Dry pharmaceutical formulations of CNP conjugates
阅读说明：本技术 Cnp缀合物的干燥药物制剂 (Dry pharmaceutical formulations of CNP conjugates ) 是由 A·R·H·什歌德斯 U·赫泽尔 C·平霍尔特 S·海尼希 于 2020-02-10 设计创作，主要内容包括：一种干燥药物制剂,其中所述药物制剂包含CNP缀合物、缓冲剂和填充剂,且其中所述CNP缀合物包含共价和可逆地缀合至聚合物部分的CNP部分。(A dry pharmaceutical formulation, wherein the pharmaceutical formulation comprises a CNP conjugate, a buffer, and a bulking agent, and wherein the CNP conjugate comprises a CNP moiety covalently and reversibly conjugated to a polymer moiety.)
1. A dry pharmaceutical formulation, wherein the pharmaceutical formulation comprises a CNP conjugate, a buffer and a filler, and wherein the CNP conjugate comprises a CNP moiety covalently and reversibly conjugated to a polymer moiety.
2. A dry pharmaceutical formulation according to claim 1, wherein the buffering agent is selected from: succinic acid, citric acid, lactic acid, acetic acid, glutamic acid, fumaric acid, aspartic acid, glutaric acid, phosphoric acid, histidine, gluconic acid, tartaric acid, malic acid, and mixtures thereof.
3. A dry pharmaceutical formulation according to claim 1 or 2, wherein the buffering agent is succinic acid.
4. A dry pharmaceutical formulation according to any one of claims 1 to 3, wherein the filler is selected from: trehalose, mannitol, sucrose, raffinose, gelatin, lactose, dibasic calcium phosphate, sorbitol, xylitol, glycine, histidine, hydroxyethyl starch, dextrose, dextran, propylene glycol, and mixtures thereof.
5. The dry pharmaceutical formulation according to any one of claims 1 to 4, wherein the filler is selected from: trehalose, sucrose and glycine.
6. A dry pharmaceutical formulation according to any one of claims 1 to 5, wherein the filler is trehalose.
7. A dry pharmaceutical formulation according to any one of claims 1 to 6, wherein the dry pharmaceutical formulation of the invention comprises one or more further excipients.
8. A dry pharmaceutical formulation according to claim 7, wherein the one or more further excipients are selected from: preservatives, stabilizers, anti-adsorbers, cryoprotectants, oxidation protectants, and other adjuvants.
9. A dry pharmaceutical formulation according to any one of claims 1 to 8, wherein the dry pharmaceutical formulation of the invention comprises a stabilizer.
10. A dry pharmaceutical formulation according to any one of claims 1 to 9, wherein the dry pharmaceutical formulation of the invention comprises a preservative.
11. A dry pharmaceutical formulation according to any one of claims 1 to 10, wherein the dry pharmaceutical formulation of the invention comprises an anti-adsorbent agent.
12. The dry pharmaceutical formulation according to any one of claims 1 to 11, wherein the dry pharmaceutical formulation of the invention comprises a cryoprotectant.
13. The dry pharmaceutical formulation according to any one of claims 1 to 12, wherein the dry pharmaceutical formulation of the present invention comprises an oxidant protectant.
14. A dry pharmaceutical formulation according to any one of claims 1 to 13, wherein the dry pharmaceutical formulation of the invention comprises further excipients selected from the group consisting of: humectants, viscosity modifiers, and antibiotics.
15. The dry pharmaceutical formulation of any one of claims 1 to 14, wherein the pharmaceutical formulation comprises a pH adjusting agent.
16. A dry pharmaceutical formulation according to claim 15, wherein the pH adjusting agent is selected from: tris, sodium hydroxide, potassium hydroxide, lysine and mixtures thereof.
17. The dry pharmaceutical formulation of claim 15 or 16, wherein the pH adjusting agent is Tris.
18. The dry pharmaceutical formulation of any one of claims 1 to 17, wherein the pharmaceutical formulation comprises a CNP conjugate, succinic acid, trehalose, and Tris.
19. The dry pharmaceutical formulation of any one of claims 1 to 18, wherein the CNP moiety has the sequence of SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25 or SEQ ID NO: 30, or a pharmaceutically acceptable salt thereof.
20. The dry pharmaceutical formulation of any one of claims 1 to 19, wherein the CNP moiety has the sequence of SEQ ID NO: 24, or a fragment thereof.
21. The dry pharmaceutical formulation of any one of claims 1 to 20, wherein the polymer portion comprises a polymer selected from the group consisting of: 2-methacryloyl-ethoxyphosphorylcholine, poly (acrylic acid), poly (acrylate), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amidoamine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), polybutylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), poly (ester), poly (ethylene glycol), poly (ethylene oxide), poly (ethyl phosphate), poly (ethyloxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl-oxazoline), poly (hydroxymethyl acrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate), Poly (hydroxypropyl oxazoline), poly (imino carbonates), poly (lactic acid), polylactic acid-co-glycolic acid, poly (methacrylamide), poly (methacrylate), poly (methyl oxazoline), poly (organophosphazene), poly (orthoester), poly (oxazoline), poly (propylene glycol), poly (siloxane), poly (urethane), poly (vinyl alcohol), poly (vinyl amine), poly (vinyl methyl ether), poly (vinyl pyrrolidone), silicone, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnouronic acid, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate based polymers, Xylan, and copolymers thereof.
22. The dry pharmaceutical formulation of any one of claims 1 to 21, wherein the formulation comprises, based on the total weight of the dry pharmaceutical formulation:
23. the dry pharmaceutical formulation of any one of claims 1 to 22, wherein the formulation comprises, based on the total weight of the dry pharmaceutical formulation:
24. the dry pharmaceutical formulation of any one of claims 1 to 23, wherein the CNP conjugate is of formula (Ia) or (Ib):
wherein the content of the first and second substances,
-D is a CNP moiety;
-L1-is a reversible linker moiety;
-L2-is a single chemical bond or a spacer;
-Z is a polymer moiety;
x is an integer selected from: 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; and is
y is an integer selected from: 1. 2, 3, 4 and 5.
