Moreover, a considerable peptide formation scarcely occurs, since the velocity of the hydrolysis reaction is higher than that of the peptide synthesis at the working conditions.
It has later become known that addition of an organic solvent to the reaction medium in an enzymatically catalyzed process remarkably increases the velocity of the peptide bond synthesis and decreases the velocity of the hydrolysis, cfr. Ingalls et al. (1975), Biotechn. Bioeng. 17, 1627, and Homandberg et al., Biochemistry 17, 5220 (1978). The concentration of the solvent in the reaction medium should be high, and in the latter literature passage it is stated that when using 1,4-butanediol as solvent the best results are obtained with a concentration of 80% of said solvent.
Realizing this, desoctapeptide-(B23-B30) insulin (DOI) was successfully coupled by trypsin catalysis with a synthetic octapeptide corresponding to the B23-B30 positions of human insulin using an excess (10:1) of the latter reactant and using an organic solvent (DMF) in a concentration of more than 50%, cfr. J. Am. Chem. Soc. 101, 751-752 (1979). The coupling proceeds with a reasonable yield, but, all things consid
REFERENCES:
patent: 3276961 (1966-10-01), Bodanszky et al.
patent: 3903068 (1975-09-01), Ruttenberg
patent: 4320196 (1982-03-01), Morihara et al.
patent: 4320197 (1982-03-01), Morihara et al.
patent: 4343898 (1982-08-01), Markussen
The Condensed Chemical Dictionary, 10th Ed., Revised by G. G. Hawley, p. 564, 1981.
Schmitt et al., Hoppe-Seyler's Z. Physiol. Chem., 359, 799-802 (1978).
Obermeier et al., Hoppe-Seyler's Z. Physiol. Chem., 357, 759-767 (1976).
Morihara et al., Biochem. and Biophys. Research Comm., V92, No. 2, 396-402 (1980).
Morihara et al., Nature, 280, 412-413 (1979).
Masaki et al., Agri. Biol. Chem., 42(7), 1443-1445 (1978).
Gattner et al., Peptides 1980 Proceedings, 372-377 (1981).
Ingalls et al., Biotech. and Bioeng., V.XVII, 1627-1637 (1975).
Homandberg et al., Biochemistry, V. 27, No. 24, 5220-5227 (1978).
Inouye et al., JACS, 101:3, 751-752 (1979).
Andresen Finn H.
Balschmidt Per
Hejnos Kim R.
Kofod Hans
Huleatt Jayme A.
Nordisk Insulinlaboratorium
Wiseman Thomas G.
LandOfFree
Process for preparing human insulin or B-30 esters thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for preparing human insulin or B-30 esters thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for preparing human insulin or B-30 esters thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-865980