Pharmaceutical preparation
09757469 · 2017-09-12
Assignee
Inventors
Cpc classification
A61P5/10
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
International classification
Abstract
Polyethylene glycol modified human chorionic gonadotropin (hCG), preparations thereof, compositions thereof, and methods of preparation and use thereof are described.
Claims
1. A polyethylene glycol modified hCG of the following formula:
CH.sub.2R—CHR—CH.sub.2—O—C(═O)—NH—(CH.sub.2).sub.2—CH.sub.2-hCG wherein each R is a polyethylene glycol (PEG) or a methoxypoly (ethylene glycol) (mPEG).
2. A polyethylene glycol modified hCG of the following formula: ##STR00007## wherein each R is a polyethylene glycol (PEG) or a methoxypolyethylene glycol (mPEG), and m is an integer in the range from 1 to 20.
3. A polyethylene glycol modified human chorionic gonadotrophin (hCG) according to the following formula:
(R).sub.n—X—Y (Ia) wherein: X is a linking group comprising a moiety according to the following formula:
(Z.sup.1CH.sub.2).sub.b—(CH.sub.2).sub.z—(CHZ.sup.2).sub.a—(CH.sub.2).sub.u—O—C(═O)—NH—(CH.sub.2).sub.t—CH.sub.2— a and b are each independently selected from 0 and 1 provided that a and b are not both 0; t, u and z are each independently 0 or an integer in the range from 1 to 10, and Z.sup.1 and Z.sup.2 each represent a bond to an R group, or Z.sup.1 and Z.sup.2 are each independently selected from groups of the formula:
(Z.sup.3CH.sub.2).sub.d—(CH.sub.2).sub.e—(CHZ.sup.4).sub.c—(CH.sub.2).sub.f—O(CH.sub.2CH.sub.2O).sub.m— Z.sup.3 and Z.sup.4 each represent a bond to an R group; c and d are each independently selected from 0 and 1; e and f are each independently 0 or an integer in the range from 1 to 10, and m is an integer in the range from 1 to 20; each R is a polyethylene glycol (PEG) or a methoxypolyethylene glycol (mPEG) group; n is 2, 3 or 4; and Y is human chorionic gonadotrophin (hCG).
4. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is recombinant hCG.
5. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a human cell line-derived recombinant hCG.
6. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG (rhCG) including α2,3- and α2,6-sialylation.
7. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG having a sialic acid content of 15 moles of sialic acid or greater per mole of hCG protein.
8. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG comprising sialylation, wherein 10% or more of the total sialylation is α2,3-sialylation.
9. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG comprising sialylation, wherein 45% to 80% of the total sialylation is α2,3-sialylation.
10. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG comprising sialylation, wherein 20% to 55% of the total sialylation is α2,6-sialylation.
11. A polyethylene glycol modified hCG according to claim 3, wherein a polyethylene glycol or methoxypolyethylene glycol is conjugated to an amino acid residue of the hCG.
12. A polyethylene glycol modified hCG according to claim 3, wherein a polyethylene glycol or methoxypolyethylene glycol is conjugated to the N-terminus or the C-terminus of the hCG.
13. A polyethylene glycol modified hCG according to claim 3, wherein the polyethylene glycol or methoxypolyethylene glycol is conjugated to the N-terminus of the hCG.
14. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG having a sialic acid content in the range from 15 to 25 moles of sialic acid per mole of hCG protein.
15. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG comprising sialylation, wherein 50% or less of the total sialylation is α2,6-sialylation.
16. A polyethylene glycol modified hCG according to claim 3, wherein the hCG is a recombinant hCG comprising sialylation, wherein 10% or more of the total sialylation is α2,3-sialylation and 50% or less of the total sialylation is α2,6-sialylation.
17. A polyethylene glycol modified hCG according to claim 3, wherein a, b and u are 1, t is 0 and z is 0.
18. A polyethylene glycol modified hCG according to claim 3, wherein a, b and u are 1, t is 2 and z is 0.
19. A polyethylene glycol modified hCG according to claim 3, wherein Z.sup.1 and Z.sup.2 are each groups of the formula (Z.sup.3CH.sub.2).sub.d—(CH.sub.2).sub.e—(CHZ.sup.4).sub.c—(CH.sub.2).sub.f—O(CH.sub.2CH.sub.2O).sub.m—, c, d and f are 1 and e is 0.
20. A polyethylene glycol modified hCG according to claim 3, comprising hCG modified with a 2-arm or a 4-arm branched methoxypolyethylene glycol moiety, wherein: the 2-arm branched methoxypolyethylene glycol moiety is of the following formula: ##STR00008## the 4-arm branched methoxypolyethylene glycol moiety is of the following formula: ##STR00009## wherein: each m is an integer and (CH.sub.2CH.sub.2O).sub.m represents repeating units of a polyethylene glycol polymer; each n is an integer wherein (CH.sub.2CH.sub.2O).sub.n represents repeating units of a polyethylene glycol polymer.