25. A dry pharmaceutical formulation according to claim 24, wherein x of formula (Ia) is an integer selected from: 1. 2, 3, 4, 6 and 8.
26. The dry pharmaceutical formulation of claim 24, wherein y of formula (Ib) is an integer selected from: 2. 3, 4 and 5.
27. The dry pharmaceutical formulation of claim 24 or 25, wherein the CNP conjugate is of formula (Ia) and x is 1.
28. A dry pharmaceutical formulation according to any one of claims 24 to 27, wherein-L 1-is linked to-D by an amide linkage.
29. The dry pharmaceutical formulation according to any one of claims 24 to 28, wherein-Z and-L2The bond between-is a stable bond.
30. A dry pharmaceutical formulation according to any one of claims 24 to 29, wherein-L2-is a spacer moiety.
31. A dry pharmaceutical formulation according to any one of claims 24 to 30, wherein-L2-has a molecular weight ranging from 14g/mol to 750 g/mol.
32. A dry pharmaceutical formulation according to any one of claims 24 to 31, wherein-L2-has a chain length of 1 to 20 atoms.
33. A dry pharmaceutical formulation according to any one of claims 24 to 32, wherein-L2-is of formula (i):
wherein the content of the first and second substances,
dotted line marked with an asterisk indicates attachment to-L1-；
An unlabeled dashed line indicates attachment to-Z;
-R1selected from: -H, C1-6Alkyl radical, C2-6Alkenyl and C2-6An alkynyl group;
n is selected from: 0. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18; and is
Wherein the moiety of formula (i) is optionally further substituted.
34. A dry pharmaceutical formulation according to claim 33, wherein n of formula (i) is selected from: 3. 4, 5, 6, 7, 8 and 9.
35. The dry pharmaceutical formulation according to any one of claims 24 to 34, wherein-Z has a molecular weight in the range of 5 to 200 kDa.
36. The dry pharmaceutical formulation according to any one of claims 24 to 35, wherein-Z comprises a polymer selected from: 2-methacryloyl-ethoxyphosphorylcholine, poly (acrylic acid), poly (acrylate), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amidoamine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), polybutylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), poly (ester), poly (ethylene glycol), poly (ethylene oxide), poly (ethyl phosphate), poly (ethyloxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl-oxazoline), poly (hydroxymethyl acrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate), Poly (hydroxypropyloxazoline), poly (iminocarbonate), poly (lactic acid), polylactic-co-glycolic acid, poly (methacrylamide), poly (methacrylate), poly (methyloxazoline), poly (organophosphazene), poly (orthoester), poly (oxazoline), poly (propylene glycol), poly (siloxane), poly (urethane), poly (vinyl alcohol), poly (vinyl amine), poly (vinyl methyl ether), poly (vinyl pyrrolidone), silicone, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonic acid, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate based polymers, Xylan, and copolymers thereof.
37. A method of preparing the dry pharmaceutical formulation of any one of claims 1-36, wherein the method comprises the steps of:
(i) mixing the CNP conjugate with at least one buffer and one bulking agent;
(ii) (ii) adjusting the pH of the mixture of step (i);
(iii) (iii) optionally, filtering the mixture from step (ii);
(iv) (iv) transferring an amount of the mixture of step (ii) or (iii) corresponding to the desired number of doses into a vessel;
(v) drying the mixture;
(vi) sealing the container; and is
Wherein the order of steps (ii) and (iii) may optionally be reversed.
38. A method of reconstituting the dry pharmaceutical formulation of any one of claims 1 to 36, wherein said method comprises the steps of:
(a) contacting the dry pharmaceutical formulation of any one of claims 1 to 36 with a reconstitution solution.
39. A reconstituted pharmaceutical formulation obtainable from the reconstitution process of claim 38.
40. A dry pharmaceutical formulation according to any one of claims 1 to 36 or a reconstituted pharmaceutical formulation according to claim 39 for use as a medicament.
41. The dry pharmaceutical formulation of any one of claims 1 to 36 or the reconstituted pharmaceutical formulation of claim 39 for use in the treatment, control, delay or prevention of one or more diseases treatable, controllable, delayed or preventable with CNP.
42. A dry pharmaceutical formulation for use according to claim 41, wherein the disease is selected from: bone-related disorders, such as skeletal dysplasia; cancer; autoimmune diseases; fibrotic diseases; inflammatory diseases; central nervous system diseases, such as neurodegenerative diseases; infectious diseases; pulmonary diseases; heart and vascular disease; metabolic diseases and ophthalmic diseases.
43. A dry pharmaceutical formulation for use according to claim 41 or 42, wherein the disease is achondroplasia.
44. A method of treating, controlling, delaying or preventing one or more diseases treatable by CNP in a patient, comprising administering to the patient a therapeutically effective amount of the reconstituted pharmaceutical formulation of claim 39.
45. The method of claim 44, wherein the disease is selected from the group consisting of: bone-related disorders, such as skeletal dysplasia; cancer; autoimmune diseases; fibrotic diseases; inflammatory diseases; central nervous system diseases, such as neurodegenerative diseases; infectious diseases; pulmonary diseases; heart and vascular disease; metabolic diseases and ophthalmic diseases.
46. The method of claim 44 or 45, wherein the disease is achondroplasia.
Examples
Materials and methods
All materials are commercially available unless otherwise indicated.
The content and purity of compound (1) was determined using RP-HPLC and the free CNP-38 was detected: mobile phase a consisted of 0.05% aqueous TFA and mobile phase B consisted of acetonitrile containing 0.04% TFA. Using a Waters Acquity CSH C18, 1.7 μm, 2.1 × 100mm chromatography column. The flow rate was set at 0.3mL/min, the detection was at 215nm wavelength, and the column run temperature was 60 ℃ (± 1 ℃). By containing-20 dilution of the sample with formulation buffer. The content was determined by comparing the peak area with a reference solution.
