WO1994010200A1 - Modifications de la somatotrophine - Google Patents

Modifications de la somatotrophine Download PDF

Info

Publication number
WO1994010200A1
WO1994010200A1 PCT/US1993/009232 US9309232W WO9410200A1 WO 1994010200 A1 WO1994010200 A1 WO 1994010200A1 US 9309232 W US9309232 W US 9309232W WO 9410200 A1 WO9410200 A1 WO 9410200A1
Authority
WO
WIPO (PCT)
Prior art keywords
replacing
somatotropin
serine
asparagine
glutamic acid
Prior art date
Application number
PCT/US1993/009232
Other languages
English (en)
Inventor
S. Russ Lehrman
Original Assignee
The Upjohn Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Upjohn Company filed Critical The Upjohn Company
Priority to EP93922424A priority Critical patent/EP0669939A1/fr
Priority to JP6511061A priority patent/JPH08502963A/ja
Priority to AU51422/93A priority patent/AU5142293A/en
Publication of WO1994010200A1 publication Critical patent/WO1994010200A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention provides novel single- or multiple-site substitution analogs of somatotropins that display enhanced stability relative to the unmodified protein.
  • Somatotropin also referred to as St or growth hormone (GH) was originally discovered in pituitary gland extracts from various animal species. These proteins are single chain molecules containing about 200 amino acids and two intramolecular disulfide bonds. Since these proteins contain no carbohydrate, lipid or other cofactors, their molecular weight is approximately 22,000 daltons. A number of mammalian, avian, and aquatic somatotropins have been well characterized with respect to their structure and biological behavior (see for example, J.L. Kostyo & A.E. Wilhemi, Metabolism 25:105-124 (1976); O.G.P. Isaksson, et al, Ann. Rev. Physiol.
  • Mammalian somatotropins are conserved molecules and similarities in amino acid sequences and protein structure are found between different species of animals.
  • conformational stability The ability of a protein to maintain this tertiary structure is referred to as conformational stability. Often, the latter structure is essential for maintaining biological activity. In these cases, loss of tertiary structure is a form of protein degradation. Further, chemical and conformational stability of a protein are interrelated. That is, reactions that modify the covalent structure of proteins can affect conformational stability, and structural changes that alter conformational stability can affect the rate of chemical degradation, often in an adverse manner.
  • somatotropins A number of chemical and conformational degradation pathways have been identified for somatotropins. For example, deamidation, hydrolysis, and rearrangement reactions of the carboxamide sidechain of asparagine have been observed at positions 13, 47, 140, and 148 (C. Secchi, et al., Int. J. Peptide Protein Res. 28: 298 (1986), at positions 99 (International publication WO 87/01708), and position 129 (Wood, D.C., et al., J. Biol. Chem. 264:14741 (1989)), of the highly homologous animal somatotropins.
  • Methionines at positions 5, 124, 149, and 179 have been shown to oxidize to the corresponding sulfoxide (O. Cascone, et al.. Int. J. Peptide Protein Res. 16:299-305 (1980)).
  • treatment of the protein with alkali under favorable conditions leads to intermolecular disulfide bridges generating reducible dimers.
  • cysteines of the short loop positions 181 and 189.
  • Conditions that lead to the loss of tertiary structure have also been defined. Denaturation of somatotropin occurs as a result of treatment with pH, guanidinium chloride, urea, and heat.
  • partially denatured somatotropin has been shown to form a non- bioactive, poorly soluble aggregate species (D.N. Brems, Biochemistry 27:4541-4546 (1988)).
  • modified forms of somatotropin that selectively stabilize the protein by minimizing individual degradation pathways while maintaining bioactivity in the target animal.
  • These compounds are obtained by using site-directed modifications to generate nucleic acid substitutions in a somatotropin cDNA.
  • the modified cDNA is then transformed into a host organism and the modified somatotropin analog expressed and purified.
  • site-directed as used here is the process of controlled modifications of the primary structure of somatotropin. Single-site modifications are changes in the somatotropin gene sequence that produce one to three amino acid residue substitutions within a short segment of somatotropin.
  • Single-site substitution analogs are designed to address a specific degradation pathway (e.g., deamidation of asparagine at position 47).
  • Multiple-site substitution analogs combine two or more single-site substitutions into somatotropin in order to further stabilize the protein.
  • Site-directed modifications enhance St stability either by directly eliminating functional groups that undergo chemical degradation, or by reinforcing the conformational stability of native tertiary structure.
  • somatotropin analogs A number of variant or modified somatotropins, also referred to as somatotropin analogs, are known. For example:
  • EP publication 0 355 460 and International publications WO 92/01789 and WO 90/02758 also report modified somatotropins by substitution of cysteine residues.
  • International publication WO 90/08164 discloses somatotropins displaying enhanced stability by replacement of the asparagine residue at position 99 of bovine somatotropin.
  • FIG. 1 shows the results of accelerated stability studies of rpSt and modified rpSt analyzed by RP-HPLC. Together with data of Figures 2-8, this data shows that standard protein analysis is not sufficiently sensitive to permit the identification of specific stability enhancement. Peak area (as a percentage of initial peak area) is plotted as a function of time. Somatotropins are dissolved in sodium carbonate buffer, pH 9.8 and incubated at 30°C for up to 30 days for these analyses. rpSt (o); A48P (D); F103E ( ⁇ ); C181.189S (v); N99S (•) ⁇ Further details are described in the Detailed Description section.
  • Figure 2 shows tryptic maps of native rpSt (top) and following 30 days of decomposition (bottom) in alkaline buffers. Peaks are identified using LC-MS (LC-MS: liquid chromatography-mass spectrometry). The numbers correspond to individual tryptic fragments of native rpSt and are identified in Table IV. Individual degradation products, labelled in Figure 2 (bottom) are identified in Table V. This approach therefore permits a more detailed and sensitive assessment of chemical stability.
  • Figure 3 shows a tryptic map of M5S following exposure to alkali buffers. This modified form of pSt is incubated as described in Figure 1. Samples are analyzed by tryptic mapping as described by J.J.Dougherty, et al. (Analytical Biochemistry 190:7-20 (1990)). In brief, this procedure calls for the incubation of somatotropin with trypsin at pH 8.3 at 5°C for 24 hours. The digests are mapped by HPLC. Additional details are described in the Detailed Description section.
  • Figure 4 shows a tryptic map of N47D following exposure to aqueous alkaline.