21. A polyethylene glycol modified hCG according to claim 20, comprising hCG modified with the 2-arm branched methoxypolyethylene glycol moiety.
22. A polyethylene glycol modified hCG according to claim 21, wherein the modified hCG is of the following formula ##STR00010##
23. A polyethylene glycol modified hCG according to claim 20, comprising hCG modified with the 4-arm branched methoxypolyethylene glycol moiety.
24. A polyethylene glycol modified hCG according to claim 23, wherein the modified hCG is of the following formula ##STR00011##
25. A pharmaceutical composition comprising a polyethylene glycol modified hCG according to claim 3.
26. A pharmaceutical composition according to claim 25, further comprising follicle stimulating hormone (FSH) or luteinizing hormone (LH), or both FSH and LH.
27. A method of treatment of infertility comprising a step of administering to a subject a composition comprising a polyethylene glycol modified hCG according to claim 3.
Description
DETAILED DESCRIPTION OF THE INVENTION
(1) The present invention will now be described in more detail with reference to the following Examples and to the attached drawings in which:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
SEQUENCE SELECTION
(10) Human hCG
(11) The coding region of the gene for the hCG alpha polypeptide was used according to Fiddes and Goodman (1979). The sequence is banked as AH007338 and at the time of construction there were no other variants of this protein sequence. The sequence is referred to herein as SEQ ID NO:1.
(12) The coding region of the gene for hCG beta polypeptide was used according to Fiddes and Goodman (1980). The sequence is banked as NP_000728 and is consistent with the protein sequences of CGbeta3, CGbeta5 and CGbeta7. The sequence is referred herein as SEQ ID NO: 3
(13) Sialyltransferase
(14) α2,3-Sialyltransferase—The coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (α2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred herein as SEQ ID NO: 5.
(15) α2,6-Sialyltransferase—The coding region of the gene for beta-galactosamide alpha-2,6-sialyltransferase 1 (α2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (1990). The sequence is banked as NM_003032 and referred herein as SEQ ID NO: 7.
(16) Plasmids
(17)
EXAMPLES
Example 1
Construction of the hCG Expression Vector
(18) The coding sequence of hCG alpha polypeptide (AH007338, SEQ ID NO: 1) and hCG beta polypeptide (NP_000728, SEQ ID NO: 3) were amplified by PCR using the primer combinations CGa-fw and CGa-rev and CGb-fw and CGb-rec respectively.
(19) TABLE-US-00001 (SEQ ID NO: 9) CGa-fw 5′-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3′ (SEQ ID NO: 10) CGa-rev 5′-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3′ (SEQ ID NO: 11) CGb-fw 5′-CCAGGCGCGCCACCATGGAGATGTTCCAGGGGCTGC-3′ (SEQ ID NO: 12) CGb-rev 5′-CCGGGTTAACTTATTGTGGGAGGATCGGGG-3′
(20) The resulting amplified hCG beta DNA was digested with the restriction enzymes AscI and HpaI and inserted into the AscI and HpaI sites on the CMV driven mammalian expression vector carrying a neomycin selection marker. Similarly the hCG alpha DNA was digested with BamHI and NheI and inserted into the sites BamHI and NheI on the expression vector already containing the hCG beta polypeptide DNA.
(21) The vector DNA was used to transform the DH5α strain of E. coli. Colonies were picked for amplification and, of the number which included the vector containing both hCG alpha and beta, twenty were selected for sequencing. All colonies selected for sequencing contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 3. Plasmid phCG A+B was selected for transfection (
Example 2
Construction of the ST3 Expression Vector
(22) The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4 (ST3, L23767, SEQ ID NO: 5) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.
(23) TABLE-US-00002 (SEQ ID NO: 13) 2,3STfw 5′-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3′ (SEQ ID NO: 14) 2,3STrev 5′-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3′
(24) The resulting amplified ST3 DNA was digested with the restriction enzymes BamHI and AflII and inserted into the BamHI and AflII sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST3#1 (
Example 3
Construction of the ST6 Expression Vector
(25) The coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6, NM_003032, SEQ ID NO: 7) was amplified by PCR using the primer combination 2,6STfw and 2,6STrev.
(26) TABLE-US-00003 (SEQ ID NO: 15) 2,6STfw 5′-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3′ (SEQ ID NO: 16) 2,6STrev 5′-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3′
(27) The resulting amplified ST6 DNA was digested with the restriction enzymes BamHI and AflII and inserted into the BamHI and AflII sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST6#11 (
Example 4
Stable Expression of phCG A+B in PER.C6® Cells. Transfection Isolation and Screening of Clones
(28) PER.C6® clones producing hCG were generated by expressing both polypeptide chains of hCG from a single plasmid (see Example 1).