Purity of compound (1) was determined using SE-HPLC: the mobile phase consisted of 15mM sodium phosphate (pH 7.40), 135mM sodium chloride, 0.2% Pluronic F-68 in water. A GE Superdex 200 Increase 10/300 GL column was used. The flow rate was set at 0.75mL/min, detection was at 215nm wavelength, and the column run temperature was room temperature. The samples were diluted with formulation buffer containing 0.05% Pluronic F-68.
The conversion of aspartic acid (28) to isoaspartic acid (28) and the oxidation of methionine (33) to methionine oxide (33) (met (o)) was evaluated using the peptide mapping of compound (1). The thermolysin digestion of (1) was carried out at pH 7.5, 37 ℃ and a thermolysin/CNP-38 ratio of 1:20(w/w) for 7 h. At the Waters Acquity UPLC HSS T3,1.8 μm, 2.1X150mm column, 0.10 vol% aqueous TFA as mobile phase A and acetonitrile containing 0.09% TFA as mobile phase B, and detecting the resulting peptide mixture at 210nm, separated by RP-HPLC. The flow rate was set at 0.28mL/min and the column run temperature was 45 ℃ (+ -1 ℃). The thermolysin fragment containing aspartate (28) and methionine (33) was characterized by LC-MS. In conventional analysis, fragments are compared to a reference mixture containing these fragment peptides, the fragments being quantified based on their respective peak areas relative to the peak area of the corresponding unmodified fragment. Under thermolysin digestion and subsequent RP-HPLC conditions, (1) can be quantified by the formation of the isoaspartic acid product of aspartic acid (28) and the oxidation of methionine (33).
Bioperformance analysis was performed by in vitro release of CNP-38 from (1) and subsequent bioperformance analysis of the released CNP-38 in a cellular assay.
CNP-38 release from (1) in vitro and RP-HPLC quantification: (1) incubate at pH 10.0 and 15 ℃ for 24 hours. The pH was adjusted by diluting the recombinant formulation with release buffer at a dilution of 1:3.5(v/v) (e.g., 50. mu.L (1) of a 3.6mg CNP-38 eq/mL solution diluted with 175. mu.L release buffer) (0.5M boric acid, 10mM methionine, 2.383g/L Pluronic-F68; pH adjusted to 10.0 using 4M aqueous NaOH solution). After incubation, the release was quenched by 1: 1(v/v) dilution with 5% (v/v) acetic acid. The amount of CNP-38 released was determined by peak area compared to a CNP-38 reference solution with known content.
The biological potency of the released CNP-38 was determined by functional cGMP stimulation in HEK293 cells: a Hek293 cell line overexpressing NPR-B was developed as follows: the coding region for the NPR-B reference sequence was cloned into a lentiviral vector under a CMV promoter for constitutive receptor expression. The bicistronic element located on a puromycin resistant vector was used as a eukaryotic selectable marker. After transduction, the stably growing cell pool was subjected to qRT-PCR to confirm receptor mRNA expression compared to the parental Hek293 cells. Stimulation of the NPR-B receptor with CNP-38 results in intracellular production of the second messenger, cGMP, which can be detected using a commercially available cGMP assay.
DMEM/Glutamax/HEPES medium containing 10% FBS and 1% puromycin solution (370. mu.g/mL) at 37 ℃ and 5% CO2Cells were cultured routinely. For each assay, cells were suspended in DMEM + 2% BSA and incubated at 37 ℃ and 5% CO2Incubate for 3 hours. Prepared in stimulation buffer (DMEM + 2% BSA +0.1mM IBMX +-20) and added to the cells (additional 1:2 dilutions of the CNP-38 dilution series). At 37 ℃ and 5% CO2After 60 minutes of incubation, cells were lysed and cGMP levels were determined using a commercially available cGMP TR-FRET assay (Cisbio, cGMP kit, cat # 62GM2 PEB). Potency was determined by parallel line analysis using four parameter logarithmic curve fitting in an effective PLA software, in comparison to the CNP-38 reference standard.
RP-HPLC was used to determine the amount of degraded CNP-38 (deamidated and asparagine variants). Mobile phase a consisted of 0.05% aqueous TFA and mobile phase B consisted of acetonitrile containing 0.04% TFA. Using a Waters Acquity HSS T3C 18,1.8 μm, 2.1 × 100mm column. Setting the flow rate at 0.5mL/min, and detectingThe column operating temperature was set at 30 ℃ at a wavelength of 215 nm. Deamidation and asparagine variants were quantified relative to the peak area of unmodified CNP-38.
Example 1: synthesis of Compound (1)
Compound (1) was synthesized as described for conjugate 11i in WO 2017/118693.
Example 2: stability test of lyophilized preparation containing Compound (1)
As shown in table 1, eight different formulations (F1-F8) containing compound (1) were prepared, and 200 μ L of each formulation was filled in a vial and lyophilized. The water content of the lyophilized product was below 0.4% in all formulations as determined by Karl-Fischer titration. Each formulation contained the appropriate amount of Compound (1) to give a concentration of 3.6mg CNP-38/ml upon reconstitution with 200. mu.L water. The formulation was placed in an incubator set to maintain 25 ℃ or 40 ℃. After 3 months (T3M), the formulation was reconstituted with water and analyzed. The results show that compound (1) has a better purity profile for the trehalose-containing formulation than the mannitol-containing formulation. In addition, it was observed that no additives were required to prevent oxidation of methionine.
TABLE 1
TABLE 2
aLOQ＝0.1％
Example 3: stability testing of liquid and lyophilized formulations containing Compound (1)
The formulation (F9) of (1) was prepared, and 200 μ L of the formulation was filled in a vial and lyophilized. The lyophilized formulation contained the appropriate amount of compound (1) to give a concentration of 3.0mg CNP-38/ml upon reconstitution with 200 μ L of water. The lyophilized product and the liquid formulation used to prepare the lyophilized product were placed in an incubator set to maintain 40 ℃.
After 21 days (T21D), the lyophilized product was reconstituted with water and the formulation was analyzed. The results show that lyophilization protects (1) from degradation like methionine oxidation and isoaspartic acid formation.