  • Figure 5 shows a tryptic map of A48P following exposure to alkaline buffers. Incubation and analyses are briefly described in the legends of Figures 1 and 3, respectively.
  • Figure 6 shows a tryptic map of N99S following exposure to alkaline buffers. Incubation and analyses are briefly described in the legends of Figures 1 and 3, respectively.
  • Figure 7 shows a tryptic map of C181,189S following exposure to alkaline buffers. Incubation and analyses are briefly described in the legends of Figures 1 and 3, respectively.
  • This invention relates to modified somatotropins, or somatotropin analogs, having one or more amino acid changes that enhance the chemical and conformational stability of the protein while maintaining levels of bioactivity which are the same as, or similar to, the native protein.
  • a first aspect of the invention relates to somatotropin analogs wherein one of the following single-site substitutions is made in the protein: serine replacing methionine at amino acid position 5 (the analog designated M5S), serine replacing asparagine at position 13 (N13S), leucine for the valine found at amino acid position 15 (V15L), histidine replacing glutamine at position 19, and arginine replacing histidine at position 20 (Q19H,H20R), glutamic acid replacing glycine at position 40 (G40E), aspartic acid replacing asparagine at position 47 (N47D), proline replacing alanine at position 48 (A48P), glutamic acid replacing phenylalanine at position 52 (F52E), asparagine replacing glycine at position 63 (G63N), phenylalanine replacing tryptophan at position 86 (W86F), glutamic acid replacing glycine at position 88 (G88E), alanine replacing threonine at position 98
  • the invention relates to analogs of porcine or bovine somatotropin, most particularly porcine, having one of the substitutions listed above.
  • a second aspect of the invention relates to multiple-site somatotropin analogs wherein two or more of the following single-site substitutions is made in the protein: serine replacing methionine at amino acid position 5 (M5S), serine replacing asparagine at position 13 (N13S), leucine replacing valine at position 15 (V15L), histidine replacing glutamine at position 19, and arginine replacing histidine at position 20 (Q19H,H20R), glutamic acid replacing glycine at position 40 (G40E), aspartic acid replacing asparagine at position 47 (N47D), proline replacing alanine at position 48 (A48P), glutamic acid replacing phenylalanine at position 52 (F52E), asparagine replacing glycine at position 63 (G63N), phenylalanine replacing tryptophan at position 86 (W86F), glutamic acid replacing glycine at position 88 (G88E), alanine replacing threonine at position 98, se
  • this aspect of the invention relates to somatotropin analogs wherein the following multiple-site substitutions are made in the somatotropin protein: (a) V15L, A48P, F52E;
  • the invention relates to analogs of porcine or bovine somatotropin, most particularly porcine, having the multiple-site substitutions listed above.
  • An additional aspect of the invention also relates to expression plasmids comprising cDNA encoding one of the somatotropin analogs of the invention.
  • a third aspect of the invention relates to host cells transformed with expression plasmids comprising cDNA encoding a porcine or bovine somatotropin of the invention.
  • Additional aspects of the invention relate to a method of increasing the growth of an animal, a method for increasing milk production in a cow, and a method for increasing lean-to- fat ratio in a pig, each of the methods of the invention comprising administering to the animal an effective amount of a somatotropin analog having single-site or multiple-site amino acid substitutions as described above.
  • one method of the invention comprises administering to an animal an effective amount of a somatotropin having one of the following single-site substitutions in the amino acid sequence of the protein: serine replacing methionine at amino acid position 5 (the analog designated M5S), serine replacing asparagine at position 13 (N13S), leucine for the valine found at amino acid position 15 (V15L), histidine replacing glutamine at position 19, and arginine replacing histidine at position 20 (Q19H,H20R), glutamic acid replacing glycine at position 40 (G40E), aspartic acid replacing asparagine at position 47 (N47D), proline replacing alanine at position 48 (A48P), glutamic acid replacing phenylalanine at position 52 (F52E), asparagine replacing glycine at position 63 (G63N), phenylalanine replacing tryptophan at position 86 (W86F), glutamic acid replacing glycine at position 88 (G88E), alanine
  • An additional method of the invention comprises administering to the animal an effective amount of a multiple-site somatotropin having two or more of the following: serine replacing methionine at amino acid position 5 (designated M5S), serine replacing asparagine at position 13 (N13S), leucine replacing valine at position 15 (V15L), histidine replacing glutamine at position 19, and arginine replacing histidine at position 20 (Q19H.H20R), glutamic acid replacing glycine at position 40 (G40E), aspartic acid replacing asparagine at position 47 (N47D), proline replacing alanine at position 48 (A48P), glutamic acid replacing phenylalanine at position 52 (F52E), asparagine replacing glycine at position 63 (G63N), phenylalanine replacing tryptophan at position 86 (W86F), glutamic acid replacing glycine at position 88 (G88E), serine replacing asparagine at position 99 (N99S), alan
  • this aspect of the method of the invention comprises administering to the animal an effective amount of a somatotropin having multiple-site substitutions selected from the group consisting of (a) V15L, A48P, F52E; (b) A48P, N99S, CI8I,189S; (c) F52E, G88E, F103E, I137E; (d) V15L, Q19H.H20R, A48P, F52E; (e) N13S, W86F, Q140S, N148S, M124L, M179L; and (f) V15L, A48P, F52E, N99S, F103E, C181.189S.
  • a somatotropin having multiple-site substitutions selected from the group consisting of (a) V15L, A48P, F52E; (b) A48P, N99S, CI8I,189S; (c) F52E, G88E, F103E, I137E; (d) V15L, Q
  • the present invention discloses somatotropin modifications that display enhanced conformational and chemical stability as compared to native pituitary or recombinantly derived somatotropins.
  • Single site modifications meeting this criteria include, for example, M5S, V15L, Q19H,H20R, A48P, G40E, N47D, F52E, G88E, F103E, I137E, and R125S.
  • Additional modifi ⁇ cations meeting this criteria and included as multiple-site analogs include, for example, N99S, Cl 81.189S. This shorthand notation shows, for example, that in M5S, methionine at position 5 of the rpSt primary structure, is replaced by serine.
  • Sequence ID: 1 shows the primary structure of pSt as isolated from pigs.