(29) To obtain stable clones a liposome based transfection agent was used with the phCG A+B construct. Stable clones were selected in PER.C6® selection media supplemented with 10% FCS and containing G418. Three weeks after transfection G418 resistant clones grew out. A total of 389 clones were selected for isolation. The isolated clones were cultured in selection medium until 70-80% confluent. Supernatants were assayed for hCG protein content using an hCG selective ELISA and pharmacological activity at the hCG receptor in cloned cell line, using a cAMP accumulation assay. Clones (118) expressing functional protein were progressed for culture expansion to 24 well, 6 well and T80 flasks.
(30) Studies to determine productivity and quality of the material from 47 clones were initiated in T80 flasks to generate sufficient material. Cells were cultured in supplemented media as previously described for 7 days and the supernatant harvested. Productivity was determined using the hCG selective ELISA. The isoelectric profile of the material was determined (using the method described in Example 6). The information from the IEF was used to select clones for metabolic clearance rate analysis. Clones with sufficient productivity and quality were selected for sialyltransferase engineering.
Example 5a
Level of Sialylation is Increased in Cells that over Express α2,3-sialyltransferase. Stable Expression of pST3 in hCG Expressing PER.C6® Cells; Transfection Isolation and Screening of Clones
(31) PER.C6® clones producing highly sialylated hCG were generated by expressing α2,3 sialyltransferase from separate plasmids (see Example 2) in PER.C6® cells already expressing both polypeptide chains of hCG (see Example 4). Clones produced from PER.C6® cells as set out in Example 4 were selected for their characteristics including productivity, good growth profile, production of functional protein, and produced hCG which included some sialylation.
(32) Stable clones were generated as previously described in Example 4. Clones from the α2,3-sialyltransferase program were isolated, expanded and assayed. The final clone number for the α2,3-study was five. The α2,3-sialyltransferase clones were adapted to serum free media and suspension conditions.
(33) As before clones were assayed using a hCG selective ELISA, functional response in an hCG receptor cell line, IEF (Example 6). They were also assessed for metabolic clearance rate (Example 9) and USP hCG Bioassay (Example 10). Results were compared to a commercially available recombinant hCG (OVITRELLE®, Serono) and the parental hCG PER.C6® cell lines. Representative samples are shown in the Examples and Figures.
(34) In conclusion expression of hCG together with α2,3-sialyltransferase in PER.C6® cells results in increased levels of sialylated hCG compared to cells expressing hCG only.
Example 5b
Stable Expression of pST3 in hCG Expressing PER.C6® Cells—a Different Method
(35) The alpha beta heterodimer produced above (Example 4) had a low level of sialylation resulting in a very basic IEF profile. As indicated above (Example 5a) expression of hCG together with α2,3-sialyltransferase in PER.C6® cells results in increased levels of sialylated hCG compared to cells expressing hCG only.
(36) A double transfection of the hCG alpha and beta subunit genes together with the α2,3 sialyltransferase enzyme gene into PER.C6® cells in suspension cell culture format was performed. Cell lines were generated by co-transfecting the hCG vector (dual alpha/beta, Example 1) and the vector encoding α2,3-sialyltransferase (Example 2) under serum free conditions. Clones produced from PER.C6® cells were selected for their characteristics including productivity, good growth profile, production of functional protein, and produced hCG which included some sialylation. Clones were isolated, expanded and assayed.
(37) As before clones were assayed using a hCG selective ELISA, functional response in an hCG receptor cell line, IEF (Example 6). They were also assessed for metabolic clearance rate (Example 9) and USP hCG Bioassay (Example 10). Results were compared to a commercially available recombinant hCG (OVITRELLE®, Serono) and the parental hCG PER.C6® cell lines. Representative samples are shown in the Examples and Figures (see Examples 6, 9, 10,
(38)
Example 6
Analysis of the Isoelectric Point pI of PER.C6® Produced hCG Isoforms by Isoelectric Focussing
(39) Electrophoresis is defined as the transport of charged molecules through a solvent by an electrical field. The mobility of a biological molecule through an electric field will depend on the field strength, net charge on the molecule, size and shape of the molecule, ionic strength and properties of the medium through which the molecules migrate.
(40) Isoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their pI. The pI is the pH at which a protein has no net charge and will not migrate in an electric field. The sialic acid content of the hCG isoforms subtly alters the pI point for each isoform, which can be exploited using this technique to visualise the PER.C6® hCG isoforms from each clone.
(41) The isoelectric points of the PER.C6® produced hCG isoforms in cell culture supernatants were analyzed using isoelectric focussing. Cell culture media from PER.C6® hCG clones were produced as described in Example 4, 5a and 5b.