TABLE 3
TABLE 4
aLOQ＝1.2％
Example 4: stability testing of formulations containing (1) and surfactant
Three different formulations (F10-F12) of (1) were prepared and 1080 μ L was filled into vials and lyophilized. Each formulation contained the appropriate amount of (1) to give a concentration of 3.5mg CNP-38/ml upon reconstitution with 1000. mu.L water. Some formulations contain polysorbate 20(PS 20) or polysorbate 80(PS 80) as a surfactant. The lyophilized formulation was placed in an incubator set to maintain 40 ℃/75% RH. After 1 month (T1M) and 3 months (T3M), the formulations were reconstituted with 1mL of water and analyzed.
Compared to F10, formulations F11 and F12 containing surfactants showed a slight increase in methionine oxidation under pressure conditions of (1) and a clear effect on the reconstitution time (no shaking) after addition of 1mL of water.
TABLE 5
TABLE 6
aLOQ＝0.1％bLOQ＝0.8-1.1％cWithout shaking
Example 5: long-term stability test of lyophilized formulations containing Compound (1)
A lyophilized formulation of compound (1) (F13) was prepared by lyophilizing 1080 μ L of compound (1) in a vial. The formulation contained a suitable amount of (1) to give a nominal concentration of 3.9mg CNP-38/vial and 3.6mg CNP-38/ml upon reconstitution with 1000. mu.L water. The formulations were placed in incubators set to maintain 5 deg.C, 25 deg.C/60% RH, 30 deg.C/65% RH and 40 deg.C/75% RH, respectively. After 3 months (T3M), 4 months (T4M), 6 months (T6M), 9 months (T9M), 12 months (T12M), 18 months (T18M) and 24 months (T24M), the formulations were reconstituted with 1000 μ L of water and analyzed.
The results show the high stability of compound (1) in lyophilized formulation F13, content, purity and bioefficacy as shown herein. Furthermore, the formulation shows good stability when tested by the relevant pharmacopoeia method. The lyophilized formulation was characterized by a white cake and no change in the appearance of the cake was detected regardless of storage. The residual moisture content after lyophilization was very low (0.03%) and only increased slightly during the study. There was no significant change in reconstitution time during storage. Visual inspection showed that throughout the study, the samples were essentially free of visible particles for all samples and a small amount of sub-visible particles, i.e. > 25 μm and 71 > 10 μm (determined by flow-through microscopy), were observed during 12 months of storage at 5 ℃ a slight change in colour from B9 to > B8, whereas during 6 months of storage at 25 ℃ and 30 ℃ or 3 months of storage at 40 ℃ no colour increase was observed (colour was assessed by spectrocolorimetry and the absolute value of the colour was determined according to european pharmacopoeia, 8 th edition, monograph 2.2. freeze-dried samples were stored for up to twelve months of turbidity (measured by turbidimeter) at 2-8 ℃, 25 ℃/60% r.h and 30 ℃/65% r.h.samples were only measured as a minimal increase in turbidity, no change in appearance was observed during storage at 5 ℃ for up to 12 months, with values less than 1NTU (nephelometric turbidity units). Values of less than 1NTU were also obtained during 6 months at 25 deg.C, 30 deg.C and 40 deg.C. The pH was not affected by storage. Finally, the osmolality of T0 was within the physiological range and no changes were detected during the study.
TABLE 7
TABLE 8
aLOQ＝0.1％,bLOQ＝1％,cDuplicate analysis
Example 6: stability testing of Low dose formulations containing Compound (1)
The lyophilized formulation of (1) (F14) was prepared by lyophilizing 1060 μ L of (1) in a vial. The formulation contained a large amount of (1) such that upon reconstitution, a nominal concentration of 0.80mg CNP-38/vial and 0.75mg CNP-38/ml was produced. The formulations were placed in an incubator set to maintain 5 deg.C, 25 deg.C/60% RH, 30 deg.C/65% RH and 40 deg.C/75% RH. After 1 month (T1M), 3 months (T3M), 6 months (T6M) and 12 months (T12M), the formulations were reconstituted with 1.0mL of water and analyzed.
The results show the high stability of compound (1) in lyophilized formulation F14, content, purity and bioefficacy as shown herein. Furthermore, the formulation shows good stability when tested by the relevant pharmacopoeia method. The lyophilized formulation was characterized by a white cake and no change in the appearance of the cake was detected regardless of storage. The residual moisture content after lyophilization was very low (< 0.31%) and only increased slightly during the study. The reconstitution time during storage did not change significantly. Visual inspection showed that at the available time points, the reconstituted solution was essentially free of visible particles (evaluated according to european pharmacopoeia 2.9.20 and USP <790 >). Furthermore, 0. gtoreq.25 μm and 5. gtoreq.10 μm (as determined by flow-through microscopy) were observed for all samples and small amounts of sub-visible particles, regardless of storage temperature and storage time. A slight increase in color from ═ WFI to < B9 was observed after 3 months of storage at 30 ℃ and 40 ℃ (determined according to european pharmacopoeia 2.2.2 using B standard (B-scale)). During storage for three months, the clarity was not affected and the reconstituted solution was found to be WFI (determined according to european pharmacopoeia 2.2.1). The pH was not affected by storage. Finally, the osmolality of T0 was within the physiological range and no change was detected within 3 months.
TABLE 9
Watch 10
aND, not detectedbLOQ＝0.10％cLOQ＝2.0％
Example 7: stability of CNP-38 at different pH values
A preformulation study was performed to evaluate the stability of CNP-38 at different pH values. CNP-38 (synthesized as described in WO 2017/118693) was incubated in a solution of about 1mg/mL in succinate buffer at pH 4.0, 4.5, 5.0, 5.5 and 6.0, 37 ℃. The amount of degraded CNP-38 (sum of deamidated and asparagine variants of CNP-38) was assessed by RP-HPLC after 7 days. The amount of degraded CNP-38 was 2.7% at pH 4.0, 1.6% at pH 4.5, 1.0% at pH 5.0, 2.9% at pH 5.5, and 3.3% at pH 6.0, indicating a preferred pH of 5.0 for CNP-38 formulations.