  • Other closely related somatotropin molecules having pSt activity have also been identified due to genetic polymorphism. Because of these changes and the slightly altered structure of homologous somatotropins, minor changes in the numbering and specific residues in the primary structure are found in the literature. The identity of specific protein modifications that enhance somatotropin stability have been made by characterizing single-site and multiple-site amino acid substitution somatotropins in chemical and bioactivity studies.
  • the strategies used to enhance the overall stability of the protein include: (i) modifications of somatotropin primary structure to enhance conformational stability of the protein, and/or (ii) specific elimination of somatotropin residues that are known to be involved in the decomposition of the protein, and/or (iii) modifications of somatotropin primary structure that permit the protein to be handled in neutral aqueous media.
  • This strategy can also result in an indirect stabilization of protein since it can now be processed, stored, and formulated under conditions that will have a much lower propensity for decomposing somatotropins.
  • Suitable amino acid substitutions which will remove this decomposition site and enhance solubility of the protein are serine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, proline, and threonine.
  • Suitable amino acid substitutions which will remove this hydrolytic degradation site are serine, glutamic acid, threonine, glutamine, aspartic acid, lysine, arginine, alanine, and histidine.
  • Suitable amino acid substitutions which will remove the conformational flexibility of the backbone include tyrosine, proline, threonine, serine, glutamic acid, asparagine, glutamine, aspartic acid, lysine, and arginine.
  • Suitable amino acid substitutions which will enhance the aqueous solubility of the protein are glutamic acid, alanine, glycine, tyrosine, proline, threonine, serine, histidine, asparagine, glutamine, aspartic acid, lysine, and arginine.
  • Suitable amino acids substitutions which will enhance the conformational stability of the protein backbone include asparagine, arginine, lysine, aspartic acid, glutamine, glutamic acid, histidine, serine, threonine, proline, tyrosine, and alanine.
  • Suitable amino acids substitutions which will remove this degradation site without substantively enhancing the hydrophilicity of the protein interior include phenylalanine, tyrosine, leucine, isoleucine, and valine.
  • Suitable amino acids which are expected to elimante secondary decomposition reactions near residue 99 while maintaining overall solubility of the protein include alanine, proline, glycine, glutamic acid, aspartic acid, lysine, and arginine.
  • Suitable amino acids which are expected to elimante secondary decomposition reactions near residue 99 while maintaining overall solubility of the protein include alanine, proline, glycine, glutamic acid, aspartic acid, lysine, and arginine.
  • Suitable amino acid substitutions which will enhance the aqueous solubility of the protein include glutamic acid, alanine, glycine, tyrosine, proline, threonine, serine, histidine, asparagine, glutamine, asparatic acid, lysine, and arginine.
  • Suitable amino acids which will eliminate the oxidative degradation of this residue include leucine, arginine, asparagine. aspartic acid, glutamic acid, glutamine, proline, serine, and threonine. Replacing arginine at position 125 with serine is also known to enhance the stability of the molecule.
  • Suitable amino acid substitutions which will enhance the aqueous solubility of rpSt include glutamic acid, alanine, glycine, tyrosine, proline, threonine, serine, histidine, asparagine, glutamine, aspartic acid, lysine, and arginine.
  • modified somatotropins of the invention are first characterized by means of the chemical and conformational studies described in more detail below.
  • a somatotropin generally exhibits an enhancement (as compared to the native somatotropin) in one of the stability or conformational studies described herein. It is contemplated that some degree of decreased bioactivity is acceptable due, in part, to the advantage gained by enhanced stability. Preferred modifications enhance both chemical and/or conformational stability as well as bioactivity in the host species.
  • Site-directed mutagenesis is a technique well known in the art of recombinant technology.
  • Several techniques for site-directed mutagenesis have been developed for introducing specific changes in a DNA sequence and may be used to produce the compounds of the instant invention (Kramer, E.,W, et al, Nucl. Acids Res. 12:9441-56 (1984); Mandecki, W., PNAS USA, 83:7177-81 (1986); Zollar, M.J. and Smith, M., Nucl. Acids Res.
  • One of the variations is to use a longer mutagenic oligonucleotide (about 40 bases) in the mutagenesis reaction and to screen candidates using a shorter oligonucleotide ( ⁇ 20 bases).
  • the other variation in the protocol is to transform the mutagenic reaction into either BHM71-18 mutS::TnlO or BST-lc, pool the colonies by scraping the colonies together, isolating vector DNA and re-transforming into either BST-lc or MCI 061. Electroporation is used when the initial transformation yields are low. Procedures leading to the MC1061 and BST-lc culture systems are described detail in International publication W088/06186. BST-IC is available from the Agricultural Research Service Culture Collection in the Northern Regional Research Center.
  • MC1061 is purchased from Clontech Laboratories, Inc., Palo Alto, CA.
  • BHM71-18 mutS::TnlQ is purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN, as a part of a Site-Directed Mutagenesis Kit (Cat. No. 1027-492). It is apparent to those skilled in the art that due to the degeneracy of the genetic code that other triplets (coding for the same amino acids) may be substituted in the primers used to make the modified somatotropins of the invention.
  • the porcine somatotropin gene (PStl) used in these studies is isolated from a porcine pituitary cDNA library constructed at the Upjohn Company and is described in International publication WO 88/06186.
  • the pURA plasmid, used for expression of the pSt gene, is described in International publication WO 88/06186.
  • the vector is modified to contain the Serratia marcescens trg promoter as described in WO 90/05186 and the ConSD ribosome- binding site as described by Curry and Tomich supra.
  • the E. coli strain and preferred host for expression of the somatotropin analogs of the invention is BST-lc.
  • Transformed cells containing the modified porcine gene are grown at the 10 liter scale. After fermentation the cell are centrifuged, and the modified porcine somatotropins are isolated using a bench scale modification of procedures described International publication WO 87/00204.
  • somatotropin analog is verified by use of a quantitative tryptic map (J.J. Dougherty, et al, Analytical Biochemistry 190:7-20 (1990)).
  • the trypsin used in these studies is previously treated with TPCK (l-tosylamide-2-phenylethyl chloromethyl ketone) in order to irreversibly inhibit chymotrypsin that may contaminate the product (Worthington Biochemical, Freehold, NJ.).
  • TPCK l-tosylamide-2-phenylethyl chloromethyl ketone