(42) PER.C6® hCG samples were separated on NOVEX® IEF Gels containing 5% polyacrylamide under native conditions on a pH 3.0-7.0 gradient in an ampholyte solution pH 3.0-7.0. Proteins were visualised using Coomassie Blue staining, using methods well known in the art.
(43)
Example 7
Analysis of the Sialic Acid Linkages of PER.C6® hCG
(44) Glycoconjugates were analyzed using a lectin based glycan differentiation method. With this method glycoproteins and glycoconjuagates bound to nitrocellulose can be characterized. Lectins selectively recognize a particular moiety, for example α2,3 linked sialic acid. The lectins applied are conjugated with the steroid hapten digoxigenin which enables immunological detection of the bound lectins.
(45) Purified PER.C6® hCG from a parental clone (no additional sialyltransferase), and a α2,3-sialyltransferase engineered clone were separated using standard SDS-PAGE techniques. A commercially available recombinant hCG (OVITRELLE®, Serono) was used as a standard.
(46) Sialic acid was analyzed using the DIG Glycan Differentiation Kit (Cat. No. 11 210 238 001, Roche) according to the manufacturer's instructions. Positive reactions with Sambucus nigra agglutinin (SNA) indicated terminally linked (2-6) sialic acid. Positive reactions with Maackia amurensis agglutinin II (MAA): indicated terminally linked (α2-3) sialic acid.
(47) In summary the parental clone contained low levels of both α2,3- and α2,6-sialic acid. The clones engineered with α2,3-sialyltransferase contained high levels of α2,3-sialic acid linkages and low levels of α2,6-sialic acid linkages. The standard control Ovitrelle only contains α2,3-sialic acid linkages. This is consistent with what is known about recombinant proteins produced in Chinese Hamster ovary (CHO) cells (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990).
(48) In conclusion, engineering of PER.C6® hCG cells with α2,3-sialyltransferase successfully increased the number of sialic acid molecules conjugated to the recombinant hCG in the sample.
Examples 8A and 8B
Quantification of Total Sialic Acid
(49) Sialic acid is a protein-bound carbohydrate considered to be a mono-saccharide and occurs in combination with other mono-saccharides like galactose, mannose, glucosamine, galactosamine and fucose. The total sialic acid on purified rhCG according to the invention was measured using a method based on the method of Stanton et. al. (J. Biochem. Biophys. Methods. 30 (1995), 37-48).
Example 8A
(50) The total sialic acid content of PER.C6® recombinant hCG modified with α2,3-sialyltransferase (e.g. Example 5a, Example 5b) was measured and found to be greater than 15 mol/mol, [expressed in terms of a ratio of moles of sialic acid to moles of protein], for example greater than 18 mol/mol, for example 19.1 mol/mol. This can be compared to OVITRELLE® which has total sialic acid content of 17.6 mol/mol.
Example 8B
(51) The total sialic acid content of PER.C6® recombinant hCG modified with α2,3-sialyltransferase 080019-19 (prepared by the methods of Example 5b above) was measured and found to be 20 mol/mol, [expressed in terms of a ratio of moles of sialic acid to moles of protein]. Again, this may be favourably compared with OVITRELLE® which has total sialic acid content of 17.6 mol/mol. This Example (080019-19) was tested to quantify the relative amounts of α2,3 and α2,6 sialic acid (Example 8C).
Example 8C
Quantification of Relative Amounts of α2,3 and α2,6 Sialic Acid
(52) The relative percentage amounts of α2,3 and α2,6 sialic acid on purified rhCG [Example (080019-19), and two other Examples prepared by the methods of Example 5] were measured using known techniques —HPLC with Normal-phase (NP).
(53) To quantify the alpha 2,3 and 2,6 sialic acid in O-link glycans the following analysis was performed. The O-linked glycans were cleaved from the hCG sample using an Orela Glycan Release Kit and separated on NP-HPLC. Samples of the extracted, pooled, glycans (extracted as above) were digested with different sialidases to determine the linkages. This Enzymatic degradation of glycans was performed using alpha 2-3,6,8 sialidase and alpha 2-3, sialidase. The enzymatic digested glycans were then re-separated on the NP column, and the O-Glycans were identified on the NP-HPLC using prepared standards. The relative percentages were calculated and are shown in the following table (SA=Sialic Acid).
(54) TABLE-US-00004 % SA 09PD84-006-3 09PD-84-04 080019-19 Structure 63 63 59 α 2, 3 SA 37 37 41 α 2, 6 SA
(55) The relative percentages were found to be in the ranges 55%-65% (e.g. 59%) for α2,3 sialylation; and 35 to 45% (e.g. 41%) for α2,6 sialylation.