Example 8: stability testing of formulations comprising Compound (1) reconstituted with Water with antioxidants and/or preservatives
A lyophilized formulation of compound (1) (F13) was prepared by lyophilizing 1080 μ L of compound (1) in a vial. The formulation contained a suitable amount of (1) to give a nominal concentration of 3.9mg CNP-38/vial and 3.6mg CNP-38/ml upon reconstitution with 1000. mu.L water. The water contained antioxidants and/or preservatives (R1-R5, table 11). The vial containing the reconstituted formulation was placed in an incubator set to maintain 5 ℃. After 4 weeks (T4W), the samples were analyzed.
The results show that compound (1) is stable after reconstitution under test conditions and is not affected by the presence of antioxidants and/or preservatives (table 12). The pH of the reconstituted solution after reconstitution is not affected by antioxidants and/or preservatives.
TABLE 11
Methionine
M-cresol
Phenol and its preparation
R1
-
-
-
R2
10mM
0.3％(w/w)
-
R3
10mM
-
0.3％(w/w)
R4
-
0.3％(w/w)
-
R5
-
-
0.3％(w/w)
TABLE 12
aLOQ＝0.10％bLOQ＝1.0％
Abbreviations
BSA-bovine serum Albumin
cGMP-cyclic guanosine monophosphate
CI-confidence interval
DMEM-Dulbecco's Modified Eagle Medium
FBS-fetal bovine serum
HEPES-4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid
IBMX-3-isobutyl-1-methylxanthine
Isoasp-isoaspartic acid
LC-MS-liquid chromatography-mass spectrometry combination
Met (O) -methionine sulfoxide
PLA-parallel line analysis
PS 20-polysorbate 20
PS 80-Polysorbate 80
RH-relative humidity
RP-HPLC-reversed phase high performance liquid chromatography
SE-HPLC-size exclusion high performance liquid chromatography
TFA-trifluoroacetic acid
TR-FRET-time resolved fluorescence energy transfer
UPLC-ultra high performance liquid chromatography
Sequence listing
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<120> dry pharmaceutical formulations of CNP conjugates
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<222> (37)..(53)
<400> 6
Asp Leu Arg Val Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu
1 5 10 15
Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly
20 25 30
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Asn
35 40 45
Ser Gly Leu Gly Cys
50
<210> 7
<211> 36
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-53 deficient in amino acids 15-31
<220>
<221> disulfide
<222> (20)..(36)
<400> 7
Asp Leu Arg Val Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Gly Leu
1 5 10 15
Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser
20 25 30
Gly Leu Gly Cys
35
<210> 8
<211> 52
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-52
<220>
<221> disulfide
<222> (36)..(52)
<400> 8
Leu Arg Val Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln
1 5 10 15
Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu
20 25 30
Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser
35 40 45
Gly Leu Gly Cys
50
<210> 9
<211> 51
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-51
<220>
<221> disulfide
<222> (35)..(51)
<400> 9
Arg Val Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu
1 5 10 15
His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser
20 25 30
Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly
35 40 45
Leu Gly Cys
50
<210> 10
<211> 50
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-50
<220>
<221> disulfide
<222> (34)..(50)
<400> 10
Val Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu His
1 5 10 15
Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys
20 25 30
Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu
35 40 45
Gly Cys
50
<210> 11
<211> 49
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-49
<220>
<221> disulfide
<222> (33)..(49)
<400> 11
Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro
1 5 10 15
Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly
20 25 30
Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly
35 40 45
Cys
<210> 12
<211> 48
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-48
<220>
<221> disulfide
<222> (32)..(48)
<400> 12
Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn
1 5 10 15
Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys
20 25 30
Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40 45
<210> 13
<211> 47
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-47
<220>
<221> disulfide
<222> (31)..(47)
<400> 13
Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala
1 5 10 15
Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe
20 25 30
Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40 45
<210> 14
<211> 46
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-46
<220>
<221> disulfide
<222> (30)..(46)
<400> 14
Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg
1 5 10 15
Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly
20 25 30
Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40 45
<210> 15
<211> 45
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-45
<220>
<221> disulfide
<222> (29)..(45)
<400> 15
Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg Lys
1 5 10 15
Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu
20 25 30
Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40 45
<210> 16
<211> 44
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-44
<220>
<221> disulfide
<222> (28)..(44)
<400> 16
Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg Lys Tyr
1 5 10 15
Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys
20 25 30
Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40
<210> 17
<211> 35
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-44 from which amino acids 14-22 are deleted
<220>
<221> disulfide
<222> (19)..(35)
<400> 17
Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Gly Leu Ser
1 5 10 15
Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly
20 25 30
Leu Gly Cys
35
<210> 18
<211> 36
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-44 from which amino acids 15-22 are deleted
<220>
<221> disulfide
<222> (20)..(36)
<400> 18
Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg Gly Leu
1 5 10 15
Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser
20 25 30
Gly Leu Gly Cys
35
<210> 19
<211> 43
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-43
<220>
<221> disulfide
<222> (27)..(43)
<400> 19
Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg Lys Tyr Lys
1 5 10 15
Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu
20 25 30
Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40
<210> 20
<211> 42
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-42
<220>
<221> disulfide
<222> (26)..(42)
<400> 20
Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly
1 5 10 15
Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp
20 25 30
Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40
<210> 21
<211> 41
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-41
<220>
<221> disulfide
<222> (25)..(41)
<400> 21
Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala
1 5 10 15
Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg
20 25 30
Ile Gly Ser Met Ser Gly Leu Gly Cys
35 40
<210> 22