  • Buffers for chromatography are: A - 0.1% TFA in water; B - 70% CH 3 CN/0.07%TFA. Following injection, the column is eluted for 5 minutes in 1% B and chromatographed using a gradient of 1% B to 64.3% B in 120 minutes. Flow rate is 1.2 ml/min. The column effluent is monitored at 214 nm with column temperature at 40°C. Because of the reproducibility of the elution profile and the resolving capacity of the HPLC analysis, it is generally easy to separate and isolate any tryptic fragments that differ in retention time from those derived from wild-type somatotropins.
  • the peptide sequence of this region can be confirmed either by mass spectrometry or by NH 2 -terminal sequence analysis. Protein purity is determined using RP-and SEC-HPLC, and isoelectric focusing. These techniques are well known by those skilled in the art.
  • Somatotropin concentrations are determined by the absorbance at 278 nm using an extinction coefficient of 15,270 M '1 (Burger, H.G., et al, J. Biol. Chem. 241:449-457 (1966)). Determination of Relative Conformational Stability
  • sample dilutions to the appropriate concentrations of protein and denaturant are made using the stock solution buffer and 8.0 M guanidine HC1.
  • the stock denaturant and buffer solutions are filtered through 0.45 ⁇ m disposable filters (Coming Inc., Coming, N.Y.) prior to use. Both the stock solutions and prepared samples are stored at 4°C. Before spectral analysis, samples are allowed to equilibrate at ambient temperature for several hours. Protein concentrations are determined using a dye- binding method (bicinchoninic acid) This method is described by Smith, P. et al., Anal. Biochem.
  • Fluorescence measurements are performed on a photon-counting spectrofluorimeter (SLM Instruments, Inc., Urbana, IL). Spectra are obtained after excitation at 295 nm by monitoring protein emission at 355 nm. 2 nd derivative UV absorption measurements are performed on scanning (Aviv Associates, Lakewood, N.J.) and diode-array spectrometers (Hewlett Packard Scientific Instruments, Palo Alto, CA). Absorption spectra are obtained between 280 and 300 nm. Subsequent data handling is performed using algorithms supplied with each vendor's software.
  • Circular dichroic (CD) measurements are performed on (JASCO 500C and 700 series spectropolarimeters (Japan Spectroscopic Company, Tokyo, Japan). Near UV (275 to 325 nm) and far UV (226 to 218 nm) data are obtained for analysis.
  • Raw data from this technique obtained in units of ellipticity (millidegrees), is expressed in units of mean residue ellipticity (deg»cm 2 /dmol) using the equation:
  • Somatotropins (2 mg/ml) are dissolved in 0.05M sodium carbonate, pH 9.8. The vials are capped, placed in an oven and incubated at 30°C for up to 30 days. Daily aliquots are taken and stored at -20°C prior to analysis. Aliquots (20 ⁇ l) are analyzed using twin pump reverse phase-HPLC with UV detection
  • Tryptic mapping is performed as described above. Determination of Relative Solubility Proteins are dissolved in 0.05M sodium carbonate pH 9.8, at a protein concentration of 50mg/ml. The pH is adjusted with 50% NaOH where necessary. After filtration, a 250ul aliquot is withdrawn for dialysis. Microdialysis is conducted in a low volume chamber, fitted with a dialysis membrane (6000-8000 molecular weight cutoff). The unit is connected to a reservoir containing 1 liter of 0.05M sodium carbonate pH 9.8. In addition, a second unit is attached to a reservoir containing 0.04M sodium phosphate pH 7.0.
  • a Minipuls 2 peristaltic pump (Gilson, Middleton, WI) is used to pump buffer through the dialysis units at a flow rate of 30ml/hr. Samples are dialyzed for 20 hours at 5°C. The dialyzed samples as well as the starting material are then centrifuged at 13,000 rpm for 6 minutes in an Eppendorf 5415 centrifuge (Brinkman Instruments, Des Plaines, EL). The protein concentration of the supematant liquid is determined using the BCA assay (Pierce Chemical Company, Rockford, IL; P.K. Smith, et al, Anal. Biochem. 150:76-85 (1985)). Determination of Relative Bioactivity
  • the compounds of the invention may be tested for bioactivity by measuring growth in hypophysectomized rats and the target species following repeated administration.
  • the hypophysectomized rat growth assay is generally recognized by those skilled in the art as an appropriate method for obtaining a relatively rapid measure of the relative potency of somatotropins.
  • the modified somatotropins of the present invention are screened using hypophysectomized rats following the procedure of Evans, H.M. and Long J.A., Anat. Rec. 21:61 (1921), and M.D. Groesbeck and A.F. Parlow, Endocrinology 120:2582-2590 (1987), as modified below. Briefly, linearity of the assay is determined via the subcutaneously injection of eight hypophysectomized rats with somatotropin at doses of 2, 10, 50, and 250 ⁇ g/rat-day.
  • the modified somatotropins are tested at 15 and 60 ⁇ g/ml.
  • the weight of the rats are measured daily on the first day and up to the day following die final injection.
  • Relative increases in total body weight are recorded using various modified somatotropins of the invention with the native form of the molecule, either natural somatotropin and/or recombinant somatotropin, serving as a positive control.
  • bioactivity of the modified somatotropins of the invention can be measured using the swine growth assay using Yorkshire pigs. In this procedure, 48 gilts and barrows are sorted according to weight, and randomly assigned to four blocks of 6 pens each. Each pen contained 2 gilts and 2 barrows. Treatment was assigned randomly to pens within a block.
  • the animals are left for a 7 day acclimation. They are then injected with somatotropin or modified somatotropin at a rate of 60 ⁇ g/kg/d for 14 days. Positive controls are administered recombinant rpSt. The pretreatment weight is compared with posttreatment weight with measurements at days 1, 15, and 22 of the experiment.
  • the compounds of the invention can be used to enhance growth characteristics in the species in which the native somatotropin has been shown to have growth-related bioactivity.
  • growth hormones are very similar in their amino acid sequences, hormones originating from one species may enhance the growth of other animal species that evolved at earlier times.
  • the compoimds of the invention are suitable for use in this manner as well.
  • somatotropins of less highly evolved species, such as cattle and pigs have not been found to be functional in more highly evolved species, such as humans.
  • Admimstration of the compounds of the invention into animals is done according to known methods using any route effective to deliver the required dosage to the animal's circulatory system. Modes of administration include those typically encoimtered for the species of choice.