Example 8D
Quantification of Relative Amounts Mono, Di, Tri and Tetra Antennary Sialylated Structures
(56) The relative percentage amounts of mono, di, tri and tetra sialylated structures on glycans extracted from purified rhCG (the three samples used in Example 8C) were measured using known techniques.
(57) Each sample of rhCG was immobilized (gel block), washed, reduced, alkylated and digested with PNGase F overnight. The N-glycans were then extracted and processed. N-glycans for NP-HPLC and WAX-HPLC analysis were labelled with the fluorophore 2AB as detailed in Royle et al.
(58) Weak anion exchange (WAX) HPLC to separate the N-glycans by charge (Example 8C) was carried out as set out in Royle et al, with a Fetuin N-glycan standard as reference. Glycans were eluted according to the number of sialic acids they contained. All samples included mono (1S), di(2S), tri(3S) and tetra(4S) sialylated structures. The relative amounts of sialylated structures were found to be in the following ratios (1S:2S:4S:4S): 0.1-4%:35-45%:0.5-8%:0-1%.
(59) A preferred example, 080019-19, included mono (1S), di(2S), tri(3S) and tetra(4S) sialylated structures. The relative amounts of sialylated structures were in the following ratios (1S:2S:4S:4S): 0.1-4%:35-45%:0.5-8%:0-1%.
Example 9
Determination of the Metabolic Clearance Rates of rhCG
(60) To determine the metabolic clearance rate (MCR) of PER.C6® hCG samples engineered using α2,3-sialyltransferase (e.g. Example 5a, 5b), conscious female rats (3 animals per clone) were injected into the tail vein at time zero with a bolus of rhCG (1-10 μg/rat, based on ELISA quantification of samples, DRG EIA 1288). Blood samples (400 μl) were taken from the tip of the tail at 1, 2, 4, 8, 12, 24 and 32 hours after test sample injection. Serum was collected by centrifugation and assayed for hCG content by ELISA (DRG EIA 1288). The MCR of PER.C6® hCG samples engineered using α2,3-sialyltransferase showed that the half life was similar to the standard (
(61)
(62)
Example 10
hCG Bioassay According to USP
(63) A hCG Bioassay was carried out, to assay the hCG specific activity. The activity was measured according to USP (USP Monographs: Chorionic Gonadotropin, USPC Official Aug. 1, 2009-Nov. 30, 2009), using OVITRELLE® as a standard. OVITRELLE® has a biological activity of 26,000 IU/mg (Curr Med Res Opin. 2005 December; 21(12): 1969-76). The acceptance limit was >21,000 IU hCG/mg. The biological activity for a sample of human cell line derived hCG recombinant hCG engineered with α2,3-sialyltransferase (having sialic acid content 19.1 mol/mol—see Example 8) was 27,477 IU hCG/mg.
Example 11
Production and Purification Overview
(64) A procedure was developed to produce recombinant hCG in PER.C6® cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several hCG-producing PER.C6® cell lines.
(65) Recombinant hCG from an α2,3- clone was purified using a using a modification of the method described by Lowry et al. (1976).
(66) For the production of PER.C6®-cell produced hCG, the cell lines were adapted to a serum-free medium, i.e., EX-CELL® 525 (JRH Biosciences). The cells were first cultured to form a 70%-90% confluent monolayer in a T80 culture flask. On passage the cells were re-suspended in the serum free medium, EX-CELL® 525+4 mM L-Glutamine, to a cell density of 0.3×10.sup.6 cells/ml. A 25 ml cell suspension was put in a 250 ml shaker flask and shaken at 100 rpm at 37° C. at 5% CO.sub.2. After reaching a cell density of >I×10.sup.6 cells/ml, the cells were sub-cultured to a cell density of 0.2 or 0.3×10.sup.6 cells/ml and further cultured in shaker flasks at 37° C., 5% CO.sub.2 and 100 rpm.
(67) For the production of hCG, the cells were transferred to a serum-free production medium, i.e., VPRO (JRH Biosciences), which supports the growth of PER.C6® cells to very high cell densities (usually >10.sup.7 cells/ml in a batch culture). The cells were first cultured to >1×10.sup.6 cells/ml in EX-CELL® 525, then spun down for 5 min at 1000 rpm and subsequently suspended in VPRO medium+6 mM L-glutamine to a density of 1×10.sup.6 cells/ml. The cells were then cultured in a shaker flask for 7-10 days at 37° C., 5% CO2 and 100 rpm. During this period, the cells grew to a density of >10.sup.7 cells/ml. The culture medium was harvested after the cell viability started to decline. The cells were spun down for 5 min at 1000 rpm and the supernatant was used for the quantification and purification of hCG. The concentration of hCG was determined using ELISA (DRG EIA 1288).
(68) Thereafter, purification of hCG was carried out using a modification of the method described by Lowry et al. (1976). This was achieved by chromatography on DEAE cellulose, gel filtration on SEPHADEX® G100, adsorption chromatography on hydroxyapatite, and preparative polyacrylamide electrophoresis.