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-40
<220>
<221> disulfide
<222> (24)..(40)
<400> 22
Arg Leu Leu Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn
1 5 10 15
Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile
20 25 30
Gly Ser Met Ser Gly Leu Gly Cys
35 40
<210> 23
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-39
<220>
<221> disulfide
<222> (23)..(39)
<400> 23
Leu Leu Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys
1 5 10 15
Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
20 25 30
Ser Met Ser Gly Leu Gly Cys
35
<210> 24
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-38
<220>
<221> disulfide
<222> (22)..(38)
<400> 24
Leu Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 25
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-37
<220>
<221> disulfide
<222> (21)..(37)
<400> 25
Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 26
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-37 mit Q1pQ (pQ = pyroglutamate)
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> X = pyroglutamate
<220>
<221> disulfide
<222> (21)..(37)
<400> 26
Xaa Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 27
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> G-CNP-37
<220>
<221> disulfide
<222> (22)..(38)
<400> 27
Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 28
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> P-CNP-37
<220>
<221> disulfide
<222> (22)..(38)
<400> 28
Pro Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 29
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> M-CNP-37
<220>
<221> disulfide
<222> (22)..(38)
<400> 29
Met Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 30
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> PG-CNP-37
<220>
<221> disulfide
<222> (23)..(39)
<400> 30
Pro Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys
1 5 10 15
Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
20 25 30
Ser Met Ser Gly Leu Gly Cys
35
<210> 31
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> MG-CNP-37
<220>
<221> disulfide
<222> (23)..(39)
<400> 31
Met Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys
1 5 10 15
Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
20 25 30
Ser Met Ser Gly Leu Gly Cys
35
<210> 32
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-37 M32N
<220>
<221> disulfide
<222> (21)..(37)
<400> 32
Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Asn
20 25 30
Ser Gly Leu Gly Cys
35
<210> 33
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> G-CNP-37 M32N
<220>
<221> disulfide
<222> (22)..(38)
<400> 33
Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Asn Ser Gly Leu Gly Cys
35
<210> 34
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> G-CNP-37 K14Q
<220>
<221> disulfide
<222> (22)..(38)
<400> 34
Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Gln Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 35
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> G-CNP-37 K14P
<220>
<221> disulfide
<222> (22)..(38)
<400> 35
Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Pro Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 36
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> G-CNP-37K 14Q, amino acid 15 deletion
<220>
<221> disulfide
<222> (21)..(37)
<400> 36
Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Gln Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 37
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> G-CNP-37 K14Q, K15Q
<220>
<221> disulfide
<222> (22)..(38)
<400> 37
Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Gln Gln
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 38
<211> 36
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-36
<220>
<221> disulfide
<222> (20)..(36)
<400> 38
Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu
1 5 10 15
Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser
20 25 30
Gly Leu Gly Cys
35
<210> 39
<211> 35
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-35
<220>
<221> disulfide
<222> (19)..(35)
<400> 39
His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser
1 5 10 15
Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly
20 25 30
Leu Gly Cys
35
<210> 40
<211> 34
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-34
<220>
<221> disulfide
<222> (18)..(34)
<400> 40
Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys
1 5 10 15
Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu
20 25 30
Gly Cys
<210> 41
<211> 33
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-33
<220>
<221> disulfide
<222> (17)..(33)
<400> 41
Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly
1 5 10 15
Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly
20 25 30
Cys
<210> 42
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-32
<220>
<221> disulfide
<222> (16)..(32)
<400> 42
Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys
1 5 10 15
Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25 30
<210> 43
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-31
<220>
<221> disulfide
<222> (15)..(31)
<400> 43
Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe
1 5 10 15
Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25 30
<210> 44
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-30
<220>
<221> disulfide
<222> (14)..(30)
<400> 44
Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly
1 5 10 15
Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25 30
<210> 45
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-29
<220>
<221> disulfide
<222> (13)..(29)
<400> 45
Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu
1 5 10 15
Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 46
<211> 28
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-28
<220>
<221> disulfide
<222> (12)..(28)
<400> 46
Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys
1 5 10 15
Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 47
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> GHKSEVAHRF-CNP-28
<220>
<221> disulfide
<222> (22)..(38)
<400> 47
Gly His Lys Ser Glu Val Ala His Arg Phe Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 48
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-27
<220>
<221> disulfide
<222> (11)..(27)
<400> 48
Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu
1 5 10 15
Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 49
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-27 K4Q, K5Q
<220>
<221> disulfide
<222> (11)..(27)
<400> 49
Gly Ala Asn Gln Gln Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu
1 5 10 15
Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 50
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-27 K4R, K5R
<220>
<221> disulfide
<222> (11)..(27)
<400> 50
Gly Ala Asn Arg Arg Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu
1 5 10 15
Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 51
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-27 K4P, K5R
<220>
<221> disulfide
<222> (11)..(27)
<400> 51
Gly Ala Asn Pro Arg Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu
1 5 10 15
Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 52
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-27 K4S, K5S
<220>
<221> disulfide
<222> (11)..(27)
<400> 52
Gly Ala Asn Ser Ser Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu
1 5 10 15
Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 53
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> GAN-CNP-27 K4P, K5R
<220>
<221> disulfide
<222> (14)..(30)
<400> 53
Gly Ala Asn Gly Ala Asn Pro Arg Gly Leu Ser Arg Gly Cys Phe Gly
1 5 10 15
Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25 30
<210> 54
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-27 K4R, K5R, K9R
<220>
<221> disulfide
<222> (11)..(27)
<400> 54
Gly Ala Asn Arg Arg Gly Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu
1 5 10 15
Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 55
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-27 K4R, K5R, K9R, M22N
<220>
<221> disulfide
<222> (11)..(27)
<400> 55
Gly Ala Asn Arg Arg Gly Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu
1 5 10 15
Asp Arg Ile Gly Ser Asn Ser Gly Leu Gly Cys
20 25
<210> 56
<211> 28
<212> PRT
<213> Artificial sequence
<220>
<223> P-CNP-27 K4R, K5R, K9R
<220>
<221> disulfide
<222> (12)..(28)
<400> 56
Pro Gly Ala Asn Arg Arg Gly Leu Ser Arg Gly Cys Phe Gly Leu Lys
1 5 10 15
Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 57
<211> 28
<212> PRT
<213> Artificial sequence
<220>
<223> M-CNP-27 K4R, K5R, K9R
<220>
<221> disulfide
<222> (12)..(28)
<400> 57
Met Gly Ala Asn Arg Arg Gly Leu Ser Arg Gly Cys Phe Gly Leu Lys
1 5 10 15
Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 58
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> human serum albumin fragment-CNP-27
<220>
<221> disulfide
<222> (22)..(38)
<400> 58
Gly His Lys Ser Glu Val Ala His Arg Phe Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 59
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> human serum albumin fragment-CNP-27M 22N
<220>
<221> disulfide
<222> (22)..(38)
<400> 59
Gly His Lys Ser Glu Val Ala His Arg Phe Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Asn Ser Gly Leu Gly Cys
35
<210> 60
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> methionine-human serum albumin fragment-CNP-27
<220>
<221> disulfide
<222> (23)..(39)
<400> 60
Met Gly His Lys Ser Glu Val Ala His Arg Phe Lys Gly Ala Asn Lys
1 5 10 15
Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
20 25 30
Ser Met Ser Gly Leu Gly Cys
35
<210> 61
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> proline-human serum albumin fragment-CNP-27
<220>
<221> disulfide
<222> (23)..(39)
<400> 61
Pro Gly His Lys Ser Glu Val Ala His Arg Phe Lys Gly Ala Asn Lys
1 5 10 15
Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
20 25 30
Ser Met Ser Gly Leu Gly Cys
35
<210> 62
<211> 26
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-26
<220>
<221> disulfide
<222> (10)..(26)
<400> 62
Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp
1 5 10 15
Arg Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 63
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-25
<220>
<221> disulfide
<222> (9)..(25)
<400> 63
Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg
1 5 10 15
Ile Gly Ser Met Ser Gly Leu Gly Cys
20 25
<210> 64
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-24
<220>
<221> disulfide
<222> (8)..(24)
<400> 64
Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile
1 5 10 15
Gly Ser Met Ser Gly Leu Gly Cys
20
<210> 65
<211> 23
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-23
<220>
<221> disulfide
<222> (7)..(23)
<400> 65
Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
1 5 10 15
Ser Met Ser Gly Leu Gly Cys
20
<210> 66
<211> 23
<212> PRT
<213> Artificial sequence
<220>
<223> R-CNP-22
<220>
<221> disulfide
<222> (7)..(23)
<400> 66
Arg Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
1 5 10 15
Ser Met Ser Gly Leu Gly Cys
20
<210> 67
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> ER-CNP-22
<220>
<221> disulfide
<222> (8)..(24)
<400> 67
Glu Arg Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile
1 5 10 15
Gly Ser Met Ser Gly Leu Gly Cys
20
<210> 68
<211> 23
<212> PRT
<213> Artificial sequence
<220>
<223> R-CNP-22 K4R
<220>
<221> disulfide
<222> (7)..(23)
<400> 68
Arg Gly Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
1 5 10 15
Ser Met Ser Gly Leu Gly Cys
20
<210> 69
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> ER-CNP-22 4KR
<220>
<221> disulfide
<222> (8)..(24)
<400> 69
Glu Arg Gly Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile
1 5 10 15
Gly Ser Met Ser Gly Leu Gly Cys
20
<210> 70
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> RR-CNP-22
<220>
<221> disulfide
<222> (8)..(24)
<400> 70
Arg Arg Gly Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile
1 5 10 15
Gly Ser Met Ser Gly Leu Gly Cys
20
<210> 71
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> HRGP fragment-CNP-22
<220>
<221> disulfide
<222> (21)..(37)
<400> 71
Gly His His Ser His Glu Gln His Pro His Gly Ala Asn Gln Gln Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 72
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> HRGP fragment-CNP-22
<220>
<221> disulfide
<222> (22)..(38)
<400> 72
Gly Ala His His Pro His Glu His Asp Thr His Gly Ala Asn Gln Gln
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 73
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> HRGP fragment-CNP-22
<220>
<221> disulfide
<222> (21)..(37)
<400> 73
Gly His His Ser His Glu Gln His Pro His Gly Ala Asn Pro Arg Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 74
<211> 36
<212> PRT
<213> Artificial sequence
<220>
<223> IgG1(FC) fragment-CNP-22
<220>
<221> disulfide
<222> (20)..(36)
<400> 74
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gly Leu
1 5 10 15
Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser
20 25 30
Gly Leu Gly Cys
35
<210> 75
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> human serum albumin-CNP-22
<220>
<221> disulfide
<222> (23)..(39)
<400> 75
Gly Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Gly Ala Asn Pro
1 5 10 15
Arg Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
20 25 30
Ser Met Ser Gly Leu Gly Cys
35
<210> 76
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> human serum albumin-CNP-22
<220>
<221> disulfide
<222> (21)..(37)
<400> 76
Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 77
<211> 35
<212> PRT
<213> Artificial sequence
<220>
<223> human ossein NPR C inhibitor fragment-CNP-22
<220>
<221> disulfide
<222> (19)..(35)
<400> 77
Phe Gly Ile Pro Met Asp Arg Ile Gly Arg Asn Pro Arg Gly Leu Ser
1 5 10 15
Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly
20 25 30
Leu Gly Cys
35
<210> 78
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<223> FGF2 heparin-binding domain fragment-CNP-22
<220>
<221> disulfide
<222> (24)..(40)
<400> 78
Gly Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly
1 5 10 15
Pro Lys Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile
20 25 30
Gly Ser Met Ser Gly Leu Gly Cys
35 40
<210> 79
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> IgG1(FC) fragment-CNP-22K 4R