  • the effective dosage range depends on the animal, as well as the age. weight, and general health of the animal to be treated. These and other parameters which are needed to determine the effective dosage range for a given species is well within the purview of one skilled in the art. For instance, in bovine the effective amount is in the range of 1.0 to 200 milligrams per animal per day. In pigs, for instance the effective amount is about 60 ⁇ g/kg/d.
  • the potential commercial use of the compounds of the invention in non-human animals derives from their enhancement of the animal's metabolism.
  • administration of an effective amount of the porcine analogs of the invention allows a pig to grow leaner in a shorter time span and requires less feed to do so.
  • administration of an effective amount of the bovine analogs of the invention allows milk producing animals, such as dairy cattle, to produce more milk for the same or lower amount of feed.
  • the compounds of the invention are identified using a letter-number-letter string which designates the location of the substitution and the native and substituted amino acid.
  • the letters of this string follow the accepted single letter designation for amino acids.
  • the single letter designation and the amino acid to which it corresponds are shown in Chart 1.
  • the first letter (X ⁇ ) designates the native amino acid found in wild-type somatotropin;
  • the second letter ( ⁇ X) designates the amino acid that is substituted in the analog.
  • the number (-0-) represents the sequential location of the amino acid in the native porcine somatotropin.
  • V15L designates a somatotropin analog of the invention where leucine is substituted for the valine residue found at position 15 of native porcine somatotropin
  • A48P designates the somatotropin analog of the invention having proline substituted for alanine at position 48 of the native porcine somatotropin.
  • an “effective amount” is the amount of a modified somatotropin of the invention which results in the desired biological effect in the target animal.
  • Effective amount includes the dosage level which is efficacious as well as the period of time such a dose is administered to attain or maintain the efficacious dose.
  • Microorganism means both single cellular prokaryotic and eukaryotic organisms such as bacteria, yeast, actinomycetes and single cells from higher plants and animals grown in cell culture.
  • Single-site means single or multiple changes in the somatotropin gene sequence which result in a one to three amino acid residue substitution within a short segment of the somatotropin protein.
  • the analogs A48P and Q19H.H20R designate single- site substitutions.
  • Multiple-site substitutions or analogs combine two or more single-site substitutions into an individual protein in order to further enhance desirable characteristics.
  • the analog V15L, A48P, F52E, N99S, F103E, C18U89S designates a multiple- site modified somatotropin.
  • Native somatotropin refers to somatotropins having amino acid sequences which are the same as the naturally-occurring forms of the molecule.
  • Native somatotropins may be derived from either natural or recombinant sources. Due to the molecular heterogeneity of somatotropins, the position numbers of amino acid residues of the various somatotropins may differ. The term native somatotropin includes these naturally occurring species. Sequence ID: 1 illustrates one species of porcine somatotropin (pSt). The numbering for other somatotropins may differ.
  • Somatotropin or “St” means an animal somatotropin and includes somatotropins derived from either the pituitary gland tissue ("natural somatotropin”) or from microorganisms transformed by recombinant genetics to produce either naturally-occurring or modified forms of somatotropin ("recombinant somatotropin”).
  • a specific mammalian source such as a somatotropin of porcine origin (porcine somatotropin or pSt)
  • the somatotropin includes those derived from either pituitary sources or from transformed microorganisms.
  • Example 1 Production of M5S.
  • a site-directed mutagenic technique for double-stranded primer extension is used to introduce altered codons for methionine at amino acid position 5 in the PStl cDNA gene.
  • the target sequence is cloned into a suitable plasmid and plasmid DNA is prepared.
  • the plasmid DNA is denatured by treatment with NaOH which causes "nicks" in the DNA molecule deoxyribose-phosphate backbone. This relaxes the DNA and permits an oligomer containing the desired sequence changes to hybridize to the target site.
  • the 3' end of the oligomer generates a primer for the DNA polymerase activity of the Polymerase A Klenow fragment which extends the primer, synthesizes a new DNA strand containing the mutagenic oligomer and displaces the normal complementary strand.
  • the extension reaction increases the probability of the incorporation by genetic recombination.
  • the DNA is transformed into competent cells and the resultant colonies are screened by colony filter hybridization. Plasmid DNA is isolated and sequenced from positive candidates.
  • the oligomers used to construct the position 5 serine change in the Pstl gene are produced by techniques described in International publication WO 88/06186 and is shown in Sequence ID:2. An oligonucleotide so produced and set forth above contains the proper change.
  • Example 2 Production of V15L Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID:3), a somatotropin analog having leucine at position 15 is also constructed.
  • Example 3 Production of G40E Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID:4), a somatotropin analog having glutamic acid at position 40 is also constructed.
  • Example 4 Production of N47D
  • Example 5 Production of A48P Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID:6), a somatotropin analog having proline at position 48 is also constructed.
  • Example 6 Production of F52E Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID:7), a somatotropin analog having glutamic acid at position 52 is also constructed.
  • Example 7 Production of E56A Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID: 8), a somatotropin analog having alanine at position 56 is also constructed.
  • Example 8 Production of G88E Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID:9), a somatotropin analog having glutamic acid at position 88 is also constructed.
  • Example 9 Production of N99S
  • Example 11 Production of R125S Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID: 12), a somatotropin analog having serine at position 125 is also constructed.
  • Example 12 Production of I137E Following the techniques of Example 1, but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID: 13), a somatotropin analog having glutamic acid at position 137 is also constructed.
  • Example 13 Production of Q19H,H20R Following the techniques of Example 1, but substituting the appropriate oligomers encoding die desired amino acids (as seen in Sequence ID: 14), a somatotropin analog having histidine at position 19 and an arginine at position 20 is also constructed.
  • Example 14 Production of C 181 , 189S