(69) During all chromatographic procedures, the presence of immunoreactive recombinant hCG was confirmed by RIA (DRG EIA 1288) and IEF (Example 6).
Example 12
PEGylation
(70) In an example of the present invention, poly (ethylene glycol) is (e.g. covalently) bound through amino acid residues of hCG (or agonist variant thereof). Preferably the poly (ethylene glycol) is bound to the N-terminus of the hCG. A number of activated poly (ethylene glycol) s having a number of different functional groups, linkers, configurations, and molecular weights are known to one skilled in the art. These may be used to synthesise PEG-hCG conjugates or PEG-hCG agonist variant conjugates, by methods which are known in the art (see e.g. Roberts M. J. et al., Adv. Drug Del. Rev. 54: 459-476, 2002), Harris J. M. et al., Drug Delivery Sytems 40: 538-551, 2001).
(71) A substantially homogeneous preparation of N-terminally pegylated hGG was synthesised using a methoxy-PEG-Hydrazine (20 kD). Methoxy-PEG-Hydrazine (20 kD) are widely available and their uses are well known in the art. A transamination reaction was used to remove the amine group at the N-terminus of the human derived recombinant hCG, to leave an aldehyde in this position. A chemical reaction between the aldehyde group and the methoxy-PEG-Hydrazine (20 kD) leads to PEGylation of the hCG at the N-terminus. The PEGylated hCG product was purified from the reaction mixture to >95% (SEC analysis) using a single ion exchange chromatography step, by methods known in the art.
(72) 1 mg/mL of purified rhCG (produced in house by the method of Example 5b and purified according to the method of Example 11) in 20 mM ammonium acetate, 150 mM NaCl (pH 8) was concentrated to 3 mg/mL, and conditioned to 50 mM Sodium acetate, 150 mM NaCl pH 5.5 buffer by 10KD-ultracentrifugal device (VIVASPIN® 20) at 4,000 rpm, 8° C.
(73) Transamination: The concentrated hCG was incubated at room-temperature for 4 hours in a solution containing 2M-sodium acetate, 0.4M acetic acid, 0.1M sodium glyoxylate and 5 mM CuSO.sub.4 (ph 5.5). The reaction was then stopped by adding EDTA to a final concentration of 20 mM. A 10KD-ultracentrifugal device was used to remove the undesirable transamination-components and the buffer exchanged to 50 mM sodium phosphate, 150 mM NaCl (pH 7.5).
(74) PEGylation: An m-PEG-Hydrazine stock solution of 10 mM was prepared by dissolving lyophilized powder (NOF) in 1 mM HCl. A sodium cyanoborohydride stock solution of 200 mM was prepared by dissolving lyophilized powder (Fluka) in water.
(75) An 11-fold molar quantity of m-PEG-Hydrazine from the 10 mM stock solution was added to a vial containing transaminated-rhCG (3 mg/mL) while stirring. Immediately afterwards 75-folds molar of sodium cyanoborohydride was added from the stock solution, as a reducing agent. The reaction mixture was stirred at room-temperature for 24 hours and the extent of hCG modification by PEG (PEGylation) was monitored by size-exclusion chromatography (SEC) HPLC column; SUPERDEX®-75 (GE healthcare). After 24 hours, the reaction was stopped and diluted 1:1 to 1 mg/mL with 400 mM glycine, 50 mM sodium phosphate, 150 mM NaCl pH 6.7 buffer and final pH adjustment was carried-out with 1M HCl. The reaction mixture was 0.2 μm filtered, divided to aliquots and stored at 4° C. The SEC HPLC analysis is shown in
(76) It can be seen from the HPLC analysis (
Example 12A
PEGylation Using 2-Branched m-PEG-Aldehyde (20 Kd)
(77) The functional group of m-PEG-Aldehyde mainly interacts with the N-terminal at pH 5.5. Experiments carried out with a linear m-PEG-Aldehyde yield PEGylated-hCG with about 6 to 7 PEG-strands rather than the expected 2-strands. This implies that m-PEG-Aldehyde interacts with other amino-acids, such as Histidine (hCG has 4 Histidine residues).
(78) The present inventors found that the use of a 2-Branched m-PEG-Aldehyde reduced or prevented unwanted PEGylations (steric hindrance suppresses access to sites other than the N-terminus) and increased the yield of a product with only N-terminal PEGylation. The procedure that was carried-out is shown in Scheme 1 and is as follows.
(79) ##STR00003##
[In schemes 1, 2 and 3 herein CH3-(CH.sub.2CH.sub.2O).sub.n is the common nomenclature representing mPEG. The integers n and m in schemes 1, 2 and 3 do not have the same meaning as those integers in the present claims.