<220>
<221> disulfide
<222> (21)..(37)
<400> 79
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Gly Ala Asn Gln Gln Gly
1 5 10 15
Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 80
<211> 36
<212> PRT
<213> Artificial sequence
<220>
<223> human serum albumin fragment-CNP-22K 4R
<220>
<221> disulfide
<222> (20)..(36)
<400> 80
Gly Val Pro Gln Val Ser Thr Ser Thr Gly Ala Asn Gln Gln Gly Leu
1 5 10 15
Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser
20 25 30
Gly Leu Gly Cys
35
<210> 81
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> fibronectin fragment-CNP-22
<220>
<221> disulfide
<222> (21)..(37)
<400> 81
Gly Gln Pro Ser Ser Ser Ser Gln Ser Thr Gly Ala Asn Gln Gln Gly
1 5 10 15
Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 82
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> fibronectin fragment-CNP-22K 4R
<220>
<221> disulfide
<222> (21)..(37)
<400> 82
Gly Gln Thr His Ser Ser Gly Thr Gln Ser Gly Ala Asn Gln Gln Gly
1 5 10 15
Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 83
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> fibronectin fragment-CNP-22K 4R
<220>
<221> disulfide
<222> (21)..(37)
<400> 83
Gly Ser Thr Gly Gln Trp His Ser Glu Ser Gly Ala Asn Gln Gln Gly
1 5 10 15
Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 84
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> Zinc finger fragment-CNP-22K 4R
<220>
<221> disulfide
<222> (21)..(37)
<400> 84
Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Ala Asn Gln Gln Gly
1 5 10 15
Leu Ser Arg Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 85
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-21
<220>
<221> disulfide
<222> (5)..(21)
<400> 85
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
1 5 10 15
Ser Gly Leu Gly Cys
20
<210> 86
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-20
<220>
<221> disulfide
<222> (4)..(20)
<400> 86
Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser
1 5 10 15
Gly Leu Gly Cys
20
<210> 87
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-19
<220>
<221> disulfide
<222> (3)..(19)
<400> 87
Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly
1 5 10 15
Leu Gly Cys
<210> 88
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-18
<220>
<221> disulfide
<222> (2)..(18)
<400> 88
Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu
1 5 10 15
Gly Cys
<210> 89
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-17
<220>
<221> disulfide
<222> (1)..(17)
<400> 89
Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly
1 5 10 15
Cys
<210> 90
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> BNP fragment-CNP-17-BNP-fragment
<220>
<221> disulfide
<222> (10)..(26)
<400> 90
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Leu Lys Leu Asp
1 5 10 15
Arg Ile Gly Ser Met Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His
20 25 30
<210> 91
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-38 L1G
<220>
<221> disulfide
<222> (22)..(38)
<400> 91
Gly Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys
1 5 10 15
Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser
20 25 30
Met Ser Gly Leu Gly Cys
35
<210> 92
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> Ac-CNP-37
<220>
<221> MOD_RES
<222> (1)..(1)
<223> acetylation
<220>
<221> disulfide
<222> (21)..(37)
<400> 92
Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 93
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> CNP-37, Xaa = K or R, with the proviso that at least one Xaa is R
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa = Lys, Arg, Pro, Ser, or gin, with the proviso that at least one of amino acids 8, 10, 14, 15, 19, or 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa = Lys, Arg, Pro, Ser, or gin, with the proviso that at least one of amino acids 8, 10, 14, 15, 19, or 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> MISC_FEATURE
<222> (14)..(14)
<223> Xaa = Lys, Arg, Pro, Ser, or gin, with the proviso that at least one of amino acids 8, 10, 14, 15, 19, or 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> MISC_FEATURE
<222> (15)..(15)
<223> Xaa = Lys, Arg, Pro, Ser, or gin, with the proviso that at least one of amino acids 8, 10, 14, 15, 19, or 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> MISC_FEATURE
<222> (19)..(19)
<223> Xaa = Lys, Arg, Pro, Ser, or gin, with the proviso that at least one of amino acids 8, 10, 14, 15, 19, or 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> disulfide
<222> (21)..(37)
<220>
<221> MISC_FEATURE
<222> (25)..(25)
<223> Xaa = Lys, Arg, Pro, Ser, or gin, with the proviso that at least one of amino acids 8, 10, 14, 15, 19, or 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<400> 93
Gln Glu His Pro Asn Ala Arg Xaa Tyr Xaa Gly Ala Asn Xaa Xaa Gly
1 5 10 15
Leu Ser Xaa Gly Cys Phe Gly Leu Xaa Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 94
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> mutant CNP-37
<220>
<221> MISC_FEATURE
<222> (14)..(14)
<223> X is selected from the group consisting of: lys, Arg, Pro, Ser, and Gln, with the proviso that at least one of the amino acids at positions 14, 15, 19, and 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> MISC_FEATURE
<222> (15)..(15)
<223> X is selected from the group consisting of: lys, Arg, Pro, Ser, and Gln, with the proviso that at least one of the amino acids at positions 14, 15, 19, and 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> MISC_FEATURE
<222> (19)..(19)
<223> X is selected from the group consisting of: lys, Arg, Pro, Ser, and Gln, with the proviso that at least one of the amino acids at positions 14, 15, 19, and 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<220>
<221> disulfide
<222> (21)..(37)
<220>
<221> MISC_FEATURE
<222> (25)..(25)
<223> X is selected from the group consisting of: lys, Arg, Pro, Ser, and Gln, with the proviso that at least one of the amino acids at positions 14, 15, 19, and 25 is selected from the group consisting of: arg, Pro, Ser and Gln
<400> 94
Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Xaa Xaa Gly
1 5 10 15
Leu Ser Xaa Gly Cys Phe Gly Leu Xaa Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 95
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> mutant CNP-37
<220>
<221> MISC_FEATURE
<222> (14)..(15)
<223> Xaa selected from the group consisting of: lys Arg, Arg Lys, Lys Pro, Pro Lys, Ser Ser Ser, Arg Ser, Ser Arg, Gln Lys, Gln
Arg, Lys Gln, Arg Gln, Arg and Gln
<220>
<221> disulfide
<222> (21)..(37)
<400> 95
Gln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Xaa Xaa Gly
1 5 10 15
Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met
20 25 30
Ser Gly Leu Gly Cys
35
<210> 96
<211> 15
<212> PRT
<213> Intelligent people
<400> 96
Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly
1 5 10 15
<210> 97
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Artificial random crimp
<400> 97
Gly Gly Pro Gly Gly Pro Gly Pro Gly Gly Pro Gly Gly Pro Gly Pro
1 5 10 15
Gly Gly Pro Gly
20
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