  • Example 16 Production of T98A,N99S,S100A Following the techniques of Example 1 , but substituting the appropriate oligomers encoding the desired amino acids (as seen in Sequence ID: 16), a somatotropin analog having alanine at position 98, serine at position 99, and alanine at position 100 is also constructed. Examples 17-22: Multiple-site modified somatotropins
  • large DNA sequences can be constructed by combining long oligonucleotide synthesis with Polymerase Chain Reaction (PCR) as has been demonstrated by R.W. Barnett and H. Erfle, Nucleic Acid Research 18:3094 (1990), P.J. Dillon and CA. Rosen, Biotechniques 9:298-299 (1990), and R.M. Horton, et al, Gene 77:61-68 (1990).
  • PCR Polymerase Chain Reaction
  • An entire gene of porcine somatotropin can be constructed containing the desired codon changes using this approach.
  • Two long oligonucleotides of about 200 bases is synthesized as described below (see, "Synthesis of Oligonucleotides").
  • the concentration of the full length oligonucleotides is low due to the inefficiency in the addition of each nucleotide.
  • These two oligonucleotides are designed to overlap by 20 to 30 bases, and when mixed together can be extended by a DNA polymerase to form a larger 400 base pair fragment. This fragment can be amplified using PCR. The fragment is then isolated, cut with restriction enzymes and cloned into an appropriate expression vector. By generating two or three sets of these fragments an entire porcine somatotropin gene can be constructed.
  • Oligonucleotides are made by the phosphoramidite method for DNA synthesis.
  • this process uses as the starting material the 5' dimethoxytrityl ether of a nucleoside bound to a silica support. This nucleoside will be the 3'-OH end of the product oligonucleotide.
  • the first step is detritylation with acid.
  • the nucleotide phosphoramidite to be added is activated.
  • the addition step occurs in less than 3 minutes and is 95 to 97% complete. Any chains which did not undergo addition is capped by acetylation so that it will not react in subsequent steps.
  • the intemucleotide linkage is then converted from the phosphite to the more stable phosphate by oxidation. This compound is detritylated and the cycle continues until the last nucleoside is added. At this point, the oligonucleotide is the bases A, G, and C The chain is then cleaved from the support with ammonium hydroxide. After, the solution containing the DNA is removed from the instrument, the protecting groups are cleaved by a 8-hour treatment with ammonium hydroxide at 55°C
  • Example 17 Production of V15L, A48P, F52E, N99S, F103E, C181.189S Following the techniques described above, a somatotropin multiple-site analog having leucine at position 15, proline at position 48, glutamic acid at position 52, serine at position 99, glutamic acid at position 103, and serine residues at positions 181 and 189 is constructed.
  • Example 18 V15L, A48P, F52E Following the techniques described above, a somatotropin multiple-site analog having leucine at position 15, proline at position 48, glutamic acid at position 52 is constructed.
  • Example 19 A48P, N99S, C181J89S
  • Example 20 F52E, G88E, F103E, I137E Following the techniques described above, a somatotropin multiple-site analog having glutamic acids at positions 52, 88, 103, and 137 is constructed.
  • Example 21 V15L, Q19H, H20R, A48P, F52E Following the techniques described above, a somatotropin multiple-site analog having leucine at position 15, histidine at position 19, arginine at position 20, proline at position 48, and glutamic acid at position 52, is constructed.
  • Example 22 N13S, W86F, Q140S, N148S, M124L, M179L Following the techniques described above, a somatotropin multiple-site analog having asparagine at position 13, phenylalanine at position 86, serines at positions 140 and 148, and leucines at positions 124, and 179 is constructed.
  • Tables I, II, and III summarize the relative conformational stability of these rpSt analogs relative to wild-type proteins. As described earlier, conformational stability is compared by equilibrium denaturation using several distinct spectral probes. (For, i.e., second derivative UV absorption, circular dichromism and fluorescent spectroscopy). These experiments are plotted in order to obtain the denaturation midpoint of unfolding.
  • the compounds of the invention are incubated in the presence of alkaline and peroxide-containing buffers in order to determine their relative susceptibility to these reagents.
  • Alkaline buffers are used in these studies since they permit the observation of hydrolytic degradation reactions in a compressed time-frame.
  • peroxide containing buffers permit the observation of oxidative degradative reactions in a compressed time-frame.
  • aliquots are subjected to trypsin digestion.
  • the tryptic fragments are separated and identified using a technique known as LC-MS. In this technique, fragments are sorted according to their affinity for reversed-phase HPLC and are then identified on the basis of their mass to charge (m/e) ratio.
  • Table IV lists the porcine St fragments that are generated by digestion by trypsin and their respective molecular weights.
  • Table V lists additional peaks that have been observed incubation of porcine St in the presence of alkaline buffers. An analog is said to have enhance chemical stability when one or more of the latter peaks do not form or form at a slower rate.
  • Example 25 Characterization of enhanced solubility in aqueous buffers at neutral pH. The modified somatotropins are characterized to determine if the solubility of these compounds in neutral, aqueous buffers could be significantly altered by single-site amino acid substitutions. Wild-type and modified somatotropins are tested via dialysis as described in Detailed Description section. The results are shown in Table IV.
  • Solubility enhancements can occur, for example, on substitution of hydrophobic amino acids with relatively hydrophilic amino acids.
  • solubility can be enhanced by changing the isoelectric point of the protein. (The isoelectric point is the pH at which the protein has no net charge).
  • Example 26 Rat growth bioassay.
  • the rat growth assay shows that the modified porcine somatotropins of the invention have equivalent bioactivity as compared to the native protein. A summary of these results are shown in Table VII.
  • Example 27 Porcine bioassay.
  • Crossbred Yorkshire pigs were utilized for this bioassay. Forty-eight gilts and 48 barrows weighing approximately 50 kg were ranked by body weight (BW). These rank orders were used to divide each gender into 4 blocks. Within each gender and block, pigs were assigned randomly to one of 6 pens, avoiding littermate assignments to the same pen. Each pen contained 2 gilts and 2 barrows. Treatment was assigned randomly to pens within a block. Following assignment to pens, the pigs were allowed at least a 7 day acclimation period before initiation of the treatments.
  • BW body weight
  • figure 2 shows the retention times for these peaks.
  • GGAGGGCATC CAGGCCCTGA TGCGGGAGCT GGAGGATGGC AGCCCCCGGG 400