(80) 1 mg/mL of purified rhCG (produced in house by the method of Example 5b and purified according to the method of Example 11) in 20 mM ammonium acetate, 150 mM NaCl pH 8 was concentrated to 3 mg/mL and conditioned to 50 mM ammonium acetate, 150 mM NaCl (pH 5.5) buffer by 10KD-ultracentrifugal device (VIVASPIN® 20) at 4,000 rpm, 8° C.
(81) A 2-Branched m-PEG-Aldehyde (40 KD) stock solution of 10 mM was prepared by dissolving lyophilized powder (NOF) in 1 mM HCL. A Sodium Cyanoborohydride stock solution of 200 mM was prepared by dissolving lyophilized powder (Fluke) in water.
(82) A 10-fold molar quantity of the 2-Branched m-PEG-Aldehyde from the 10 mM stock solution was added to a vial containing rhCG (3 mg/mL), and stirred. Immediately afterwards a 75-folds molar quantity of sodium cyanoborohydride, as reducing agent, was added from the stock solution. The reaction mixture was stirred at room-temperature for 24 hours and the extent of modification of hCG by the PEG (PEGylation) was monitored by size-exclusion chromatography (SEC) HPLC column; SUPERDEX®-200 (GE healthcare). After 24 hours the reaction was stopped and diluted 1:1 to 1 mg/mL with 400 mM glycine, 50 mM sodium phosphate, 150 mM NaCl (pH 6.7 buffer). The final pH adjustment was carried-out with 1M HCl. The reaction mixture was 0.2 μm filtered, divided to aliquots and stored at 4° C.
(83) The SEC (Superdex SUPERDEX®-200 10/300 mm GL) HPLC Analysis is shown in
(84) It will be appreciated that the use of a higher branched m-PEG reagent (for example a 4-Branched m-PEG-Aldehyde such as that represented below) is more likely to produce a single population of PEG-hCG with lower PEG-strands (Peak no.2 on
(85) ##STR00004##
A suitable scheme for preparing a 4-arm branched mPEG Aldehyde by a similar method to that shown in scheme 1 above is shown in scheme 2
(86) ##STR00005##
Example 12B
(87) The rhCG (produced in house by the method of Example 5b and purified according to the method of Example 11) was modified with a linear m-PEG-Aldehyde (10 KD), using the method of Example 12A. The procedure that was carried-out is shown in Scheme 3 and is as follows:
(88) ##STR00006##
The SEC-HPLC analysis of the product linear-PEGylated hCG is presented in
Example 13
Activated-PEG hCG Bioassay
(89) A recombinant hCG was produced by the method of Example 5b and purified according to the method of Example 11. 21 rats were allocated into 3 groups (7 rats per group), and each rat was injected three times, on three separate days (each injection at different day), with one of the following doses of the recombinant hCG (1 group=1 dose): 4.3 ng (rats in Group A), 8.6 ng (rats in Group B); and 17.1 ng (rats in Group C). After 5 days the rats were sacrificed and the uterus weighed for potency determination, according to a known and routine Bioassay.
(90) A rat which has been injected with 3 daily doses totalling 4.3 ng of the recombinant hCG has average Uterus weight of 30-40 mg after 5 days. A rat which has been injected with 3 daily doses totalling 8.6 ng rhCG has average Uterus weight of 60-70 mg after 5 days. A rat which has been injected with 3 daily doses of 17.1 ng rhCG has average Uterus weight of 110-120 mg after 5 days (see Table below).
(91) The inventors tested the products of the invention for potential to provide a long acting formulation which would allow a single weekly dose.
(92) The present inventors used the same Bioassay to measure the uterus weight after 5 days of rats treated with:
(93) (i) a single injection of Recombinant hCG produced in house by the method of Example 5b and purified according to the method of Example 11, which was administered at ten times the concentration of the highest of the three daily injection doses, that is at ten times the concentration (170 ng);
(94) (ii) a single injection of Branched-PEGylated recombinant hCG of the invention produced by the method of Example 12A which was administered at ten times the concentration of the highest of the three daily injection doses, that is at ten times the concentration (170 ng); and
(95) (iii) a single injection of Linear-PEGylated recombinant hCG of the invention produced by the method of Example 12 which was administered at ten times the concentration of the highest of the three daily injection doses, that is at ten times the concentration (170 ng).
(96) The results are shown in the following Table.
(97) It can be seen that the branched PEG-rhCG Bioassay example (Column 5 of the Table) demonstrates that a single injection of the branched PEGylated rhCG of the invention provides an average uterus weight (mg) of 134.11, roughly equivalent to 3 single injections of the highest dose of 17.1 ng rhCG. This strongly indicates that a “one-week sustained release” formulation would be viable using the PEGylated hCG of the invention. This ability to provide a one week formulation of recombinant hCG represents a significant advantage over the known hCG formulations.