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Endocrinology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Reproductive Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pregnancy & Childbirth (AREA)
  • Gynecology & Obstetrics (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Fodder In General (AREA)

Abstract

L'invention se rapporte à des modifications de la somatotrophine qui ont entraîné des améliorations inattendues dans sa stabilité structurelle et chimique sans perte de bioactivité. Les changements de ces caractéristiques ont été obtenus par la modification des somatotrophines par mutogenèse dirigée sur le site. Ces changements augmentent la stabilité de conservation de ces protéines. Les modifications typiques sont celles des somatotrophines du porc comportant des substitutions de glutamine et d'histidine arginine aux positions 19 et 20, respectivement (Q19H, H20R); l'acide glutamique est substitué à la phénylalamine à la position 52 (F52E), et l'acide glutamique est substitué à la leucine à la position 137 (I137E). Les somatotrophines modifiées sont utiles pour améliorer les caractéristiques de croissance chez des espèces animales, notamment pour augmenter le rapport muscle/graisse chez le porc et la production de lait chez la vache.
PCT/US1993/009232 1992-10-28 1993-10-04 Modifications de la somatotrophine WO1994010200A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP93922424A EP0669939A1 (fr) 1992-10-28 1993-10-04 Modifications de la somatotrophine
JP6511061A JPH08502963A (ja) 1992-10-28 1993-10-04 ソマトトロピン修飾物
AU51422/93A AU5142293A (en) 1992-10-28 1993-10-04 Somatotropin modifications

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US97109892A 1992-10-28 1992-10-28
US07/971,098 1992-10-28

Publications (1)

Publication Number Publication Date
WO1994010200A1 true WO1994010200A1 (fr) 1994-05-11

Family

ID=25517926

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/009232 WO1994010200A1 (fr) 1992-10-28 1993-10-04 Modifications de la somatotrophine

Country Status (7)

Country Link
EP (1) EP0669939A1 (fr)
JP (1) JPH08502963A (fr)
AU (1) AU5142293A (fr)
CA (1) CA2145546A1 (fr)
MX (1) MX9306676A (fr)
WO (1) WO1994010200A1 (fr)
ZA (1) ZA936811B (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999026069A1 (fr) * 1997-11-14 1999-05-27 Generic Biologicals Limited Ameliorations relatives a la detection de molecules dans des echantillons
GB2334580A (en) * 1997-11-14 1999-08-25 Generic Biolog Limited Method of detecting and differentiating a tagged exogenously applied protein from the endogenous protein
US6844322B2 (en) 2000-07-21 2005-01-18 Monsanto Technology Llc Method for induced lactation
US8513192B2 (en) 2009-01-22 2013-08-20 Novo Nordisk A/S Stable growth hormone compounds resistant to proteolytic degradation
US8779109B2 (en) 2010-01-22 2014-07-15 Novo Nordisk Health Care Ag Growth hormones with prolonged in-vivo efficacy
US8841249B2 (en) 2009-08-06 2014-09-23 Novo Nordisk A/S Growth hormones with prolonged in-vivo efficacy
US8865868B2 (en) 2008-08-06 2014-10-21 Novo Nordisk Healthcare Ag Conjugated proteins with prolonged in vivo efficacy
US9211342B2 (en) 2010-01-22 2015-12-15 Novo Nordisk Healthcare Ag Stable growth hormone compounds resistant to proteolytic degradation
US11045523B2 (en) 2013-04-05 2021-06-29 Novo Nordisk Healthcare Ag Formulation of growth hormone albumin-binder conjugate

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RS59977B1 (sr) * 2011-06-10 2020-03-31 Novo Nordisk As Polipeptidi
WO2015070911A1 (fr) * 2013-11-14 2015-05-21 Lysando Ag Séquence d'endolysine kz144 modifiée

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0355460A2 (fr) * 1988-08-24 1990-02-28 American Cyanamid Company Stabilisation de somatotropines par modification du résidu cystéine en utilisant un site de mutagénèse dirigée ou par modification chimique
WO1990008164A1 (fr) * 1989-01-19 1990-07-26 The Upjohn Company Analogues de somatotrophine
WO1991000870A1 (fr) * 1989-07-10 1991-01-24 The Upjohn Company Analogues de somatotrophine
EP0488279A2 (fr) * 1990-11-30 1992-06-03 American Cyanamid Company Somatotropines ayant des modifications au niveau de la région 3 de l'hélice alpha, différentes combinaisons de ces modifications et avec d'autres mutations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0355460A2 (fr) * 1988-08-24 1990-02-28 American Cyanamid Company Stabilisation de somatotropines par modification du résidu cystéine en utilisant un site de mutagénèse dirigée ou par modification chimique
WO1990008164A1 (fr) * 1989-01-19 1990-07-26 The Upjohn Company Analogues de somatotrophine
WO1991000870A1 (fr) * 1989-07-10 1991-01-24 The Upjohn Company Analogues de somatotrophine
EP0488279A2 (fr) * 1990-11-30 1992-06-03 American Cyanamid Company Somatotropines ayant des modifications au niveau de la région 3 de l'hélice alpha, différentes combinaisons de ces modifications et avec d'autres mutations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
S NISHIKAWA ET AL.: "STRUCTURE AND ACTIVITY OF ARTIFICIAL MUTANT VARIANTS OF HUMAN GROWTH HORMONE", PROTEIN ENGINEERING, vol. 3, no. 1, October 1989 (1989-10-01), ENGLAND GB, pages 49 - 53, XP000068020 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999026069A1 (fr) * 1997-11-14 1999-05-27 Generic Biologicals Limited Ameliorations relatives a la detection de molecules dans des echantillons
GB2334580A (en) * 1997-11-14 1999-08-25 Generic Biolog Limited Method of detecting and differentiating a tagged exogenously applied protein from the endogenous protein
GB2334580B (en) * 1997-11-14 2002-12-18 Generic Biolog Ltd Improvements in or relating to detection of exogenously administered polypeptides
US6680207B1 (en) 1997-11-14 2004-01-20 Generic Biologicals Limited Detection of molecules in samples
US6844322B2 (en) 2000-07-21 2005-01-18 Monsanto Technology Llc Method for induced lactation
US8865868B2 (en) 2008-08-06 2014-10-21 Novo Nordisk Healthcare Ag Conjugated proteins with prolonged in vivo efficacy
US8513192B2 (en) 2009-01-22 2013-08-20 Novo Nordisk A/S Stable growth hormone compounds resistant to proteolytic degradation
US8841249B2 (en) 2009-08-06 2014-09-23 Novo Nordisk A/S Growth hormones with prolonged in-vivo efficacy
US8779109B2 (en) 2010-01-22 2014-07-15 Novo Nordisk Health Care Ag Growth hormones with prolonged in-vivo efficacy
US9211342B2 (en) 2010-01-22 2015-12-15 Novo Nordisk Healthcare Ag Stable growth hormone compounds resistant to proteolytic degradation
US9695226B2 (en) 2010-01-22 2017-07-04 Novo Nordisk Healthcare Ag Growth hormones with prolonged in-vivo efficacy
US11045523B2 (en) 2013-04-05 2021-06-29 Novo Nordisk Healthcare Ag Formulation of growth hormone albumin-binder conjugate

Also Published As

Publication number Publication date
MX9306676A (es) 1994-04-29
JPH08502963A (ja) 1996-04-02
ZA936811B (en) 1995-03-15
CA2145546A1 (fr) 1994-05-11
AU5142293A (en) 1994-05-24
EP0669939A1 (fr) 1995-09-06

Similar Documents

Publication Publication Date Title
DE60038737T2 (de) Modifiziertes cytokin für seine stabilizierung
US5350836A (en) Growth hormone antagonists
KR0184235B1 (ko) 거핵구 형성 인자
KR920000849B1 (ko) 생물학적으로 활성인 단백질의 제조방법
AU634517B2 (en) Somatotropin analogs
US5994518A (en) Method of producing a polypeptide having human granulocyte colony stimulating factor activity
EP0669939A1 (fr) Modifications de la somatotrophine
CN1197461A (zh) 截短的神经胶质细胞系来源的神经营养因子
CA2285673C (fr) Analogues cycliques de composes d'histatine
AU639245B2 (en) Somatotropin analogs
CA2056467C (fr) Somatotrophines presentant des alterations dans la region 1 de l'alpha-helice, et combinaisons avec d'autres mutations
WO1992019736A1 (fr) Antagonistes de l'hormone de croissance
US5663305A (en) Somatotropin analogs
WO1988007084A1 (fr) Analogues de somathormone bovine
CA1277933C (fr) Analogues d'hormone de croissance porcines
US6465616B1 (en) Interleukin-5 antagonist
WO1997045448A1 (fr) Antagoniste d'interleukine-5
US7358338B2 (en) Heme peptide
JPH03502758A (ja) ポリペプチド類においてノルロイシン含有量を制御するための発酵培地および方法
Skryabin et al. Growth hormones: expression, structure and protein engineering
Skryabin¹ et al. GROWTH HORMONES: EXPRESSION, STRUCTURE AND PROTEIN ENGINEERING
CA2216755A1 (fr) Peptides avec des proprietes favorisant la croissance
DE19649233A1 (de) Prozessierte Polypeptide mit IL-16 Aktivität, Verfahren zu ihrer Herstellung und Verwendung
JPH0259599A (ja) 改良エリスロポイエチン組成物

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR BY CA CH CZ DE DK ES FI GB HU JP KP KR KZ LK LU LV MG MN MW NL NO NZ PL PT RO RU SD SE SK UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2145546

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1993922424

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1993922424

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: 1993922424

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1993922424

Country of ref document: EP