(98) TABLE-US-00005 Average Uterus Weight (mg) Branch PEG-rhCG rhCG QS-MP QS-MP 09PD-84-012 Linear PEG- 09PD-84- 3 injections rhCG QS-MP rhCG QS-MP 012 of 4.3 ng 09PD-84-012 09PD-84-012 X 10 (Group A), X 10 Conc. X 10 Conc. Conc. 1 8.6 ng (Group 1 Injection 1 Injection Injection B) and 17.1 ng (170 ng) (170 ng) (170 ng) Group (Group C) Group (i) Group (iii) Group (ii) Buffer A 38.81 B 57.28 C 117.23 64.05 35.22 134.11 41.9 rhCG = recombinant hCG
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(100) TABLE-US-00006 Human chorionic gonadotropin alpha polypeptide Accession number AH007338 Nucleotide sequence of hCG alpha SEQ ID 1 1 ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT 61 GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA 121 TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA 181 TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG 241 TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG 301 GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA A Protein sequence of hCG alpha 1 MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QCMGCCFSRA 61 YPTPLRSKKT MLVQKNVTSE STCCVAKSYN RVTVMGGFKV ENHTACHCST CYYHKS Human Chorionic Gonadotrophin beta polypeptide Accession number NP_000728 Nucleotide sequence of hCG beta Nucleotide sequence SEQ ID 2 1 ATGGAGATGT TCCAGGGGCT GCTGCTGTTG CTGCTGCTGA GCATGGGCGG GACATGGGCA 61 TCCAAGGAGC CGCTTCGGCC ACGGTGCCGC CCCATCAATG CCACCCTGGC TGTGGAGAAG 121 GAGGGCTGCC CCGTGTGCAT CACCGTCAAC ACCACCATCT GTGCCGGCTA CTGCCCCACC 181 ATGACCCGCG TGCTGCAGGG GGTCCTGCCG GCCCTGCCTC AGGTGGTGTG CAACTACCGC 241 GATGTGCGCT TCGAGTCCAT CCGGCTCCCT GGCTGCCCGC GCGGCGTGAA CCCCGTGGTC 301 TCCTACGCCG TGGCTCTCAG CTGTCAATGT GCACTCTGCC GCCGCAGCAC CACTGACTGC 361 GGGGGTCCCA AGGACCACCC CTTGACCTGT GATGACCCCC GCTTCCAGGA CTCCTCTTCC 421 TCAAAGGCCC CTCCCCCCAG CCTTCCAAGT CCATCCCGAC TCCCGGGGCC CTCGGACACC 481 CCGATCCTCC CACAATAA Protein sequence of hCG beta 1 MEMFQGLLLL LLLSMGGTWA SKEPLRPRCR PINATLAVEK EGCPVCITVN TTICAGYCPT 61 MTRVLQGVLP ALPQVVCNYR DVRFESIRLP GCPRGVNPVV SYAVALSCQC ALCRRSTTDC 121 GGPKDHPLTC DDPRFQDSSS SKAPPPSLPS PSRLPGPSDT PILPQ Beta-galactoside alpha-2,3-sialyltransferase 4 Accession Number L23767 Nucleotide sequence of ST3GAL4 SEQ ID 3 1 ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG 61 TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG 121 AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC 181 TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA 241 TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA 301 GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG 361 GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC 421 GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC 481 AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC 541 AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG 601 ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC 661 TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT 721 GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG 781 GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT 841 GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC 901 AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG 961 CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA Protein Sequence of ST3GAL4 1 MCPAGWKLLA MLALVLVVMV WYSISREDRY IELFYFPIPE KKEPCLQGEA ESKASKLFGN 61 YSRDQPIFLR LEDYFWVKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV 121 VVGNGHRLRN SSLGDAINKY DVVIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN 181 NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA 241 DKLLSLPMQQ PRKIKQKPTT GLLAITLALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL 301 KSMAGSGHNV SQEALAIKRM LEMGAIKNLT SF Beta-galactosamide alpha-2,6-sialyltransferase 1 Accession number NM_003032 Nucleotide sequence of ST6GAL1 SEQ ID 4 1 ATGATTCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT 61 GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG 121 CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC 181 CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT 241 CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC 301 TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC 361 AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG 421 CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC 481 AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT 541 TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA 601 GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA 661 CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG 721 AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGGAATCC TAATTGTATG GGACCCATCT 781 GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC 841 TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG 901 CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC 961 CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT 1021 TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC 1081 GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA TTTGGTGAAG 1141 CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC 1201 TTCCGGACCA TTCACTGCTA A 0p- Protein Sequence of ST6GAL1 1 MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS 61 QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN 121 KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP 181 WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE 241 KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM 301 PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF 361 DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC