KR20050044858A - Chemically-modified human growth hormone conjugates - Google Patents

Abstract

The present invention provides a chemically modified human Growth Hormone (hGH) prepared by binding a water soluble polymer to the protein. The chemically-modified protein according to the present invention may have a much longer lasting hGH activity than that of the unmodified hGH, enabling reduced dose and scheduling opportunities.

Description

Chemically Modified Human Growth Hormone Conjugate {CHEMICALLY-MODIFIED HUMAN GROWTH HORMONE CONJUGATES}

This application claims priority to US Provisional Application No. 60 / 331,907, filed November 20, 2001, pursuant to United States Code §119, Chapter 35, which is incorporated by reference in its entirety as described herein. do.

The present invention relates to chemical modification of hGH and its agonist variants, which can alter the chemical and / or physiological properties of human growth hormone (hGH). PEG-modified hGH can have an increase in the duration of plasma retention, a decrease in clearance, an increase in stability, a decrease in antigenicity, or a combination thereof. The invention also relates to a method for the modification of hGH. The invention also relates to pharmaceutical compositions comprising modified hGH. A further aspect is the use of modified hGH for the treatment of growth and developmental disorders.

Background of the Invention

Human growth hormone (hGH) is a protein comprising a single chain of 191 amino acids bridged by two disulfide bridges, the monomeric form having a molecular weight of 22 kDa. Human GH is secreted from the pituitary gland and may be produced by recombinant genetic engineering. hGH will cause growth in all body tissues that can grow. Recombinant hGH has been on the market for many years. The recombinant hGH preparations useful as the two types of therapeutic is currently commercially available: the original, for example, to pins in note (Genotropin) ^TM, or neutroavidin pin (Nutropin) similarly having a methionine residue added to the N- terminus and ^TM Chains such as Somatonorm ^™ . hGH is used to stimulate linear growth in patients with pituitary hypoplasia dwarfism, also called growth hormone deficiency (GHD) or Turner's syndrome, but long-term treatment of poor growth in children born with low birth weight (SGA) Other indications have also been proposed, including the treatment of Praer-Willi syndrome (PWS), chronic renal failure (CRI), AIDS wasting syndrome, and aging patients.

The main biological effect of growth hormone (GH) is to promote the growth of young mammals and the maintenance of tissues in older mammals. Affected organ systems include skeletal, connective tissue, muscle and intestines such as liver, small intestine and kidney. Growth hormones exert their effects through interactions with specific receptors on target cell membranes. hGH is a member of a family of homologous hormones including placental lactogen, prolactin and other genetic and species variants or growth hormones (Nicoll, C. S., et al. (1986) Endocrine Reviews 7: 169). hGH exhibits broad species specificity and binds to cloned somatogens (Leung, DW, et al. [1987] Nature 330; 537) or prolactin receptors (Boutin, JM, et al. [1988] Cell; 53: 69). It has a characteristic in these in point. Genes cloned for hGH were expressed in secreted form from Escherichia coli (Chang, CN, et al. [1987] Gene 55: 189), and their DNA and amino acid sequences were reported (Goeddel, et al. [ 1979 Nature 281: 544; Gray, et al. [1985] Gene 39: 247).

Human growth hormone (hGH) is involved in much of the regulation of normal human growth and development. These pituitary hormones exhibit a variety of biological effects, including linear growth (somatogenesis), milk secretion, macrophage activation, insulin-like and diabetic effects (Chawla, RK (1983) Ann. Rev. Med. 34, 519; Edwards, CK et al. (1988) Science 239, 769; Thomer, MO, et al. (1988) J. Clin. Invest. 81: 745). Growth hormone deficiency in children causes dwarfism, which has been successfully treated for more than 10 years by external administration of hGH.

Human growth hormone (hGH) is a single chain polypeptide consisting of 191 amino acids (molecular weight 21,500). Disulfide bonds link positions 53 and 165 and positions 182 and 189 (Niall, Nature, New Biology, 230: 90 (1971)). hGH is a powerful assimilation agent, in particular due to the retention of nitrogen, phosphorus, potassium and calcium. Treatment of pituitary excised rats with GH can restore at least a portion of the rat's growth rate (Moore et al., Endocrinology 122: 2920-2926 (1988)). Among the most pronounced effects in patients with pituitary hypoplasia (GH-deficiency) are the acceleration of the linear growth of bone-growth-plate-cartilage that causes height growth (Kaplan, Growth Disorders in Children and Adolescents (Springfield, IL: Charles C. Thomas, 1964).

hGH produces a variety of physiological and metabolic effects, including linear bone growth, milk secretion, macrophage activation, insulin-like and diabetic effects, in various animal models (RK Chawla et al., Annu. Rev. Med. 34: 519 (1983); 0. GP Isaksson et al., Annu. Rev. Physiol. 47, 483 (1985); CK Edwards et al., Science 239, 769 (1988); M. 0. Thomer and ML Vance , J. Clin. Invest. 82: 745 (1988); JP Hughes and HG Friesen, Ann. Rev. Physiol. 47: 469 (1985)). It has been reported that GH secretion decreases with age, especially in postmenopausal women [Millard et al., Neurobiol. Aging, 11: 229-235 (1990); Takahashi et al., Neuroendocrinology M, L6-137-142 (1987)]. See also, Rudman et al., J. Clin. Invest., 67: 1361-1369 (1981) and Blackman, Endocrinology and Aging, 16: 981 (1987). Moreover, there are reports that some of the signs of aging, including loss of lean body weight, dilation of adipose tissue size, and thinning of the skin, can be reduced by GH treatment three times a week. See, eg, Rudman et al., N. Eng. J. Med., 323: 1-6 (1990) and in a paper by Dr. Vance (pp. 52-54) in the same journal. This biological effect results from the interaction between hGH and specific cellular receptors. Two different human receptors have been cloned, which are the hGH liver receptor (DW Leung et al., Nature 330: 537 (1987)) and the human prolactin receptor (JM Boutin et al., Mol. Endocrinology. 3: 1455 (1989)). to be. However, other receptors, including human placental lactogen receptors, are also likely to exist (M. Freemark, M. Comer, G. Komer, and S. Handwerger, Endocrinol. 120: 1865 (1987)). This homologous receptor contains glycosylated extracellular hormone binding domains, single transmembrane domains, and cytoplasmic domains that differ significantly in sequence and size. It is assumed that one or more receptors play a decisive role in the physiological response to hGH.

In general, it is observed that physiologically active proteins administered in the body may exhibit pharmacological activity only for a short period of time due to the high removal rate in the body. In addition, the relative hydrophobicity of these proteins can place limitations on their stability and / or solubility.

Several methods have been proposed for chemically modifying proteins with water soluble polymers for the purpose of reducing the removal rate of the therapeutic protein, improving stability or removing antigenicity. This type of chemical modification can effectively prevent proteolytic enzymes from physically contacting the protein backbone itself, preventing degradation. Chemical attachment of certain water soluble polymers can effectively reduce the rejection rate due to an increase in the hydrodynamic volume of the molecule. Additional advantages include, in certain cases, increasing the stability and circulation time of the therapeutic protein, increasing solubility, and reducing immunogenicity. Poly (alkylene oxides), in particular poly (ethylene glycol) (PEG), are such chemical moieties used in the preparation of therapeutic protein products (the verb “pegylate” attaches one or more PEG molecules). Means to make). Attachment of poly (ethylene glycol) has been shown to protect against proteolysis [Sada, et al., J. Fermentation Bioengineering 71: 137-139 (1991)], and methods of attachment of certain poly (ethylene glycol) moieties It is available. US Patent No. 4,179,337 to Davis et al., Issued December 18, 1979, entitled “Non-Immunogenic Polypeptides”; And US Pat. No. 4,002,531 to Royer, filed Jan. 11, 1977, entitled “Modification of Enzymes with Polyethylene Glycol and Products Produced thereby”. For a review see Abuchowski et al., In Enzymes as Drugs . See JS Holcerberg and J. Roberts, eds. Pp. 367-383 (1981).

Copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, poly (vinyl alcohol), poly (vinyl pyrrolidone), poly (-1,3-dioxolane), poly (-1,3,6- Other water soluble polymers such as trioxane), ethylene / maleic anhydride copolymers, poly-amino acids (homopolymers or random copolymers) have been used.

Many examples of therapeutic proteins with PEG attached have been described. Adagen® (ADAGEN®), a PEG attached agent of adenosine deaminase, has been approved for the treatment of severe combined immunodeficiency diseases. ONCASPAR®, a PEG-attached L-asparaginase, is approved for the treatment of irritable ALL patients. PEG-attached peroxide dismutase is in clinical trials for the treatment of head wounds. Α-interferon with PEG (US Pat. Nos. 5,738,846, 5,382,657) is licensed for the treatment of hepatitis; Glucocerebrosidase with PEG and hemoglobin with PEG have been reported to be in preclinical testing. Another example is IL-6 with PEG attached to EF 0 442 724, entitled “Modified hIL-6,” which discloses a poly (ethylene glycol) molecule added to IL-6.

Another specific therapeutic protein that has been chemically modified is granulocyte colony stimulating factor (G-CSF). G-CSF provides a therapeutic effect against infection by inducing release of neutrophil granulocytes into the bloodstream and rapid proliferation. European Patent Publication EP 0 401 384, issued December 12, 1990, entitled “Chemically Modified Granulocyte Colony Stimulating Factor”, discloses a process for preparing G-CSF with poly (ethylene glycol) molecules attached. And materials. Modified G-CSFs and analogs thereof have also been reported in EO 0 473 268, published March 4, 1992, entitled “Continuous Release Pharmaceutical Compositions Containing Polypeptides Covalently Conjugated to Water-Soluble Polymers,” This document describes the use of various G-CSFs and derivatives covalently conjugated to water soluble particle polymers such as poly (ethylene glycol). Modified polypeptides having human granulocyte colony stimulating factor activity were reported in EP 0 335 423, published October 4, 1989. U. S. Patent No. 5,824, 784 provides a method of modifying a protein or an analog thereof at the N-terminus and a resulting composition comprising the protein or an analog thereof and a novel G-CSF composition chemically modified at the N-terminus. do. U.S. Patent 5,824,778 discloses chemically modified G-CSF.

For poly (ethylene glycol), various methods are used to attach poly (ethylene glycol) molecules to proteins. In general, poly (ethylene glycol) molecules are linked to the protein through reactive groups found in the protein.

Amino groups such as on lysine residues or at the N-terminus are convenient for this attachment. For example, Royer (US Pat. No. 4,002,531, supra) described the use of reductive alkylation to attach poly (ethylene glycol) molecules to enzymes. In EP 0 539 167, published April 28, 1993, entitled “Peg Imidate and Protein Derivatives thereof,” organic compounds and peptides having free amino groups are described as imidate derivatives or related compounds of PEG. It is described as modifying with a water soluble organic polymer. U.S. Patent 5,298,643 and U.S. Patent 5,637,749 disclose PEG aryl imidates.

Chamow et al., Bioconjugate Chem. 5: 133-140 (1994), reports on the modification of CD4 immunoadhesin to monomethoxypoly (ethylene glycol) aldehyde via reductive alkylation. The authors report that 50% of CD4-Ig was MePEG-modified under conditions that could control the extent of PEG attachment (same document, page 137). The authors also report that the in vitro binding capacity (for protein gp 120) of the modified CD4-Ig decreases at a rate associated with the degree of MePEG modification (same literature). Shaw, US Pat. No. 4,904,584, filed February 27, 1990, relates to altering the number of lysine residues in a protein for the attachment of poly (ethylene glycol) molecules through reactive amine groups.

Many methods of attaching polymers to proteins involve the use of moieties that act as linking groups. However, such moieties may be antigenic. A tresyl chloride method that does not involve a linker can be used, but this method can be difficult to use to produce therapeutic products because the use of tresyl chloride can produce toxic byproducts. Francis et al., In: Stability of protein pharmaceuticals: in vivo pathways of degradation and strategies for protein stabilization (Eds. Ahern, T. and Manning, MC Plenum, New York, 1991) and Delgado et al. , "Coupling of PEG to Protein By Activation With Tresyl Chloride, Applications In Immunoaffinity Cell Preparation", in Separations Using Aqueous Phase Systems, Applications In Cell Biology and Biotechnology , Fisher et al., Eds. Plenum Press, New York, NY, 1989 pp. 211-213.

See also, Rose et al., Bioconjugate Chemistry 2: 154-159 (1991), which reports the selective attachment of the linking carbohydrazide to the C-terminal carboxyl group of the protein substrate (insulin).

WO 93/00109 relates to methods of stimulating GH reactive tissues in mammals or birds, including maintaining continuous, effective plasma GH concentrations for at least three days. One method of achieving such plasma concentrations has been described using GH coupled to macromolecular materials such as PEG (polyethylene glycol). Coupling to macromolecular materials is said to show improved half-life. Human growth hormones with PEG are reported in WO 93/00109 using mPEG aldehyde-5000 and mPEGN-hydroxysuccinimidyl ester (mPEG-NHS-5000). Using mPEG-NHS produces a heterogeneous mixture of hGH in the form of multiple PEG attached. WO 93/00109 also discloses the use of PEG attached cysteine hGH variants of mPEG-maleimide.

WO 99/03887 discloses cysteine variant growth hormones with PEG attached. This conjugate, named BT-005, is claimed to be more effective at stimulating weight gain in growth hormone deficient rats and to have a longer half-life than hGH.

PEG-attached human growth hormones are also reported in Clark et al., Journal of Biological Chemistry 271: 21969-21977, 1996, using succinimidyl esters of carbomethylated PEG. Clark et al. Describe hGH derivatives of increased size using mPEG-NHS-5000 optionally conjugated to primary amines. Increasing PEG modification levels decrease the affinity for that receptor and increase the EC ₅₀ in cell-based assays by 1500-fold. Olson et al. Polymer Preprints 38: 568-569, 1997 describe the use of N-hydroxysuccinimide (NHS) PEG and succinimidyl propionate (SPA) PEG to attach multiple PEGs. Initiate obtaining hGH species.

International Publication No. WO 94/20069 describes anticipated PEG-attached hGH as part of the formulation for pulmonary delivery.

US Pat. No. 4,179,337 discloses a method for attaching PEG to enzymes and hormones to obtain physiologically active non-immunogenic, water soluble polypeptide conjugates. GH is mentioned as an example of the hormone to which PEG is attached.

EP publication 458064 A2 discloses attaching PEG to cysteine residues that are introduced or naturally present in somatotropin. EP publication 458064 A2 also refers to the introduction of two cysteine residues into a loop named omega loop which has been shown to be located at residues 102-112 of wild type bovine somatotropin. More specifically, EP Publication No. 458064 A2 discloses that the residues designated bovine somatotropin, numbers 102 and 112, are substituted for Ser to Cys and Tyr to Cys, respectively.

WO 95/11987 proposes attaching PEG to thiol groups of cysteine residues present in the parent molecule or introduced by site-directed mutations. WO 95/11987 relates to the attachment of PEG to protease nexin-1, but it is also proposed to attach PEG generally to hGH and other proteins.

WO 99/03887 discloses modified growth hormones, for example, by inserting additional cysteines for serine residues and attaching PEG to the introduced cysteine residues.

WO 00/42175 relates to the preparation of proteins containing free cysteine residues for PEG attachment. WO 00/42175 discloses the following hGH mutations T3C, S144C and T148C and their cysteine PEG attachment.

International Publication No. WO 9711178 (and U.S. Pat.Nos. 5,584,535,6004931, and 602711) relates to the use of GH variants as agonists or antagonists of hGH. WO 9711178 also discloses PEG attachment of hGH, including lysine PEG attachment and the introduction or replacement of lysine (eg, K168A and K172R). International Publication No. WO 9711178 also discloses a G120K substitution.

See Knauf, M. J. et al., J. Biol. Chem. 263: 15064-15070,1988, previous reports on PEG-attached hGH require multiple PEG attachments, which result in undesirable product non-uniformity resulting in 70K molecular weight of kidney filtration. A hydrodynamic volume greater than the cut-off is obtained.

GH molecules with longer half-lives will reduce the number of administrations required and will provide a more optimal therapeutic hGH concentration and concomitantly increased therapeutic effect.

The present invention provides chemically modified hGH conjugates having reduced heterogeneity, reduced clearance, increased plasma retention, improved solubility, increased stability, reduced antigenicity, or a combination thereof.

Summary of the Invention

The present invention provides a chemically modified hGH having one or more improved chemical or physiological properties selected from, but not limited to, reduced clearance, increased plasma retention period, increased stability, improved solubility and reduced antigenicity. And agonist variants thereof. Thus, as described in more detail below, the present invention has numerous aspects regarding chemical modification of hGH and its agonist variants as well as specific modifications using various poly (ethylene glycol) residues.

The present invention also relates to methods of preparing chemically modified hGH and agonist variants thereof.

The invention also relates to compositions comprising chemically modified hGH and agonist variants thereof.

Modified hGH and its agonist variants of the present invention can be used to treat dwarfism (GHD), adult GHD, Turner syndrome, long-term treatment of growth insufficiency in small babies (SGA) compared to gestational age, and Freuder-Willi syndrome (PWS). It may be useful in the treatment of patients suffering from chronic renal failure (CRI), AIDS related wasting, aging, end stage renal failure, and cystic fibrosis.

Brief description of the drawings

1 is a photograph of reduced and non-reducing SDS-PAGE analysis and anion exchange purified 20K PEG-ALD hGH of a reaction product of hGH with 20K PEG-ALD. Lane 1. MW protein standard; Lane 2. reduced hGH-10 ug; Lane 3. reduced 20 K linear PEG-ALD hGH reaction mix-10 ug; Lane 4. reduced anion exchange purified 20 K straight PEG-ALD hGH-10 ug lane 5. blank; Lane 6. non-reduced hGH-10 ug; Lane 7. non-reduced 20 K straight chain PEG-ALD hGH reaction mix-10 ug; Lane 8. non-reduced anion exchange purified 20 K straight chain PEG-ALD hGH-10 ug; Lane 9. blank; Lane 10. MW Protein Standard.

FIG. 2 is a photograph of non-reducing SDS-PAGE analysis of various anion exchange purified PEG attached hGH molecules. Lane 1. MM protein standard; Lane 2. hGH-10 ug; Lane 2. 4-6 x 5K PEG-SPA hGH-10 ug; Lane 3. 20 K straight-chain PEG-ALD hGH-10 ug; Lane 4.20 K branched PEG-ALD hGH-10 ug lane 5.40 K branched PEG hGH-10 ug.

3 is a photograph of the RP-HPLC elution profile for trypsin digests of hGH, 40K Br PEG-ALD hGH and 40K Br PEG-NHS hGH. PEG coupled primarily to the N-terminus of hGH (as shown in 40K Br ALD hGH) reduces the N-terminus (T1) fragment peak with the generation of a new PEG attached T1 peak.

FIG. 4 shows weight gain in hypophystomized rats for 11 days, with PEG for subcutaneous (SC) injection (1.8 mg / Kg) every 6 days with hGH attached to a single PEG In vivo biological activity is compared with daily administration of hGH without (0.3 mg / Kg / day).

FIG. 5 shows weight gain in rats without pituitary gland for 11 days, with 4-6 × 5K PEG-SPA-hGH, 20K branched PEG-ALD hGH with single PEG attached, and single PEG attached, respectively. For subcutaneous (SC) injections (1.8 mg / Kg) every 40 days of a 40K branched PEG-ALD hGH dosed daily (0.3 mg / Kg / day) with daily SC administration without PEG-attached hGH It is a comparison of my biological activity.

FIG. 6 shows weight gain in rats without pituitary gland for 11 days, with 4-6 × 5K PEG-CMHBA-hGH, 20K straight ALD with single PEG attached, 30K straight ALD with single PEG attached, No PEG attached for 20K branched PEG-ALD hGH with single PEG attached and 40K branched PEG-ALD hGH with single PEG attached subcutaneously (SC) injection (1.8 mg / Kg) every 6 days This is a comparison of biological activity in vivo with daily SC administration (0.3 mg / Kg / day).

FIG. 7 shows an increase in plasma IGF-1 levels in rats without pituitary gland over 9 days, with hGH without PEG attached, 5K straight PEG-ALD hGH with single PEG attached, 20K with single PEG attached. Straight PEG-ALD hGH, 20K branched PEG-ALD hGH with single PEG, 20K straight PEG-hydrazide hGH with single PEG, 30K straight PEG-ALD hGH with single PEG, 40K with single PEG Comparison of the biological activity in vivo with a single dose of branched PEG-ALD hGH, 4-6 × 5K PEG SPA hGH, 4-6 × 5K PEG-CMHBA, 1.8 mg / Kg SC.

hGH and its agonist variants are members of the recombinant protein class described in US Pat. Nos. 4,658,021 and 5,633,352. Their recombinant production and use methods are described in detail in US Pat. Nos. 4,342,832, 4,601,980, 4,898,830, 5,424,199 and 5,795,745.

E. transformed or transfected using recombinant genetic techniques. Any purified and isolated hGH or agonist variant thereof produced by host cells such as coli and animal cells can be used in the present invention. Additional hGH variants are described in US Patent Application Serial Nos. 07 / 715,300 (filed June 14, 1994) and 07 / 743,614 (August 9, 1991), and WO 92/09690 (1992). Published June 11, 2011). Among these, transformed E. coli. Particularly preferred are hGH or agonist variants thereof produced by E. coli. Such hGH or agonist variants thereof can be obtained in large quantities with high purity and uniformity. For example, the hGH or agonist variant thereof can be prepared according to the methods disclosed in US Pat. Nos. 4,342,832, 4,601,980, 4,898,830, 5,424,199 and 5,795,745. The term "substantially has the following amino acid sequence" means that the amino acid sequence is one or more amino acid changes (deleted, added, inserted or replaced) unless it causes any adverse non-similarity in its action on hGH or agonist variants thereof. ) Can be included. The use of hGH or agonist variants substantially having an amino acid sequence comprising at least one lysine, aspartic acid, glutamic acid, unpaired cysteine residues, free N-terminal α-amino groups or free C-terminal carboxyl groups More preferred.

According to the present invention, poly (ethylene glycol) is covalently linked via amino acid residues of hGH or agonist variants thereof. Various activated poly (ethylene glycols) having a number of different functional groups, linking groups, structures, and molecular weights are known in the art and used to prepare PEG-hGH conjugates or PEG-hGH agonist variant conjugates. Roberts MJ et al., Adv. Drug Del. Rev. 54: 459-476, 2002), Harris JM Et al., Drug Delivery Sytems 40: 538-551,2001). The amino acid residue can be, for example, any having a free amino, carboxyl, sulfhydryl (thiol), hydroxyl, guanidinyl, or imidizoyl group to which the terminal reactive groups of the activated poly (ethylene glycol) can be bound. It may be a reactive amino acid residue. Amino acid residues having free amino groups may comprise lysine and / or N-terminal amino acid residues, amino acid residues having free carboxyl groups may comprise aspartic acid, glutamic acid and / or C-terminal amino acid residues , Amino acid residues with free sulfhydryl (thiol) such as cysteine, amino acid residues with free hydroxyl such as serine or tyrosine, amino acid residues with free guanidinyl such as arginine, and free imidizoyl such as histidine. Amino acid residues having;

In another embodiment, oxime chemistry (see Lemieux & Bertozzi Tib Tech 16: 506-513,1998) is used to target the N-terminal serine residues.

The poly (ethylene glycol) used in the present invention is not limited to any particular form or molecular weight range. The poly (ethylene glycol) molecular weight is between 500 and 100,000. Typically, the molecular weight is 500-60,000, preferably 1,000-40,000. More preferably, molecular weights greater than 5,000 up to about 40,000 are used.

In another embodiment, the poly (ethylene glycol) is a branched PEG with one or more PEG residues attached. Preferred examples of branched PEGs include U.S. Patents 5,932,462, 5,342,940, 5,643,575, 5,919,455, 6,113,906, 5,183,660; International Publication No. WO 02/09766; Kodera Y., Bioconjugate Chemistry 5: 283-288 (1994); And Yamasaki et al., Agric. Biol. Chem., 52: 2125-2127, 1998. In a preferred embodiment, the molecular weight of each poly (ethylene glycol) of branched PEG is 5,000 to 20,000.

Poly (alkylene oxide), in particular poly (ethylene glycol), is coupled with hGH or agonist variants through terminal reactive groups which may or may not provide a binding moiety (spacer) between PEG and the protein. To form the hGH conjugates or agonist variants thereof of the invention, polymers such as poly (alkylene oxides) are converted into active forms known to those skilled in the art. The reactive group is, for example, a terminal reactive group that mediates the bond between chemical moieties on the protein, such as amino, carboxyl or thiol groups and poly (ethylene glycol). Typically one or both of the terminal polymeric hydroxy end-groups (ie, alpha and omega terminal hydroxyl groups) are converted to reactive functional groups that allow covalent conjugation. The process is often referred to as "activation" and the poly (ethylene glycol) product with reactive group is referred to as "activated poly (ethylene glycol)" below. Polymers containing both α and ε bonding groups are referred to as "bis-activated poly (alkylene oxides)" and "difunctional groups". Polymers containing the same reactive groups on the α and ε terminal hydroxyls are often referred to as “homofunctional groups” or “homobis-activation”. Polymers containing different reactive groups on the α and ε terminal hydroxyls are often referred to as “heterofunctional groups” (see eg WO 01/26692) or “heterobis-activation”. Polymers containing a single reactive group are referred to as "mono-activated" polyalkylene oxides or "single-functional groups". Other substantially non-antigenic polymers are similarly "activated" or "functionalized".

Thus, the activating polymer is suitable for mediating a bond between chemical moieties such as α- or ε-amino, carboxyl or thiol groups and poly (ethylene glycol) on the protein. Bis-activated polymers can be reacted in this manner with two protein molecules or one protein molecule and reactive small molecules in one other embodiment to effectively form a protein polymer or protein-small molecule conjugate through crosslinking.

A functional group capable of reacting with either the amino terminal α-amino group or ε-amino group of lysine found on hGH or its agonist variant is N-hydroxysuccinimidyl ester, carbonate, for example p Nitrophenyl or succinimidyl (US Pat. No. 5,808,096, US Pat. No. 5,612,460, US Pat. No. 5,324,844, US Pat. No. 5,5,122,614); Carbonyl imidazole; Azlactones (US Pat. No. 5,321,095, US Pat. No. 5,567,422); Cyclic imide thiones (US Pat. Nos. 5,405,877, 5,349,001); Isocyanates or isothiocyanates (Greenwald R. B., J. Org. Chem., 60: 331-336, 1995); Tresyl chloride (EP 714 402, EP 439 508); Halogen formate (WO 96/40792) and aldehydes.

The functional groups capable of reacting with carboxylic acid groups, reactive carbonyl groups and oxidized carbohydrate moieties of hGH or agonist variants thereof include primary amines; And hydrazine and hydrazide functional groups such as acyl hydrazide, carbazate, semicarbamate, thiocarbazate and the like (WO 01/70685).

Mercapto groups available on hGH or agonist variants thereof also include reactive groups such as thiols; It can be used as an attachment site for a suitably activated polymer having maleimide, sulfone and phenyl glyoxal groups (see, eg, US Pat. No. 5,093,531, which is incorporated herein by reference). Other nucleophiles capable of reacting with an electrophilic center include, but are not limited to, hydroxyl, amino, carboxyl, thiol, active methylene and the like.

See also US Pat. No. 5,359,030; US Patent No. 5,681,811; U.S. Patent 5,438,040; And polymers comprising lipophilic and hydrophilic moieties disclosed in US Pat. No. 5,359,030.

Also disclosed in WO 98/32466 are halogenated PEGs which can react with amino, thiol and aromatic hydroxy groups that directly covalently bond PEG to proteins. In one preferred embodiment of the invention, secondary amine or amide bonds are formed using the N-terminal α-amino group or ε-amino group of lysine of hGH or its agonist variant and activating PEG. In another preferred embodiment of the present invention the secondary amine linkage is NaCNBH (as disclosed in Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) and US Pat. No. 5,824,784). Reduction with an appropriate reducing agent such as ₃ , NaBH ₃ , pyridine borane and the like is formed between the N-terminal primary α- or ε-amino group of the hGH or its agonist variant with a straight or branched PEG aldehyde.

In a preferred embodiment, at least 70%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85% , Preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, Preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 96%, preferably at least 97%, most preferably at least 98% poly (Ethylene glycol) is present on the amino terminal α-amino group.

In another preferred embodiment of the invention, a polymer activated with an amide-forming linker, such as succinimidyl ester, cyclic imide thione, etc., has an effect on the binding and polymer between hGH or its agonist variant. (See, eg, US Pat. No. 5,349,001; US Pat. No. 5,405,877 and Greenwald et al . , Crit . Rev. Ther . Drug Carrier Syst. 17 : 101-161, 2000, which are herein incorporated by reference). Inserted as a citation). One preferred activated poly (ethylene glycol) that can be bound to the free amino group of hGH or its agonist variant includes straight or branched chain N-hydroxysuccinylimide poly (ethylene glycol), N-hydroxysuccinic It can be prepared by activating succinic acid ester of poly (ethylene glycol) with nilimide.

Another preferred embodiment of the present invention involves the use of other activating polymers to form covalent bonds of the polymer using hGH or agonist variants thereof via ε-amino or other groups. For example, isocyanate or isothiocyanate forms of terminally activated polymers can be used to form urea and thiourea-based bonds using lysine amino groups (Grinwald RB, J. Org. Chem. , 60: 331-336, 1995).

Another preferred aspect of the invention is that carbamate (urethane) linkages can be prepared using protein amino groups as disclosed in US Pat. Nos. 5,122,614, 5,324,844 and 5,612,640, which are incorporated herein by reference. Is formed. Examples include N-succinimidyl carbonate, para-nitrophenyl carbonate and carbonyl imidazole activating polymers. In another preferred embodiment of the invention, the benzotriazole carbonate derivative of PEG is linked to an amino group on hGH or agonist variant thereof.

Another aspect of the invention is that a hGH or agonist is expected to be degraded by enzymatic or pH directed hydrolysis that releases free hGH or agonist variant thereof or another hGH or agonist variant derivative thereof. Provided are prodrugs or delayed release forms of hGH or agonist variants thereof comprising a water soluble polymer, such as poly (ethylene glycol), attached to the variant molecule. Prodrugs may also be “double prodrugs” (Bundgaard in Advanced Drug Delivery Reviews 3 : 39-65, 1989), which include the use of multistage latents. In such a system, the hydrolysis reaction comprises an initial rate-limiting (low speed) enzymatic or pH indicating step and a second step comprising fast non-enzymatic hydrolysis that occurs only after the first step occurs. Such releasable polymers provide a protein conjugate that is temporary and can act as a reservoir for continuously releasing hGH or agonist variants thereof. Such functional linking groups are described in US Pat. No. 5,614,549; US Patent No. 5,840,900; US Patent No. 5,880,131; U.S. Patent 5,965,119; US Patent No. 5,965,565; US Patent No. 6,011,042; US Patent No. 6,153,655; US Patent No. 6,180,095B1; US Patent No. 6,413,507; Greenwald RB et al ., J. Med. Chem. 42 ; 3657-3667, 1999; Lee, S. et al., Bioconjugate Chem. 12 : 163-169, 2001; Garman AJ et al ., FEBS Lett. 223 : 361-365,1987; Woghiren C. et al., Bioconjugate Chem. 4: 314-318, 1993; Roberts MJ, J. Pharm. Sci. 87 ; 1440-1445,1998; Zhao X., Ninth Int. Symp. Recent Adv. Drug Delivery Syst. 199; Greenwald RB et al ., J. Med. Chem. 43 : 475-487, 2000 and Greenwald RB, Crit. Rev. Ther. Drug Carrier Syst. 17 : 101-161, 2000, Zalipsky et al., 28th Int. Symp. On controlled Release of Bioactive Materials 1; 73-74, 2001.

Conjugation reactions, called PEG attachment reactions, have historically been carried out where polymers can be attached to proteins in solutions with excess polymers. However, this general technique has proven inadequate for conjugating non-antigenic polymers and bioactive proteins while retaining sufficient bioactivity. One way to maintain hGH or agonist variant bioactivity is to substantially prevent conjugation of the hGH or agonist variant reactive group with the receptor binding site in a polymer coupling method. Another aspect of the present invention is to provide a method of conjugating poly (ethylene glycol) with hGH or agonist variants thereof while maintaining high levels of retention activity.

Chemical modification via covalent bonds can be carried out under any suitable conditions generally employed in the reaction of bioactive materials with activated poly (ethylene glycol). The conjugation reaction is performed under relatively mild conditions to prevent inactivating hGH or agonist variants thereof. Mild conditions include maintaining the pH of the reaction solution in the range of 3 to 10 and the reaction temperature in the range of about 0 ° C to 37 ° C. If the reactive amino acid residue in the hGH or agonist variant thereof has a free amino group, the modification is, among other non-limiting kinds of suitable buffers (pH 3-10) comprising phosphate, MES, citrate, acetate, succinate or HEPES. It is preferably carried out at a temperature of 4 ° C. to 37 ° C. for 1 to 48 hours. When targeting an N-terminal amino group with a reagent such as PEG aldehyde, preferably pH 4-8 is maintained. The activated poly (ethylene glycol) may be used at about 0.05 to 100 times the moles of the number of free amino groups of the hGH or agonist variant thereof, preferably 0.01 to 2.5 times. On the other hand, if the reactive amino acid residue in hGH or its agonist variant has a free carboxyl group, the modification is preferably carried out at a pH of about 3.5 to about 5.5, and the modification with poly (oxyethylenediamine) is 4 ° C. It is carried out in the presence of carbodiimide (pH 3.5-5) for 1 to 24 hours at 37 ℃. Activated poly (ethylene glycol) may be used at 0.05 to 300 times the moles of the free carboxyl groups of hGH or agonist variants thereof.

In a separate embodiment, the upper limit for the amount of polymer involved in the conjugation reaction is such that it is possible to react the activating polymer and the hGH or agonist variant thereof without forming a substantial amount of high molecular weight material, i.e. hGH or its Greater than about 1: 1 with at least about 20% of conjugates containing at least about 1 polymer strand per molecule of agonist variant. For example, taking this aspect of the invention into account, a ratio of about 6: 1 or less can be used to form a significant amount of the desired conjugate that can be isolated from any high molecular weight materials in the next step.

In another aspect of the invention, bifunctional activating PEG derivatives can be used to produce polymeric hGH or agonist variant-PEG molecules in which multiple hGH or agonist variant molecules are crosslinked via PEG. While the reaction conditions described herein can provide a significant amount of unmodified hGH or agonist variants thereof, the unmodified hGH or agonist variants thereof can be easily recycled to the next batch for further conjugation reactions. . The process of the invention produces surprisingly very small amounts, ie less than about 30%, more preferably less than about 10%, of high molecular weight materials and materials containing at least one polymeric strand per hGH or agonist variant thereof. The reaction conditions are in contrast to the conditions normally used in polymer conjugation reactions, where the active ring polymer is present in an excess molar number of times the desired material. In another aspect of the invention, the polymer is present in an amount from about 0.1 / amino groups to about 50 equivalents per equivalent of hGH or agonist variant thereof. In another aspect of the invention, the polymer is present in an amount of about 1 to about 10 equivalents per equivalent of hGH or agonist variant thereof.

The conjugate reaction of the present invention initially provides a reaction mixture or pool containing mono- and di-PEG-hGH conjugates, unreacted hGH, unreacted polymers, and usually less than about 20% high molecular weight species. High molecular weight species include conjugates containing one or more agonist variant species of the polymer strand and / or polymerized PEG-hGH. After unreacted species and high molecular weight species are removed, the composition containing the conjugate with mono- and di-polymer-hGH or agonist variants thereof is recovered first. In view of the fact that the conjugate for the majority includes a single polymer strand, the conjugate is substantially homogeneous. These modified hGH or agonist variants thereof are standard FDC-P1 cell proliferation assays (Clark et al., Journal of Biological Chemistry 271: 21969-21977, 1996), receptor binding assays (US 5,057,417) or pituitary excised rats. Have at least 0.1% in vitro biological activity with respect to natural or unmodified hGH or agonist variants as measured using growth (Clark, Journal of Biological Chemistry 271: 21969-21977, 1996). . However, in a preferred aspect of the present invention, the modified hGH or agonist variant thereof has about 25% of the biological activity in vitro, and preferably the modified hGH or agonist variant thereof has about 50% of the biological activity in vitro. %, More preferably the modified hGH or agonist variant has about 75% of the biological activity in vitro, and very preferably the modified hGH or agonist variant is equivalent or improved in vitro biological Have activity.

The method of the invention preferably comprises a somewhat limited ratio of polymer to hGH or agonist variant thereof. Therefore, it was considered that conjugates with hGH or agonist variants thereof are mainly limited to species containing only one strand of polymer. Moreover, the attachment of the polymer to the hGH or its agonist variant reactive groups is substantially less random than when more molar excess of the polymer linker is used. Unmodified hGH or agonist variants thereof present in the reaction pool can be regenerated to the next reaction using ion exchange or size-specific exclusion chromatography or similar separation techniques after the conjugate reaction is quenched.

Poly (ethylene glycol) -modified hGH or agonist variants thereof, ie chemically modified proteins according to the invention, are used in the purification of proteins, for example dialysis, salting out, ultrafiltration, ion exchange chromatography, hydrophobic interactions. It can be purified from the reaction mixture by action chromatography (HIC), gel chromatography and electrophoresis. Ion exchange chromatography is particularly effective at removing unreacted poly (ethylene glycol), and hGH or agonist variants thereof. In a further aspect of the invention, mono- and di-polymer-hGH or agonist variant species thereof are isolated from the reaction mixture to remove high molecular weight species and unmodified hGH or agonist variants thereof. The mixed species are separated by placing them in a buffer solution containing about 0.5-10 mg / ml of hGH or an agonist variant-polymer conjugate thereof. Suitable solutions have a pH of about 4 to about 8. The solution preferably contains at least one buffer salt selected from KCl, NaCl, K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaHCO ₃ , NaBO ₄ , CH ₃ CO ₂ H and NaOH. do.

Depending on the reaction buffer, a conjugate solution of hGH or its agonist variant with the polymer may first be subjected to buffer exchange / ultrafiltration to remove any unreacted polymer. For example, ultrafiltration of a conjugated solution with PEG-hGH or its agonist variant through a low molecular weight blocking (10,000 to 30,000 Dalton) semipermeable membrane removes most of the unwanted material, such as unreacted polymers, surfactants if present. do.

The distribution of the conjugate to the pool containing the desired species is preferably performed using ion exchange chromatography media. The mediator can selectively bind conjugates with PEG-hGH or agonist variants thereof through a difference in charge that changes somewhat predictably. For example, the surface charge of hGH or its agonist variant is determined by the number of possible charge groups on the surface of the protein. These charged groups typically serve as points of potential attachment of the poly (alkylene oxide) polymer. Thus, conjugates with hGH or agonist variants thereof will have a different charge than other species and allow for selective isolation.

Strong polar anion or cation exchange resins such as quaternary amine or sulfopropyl resins respectively are used for the process of the present invention. Ion exchange resins are particularly preferred. Commercially available cation exchange resins suitable for use in the present invention include, but are not limited to, SP-hitrap®, SP Sepharose HP®, and SP three. Paros® fast flow. Other suitable cation exchange resins such as S and CM resins may also be used. Non-limiting listings of commercially available anion exchange resins suitable for use in the present invention include Q-Hraplab®, Q Sepharose® HP, and Q Sepharose® Fast Flow. Other suitable anion exchange resins such as DEAE resin can also be used.

For example, the anion or cation exchange resin is preferably filled in a column and can be equilibrated by conventional means. The same pH and osmotic buffer as the polymer conjugated hGH or agonist variant solution thereof is used. The elution buffer preferably contains one or more salts selected from KCl, NaCl, K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaHCO ₃ , NaBO ₄ , and (NH ₄ ) ₂ CO ₃ . It contains. The conjugate-containing solution is then adsorbed with the unreacted polymer on the column and some high molecular weight species are not retained. At the end of the loading, a flow that is gradient to an increasing concentration of salt is added to the column to elute the fraction of the desired polyalkylene oxide-conjugated hGH or agonist variant thereof. The pooled fractions eluted after the cation or anion exchange separation step are preferably limited to homogeneous polymer conjugates. Any non-conjugated hGH or agonist variant species thereof can then be post washed from the column by conventional techniques. If desired, mono and multiple PEG attached hGH or agonist variant species can be further separated from one another via additional ion exchange chromatography or size-specific exclusion chromatography.

Techniques using multiple isocratic steps that increase salt concentration or pH may also be used. Multiple isocratic elution steps of increasing concentrations can result in continuous elution of the conjugates of the di- and then mono-hGH or agonist variant-polymers thereof.

The temperature range for elution is from about 4 ° C to about 25 ° C. Preferably, the elution is carried out at a temperature of about 4 ° C to about 22 ° C. For example, elution of PEG-hGH or agonist variant fractions thereof is detected at ultraviolet absorbance at 280 nm. Fraction collection can be achieved through a simple time elution profile.

Surfactants may be used in the process of conjugating poly (ethylene glycol) polymers with hGH or agonist variant residues thereof. Suitable surfactants include ionic agents such as sodium dodecyl sulfate (SDS). Other ionic surfactants such as lithium dodecyl sulfate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decane sulfonic acid, and the like can also be used. Nonionic surfactants may also be used. For example, materials such as poly (oxyethylene) sorbitan (twins), poly (oxyethylene) ethers (tritones) can be used. See also, Neugebauer, A Guide to the Properties and Uses of Detergents in Biology and Biochemistry (1992), Calbiochem Corp. The only limitation to the surfactants used in the present invention is that they are used at concentrations and conditions that do not cause substantial irreversible denaturation of hGH or its agonist variants and do not completely inhibit the polymer conjugate. The surfactant is present in the reaction mixture in an amount of about 0.01-0.5%, preferably 0.05-0.5%, and very preferably about 0.075-0.25%. Mixtures of surfactants are also contemplated.

It is believed that the surfactant provides a temporary, reversible protection system during the polymer conjugate process. Surfactants have been shown to be effective at selectively interfering with polymer aggregation while allowing lysine-based or amino terminal based conjugates to proceed.

The present poly (ethylene glycol) -modified hGH or agonist variants thereof have a more sustained pharmacological effect, possibly due to extended half-life in vivo.

In addition, this poly (ethylene glycol) modified hGH or agonist variant thereof may be used for growth hormone deficiency (GHD), adult growth hormone deficiency, Turner syndrome, growth failure of small born children during pregnancy (SGA), Prader Willy syndrome ( PWS), chronic renal insufficiency (CRI), AIDS weakness and aging.

To administer it to a patient, the present poly (ethylene glycol) -modified hGH or agonist variant thereof may be formulated into a pharmaceutical formulation containing a pharmaceutically acceptable diluent, preparation for the preparation of isotonic solutions, pH-adjusting agents, and the like. .

The pharmaceutical can be administered subcutaneously, intramuscularly, intravenously, pulmonary, intradermal, or orally depending on the therapeutic purpose. The dosage can be based on the condition and type of disease of the patient to be treated, and can usually be from 0.1 mg to 5 mg infusion and 0.1 mg to 50 mg oral dosage for adults.

The polymeric material included may also be water soluble, preferably at room temperature. Non-limiting examples of such polymers include water solubility of poly (alkylene oxide) homopolymers such as poly (ethylene glycol) or poly (propylene glycol), poly (oxyethylenated polyols), their co- and block co-polymers. On their condition that they are retained.

Effective non-antigens such as dextran, poly (vinyl pyrrolidone), poly (acrylamide), poly (vinyl alcohol), carbohydrate based polymers and the like can be used as substitutes for PEG-based polymers. Indeed, activation of the α- and ω-terminal groups of these polymeric materials can be done in a manner similar to that used to convert poly (alkylene oxides) and will be apparent to those skilled in the art. Those skilled in the art will be able to anticipate all polymeric materials having the amounts described herein above are illustrative only. For the purposes of the present invention, "effectively non-antigenic" means any substance which is understood in the art and which does not elicit an apparent antigenic response in mammals.

Definition of Terms

The following is a list of abbreviations and their corresponding meanings used interchangeably herein:

g gram

mg milligram

ml or mL milliliters

RT room temperature

PEG poly (ethylene glycol)

The complete contents of all publications, patents, and patent applications disclosed herein are incorporated by reference as if specifically and individually indicated that each individual publication, patent, or patent application is incorporated by reference.

Although the foregoing invention has been described in detail by way of examples and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention in light of the teachings of the invention. . The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention described in its broadest sense.

In the following examples, hGH is SEQ ID NO (SEQ ID) first. PEG or other members of the hGH or agonist variant thereof of the polypeptide group may be attached in the same manner as illustrated in the following examples.

All references, patents, or applications cited herein are incorporated as if written herein in their entirety.

The invention will be further illustrated by the following examples which are not to be construed as limiting the scope of the invention.

Example 1

PEG-ALD hGH with a linear molecular weight of 20,000

Example 1 shows a method for producing substantially homogeneous hGH with one PEG attached to the N-terminus by reductive alkylation. Methoxy- straight PEG having a molecular weight of about 20,000 through reductive amination using the difference between the relative pKa value of the primary amine at the N-terminus of the lysine residue and the relative pKa value of the primary amine at the ε-amino position. Propionaldehyde reactant (Shearwater Corp.) was selectively coupled to the N-terminus of hGH. 25 mM MES at pH 6.0 (Sigma Chemical, St. Louis, MO), 25 mM Hepes at pH 7.0 (Sigma Chemical, St. Louis, MO), or 10 mM sodium acetate at pH 4.5 (Sigma Chemical, St. Louis, M-PEG-ALD was added to hGH protein dissolved at 10 mg / mL in MO) and reacted with methoxy-PEG-propionaldehyde and M-PEG-ALD (Shearwater Corp., Huntsville, AL) to PEG for amines. A relative molar ratio of: hGH of 0.1: 0.7 was obtained (optionally 8% acetonitrile may be added). 1 M NaCNBH ₄ mother liquor (Sigma Chemical, St. Louis, MO) dissolved in H ₂ 0 was added to catalyze the reaction to give a final concentration of 10-50 mM. The reaction was carried out in the dark at 4 ° C. to room temperature for 18-24 hours. 1 M Tris (Sigma Chemical, St. Louis, Mo.), pH 7.6, was added to a final concentration of 50 mM to terminate the reaction or diluted in appropriate buffer for subsequent purification.

Example 2

PEG-ALD hGH with a linear molecular weight of 30,000

The method described in Example 1 was used to couple PEG-propionaldehyde reactants (Shearwater Corp.) with a methoxy- straight chain molecular weight of 30,000 to the N-terminus of hGH.

Example 3

PEG-ALD hGH with a linear molecular weight of 5,000

The PEG-propionaldehyde reactant (Fluka) of methoxy- straight chain molecular weight 5,000 was coupled to the N-terminus of hGH using the method described in Example 1.

Example 4

PEG-ALD hGH with a branched chain molecular weight of 40,000

The method described in Example 1 was used to couple a PEG-aldehyde (PEG2-ALD) reactant (Shearwater Corp.) with a methoxy-branched molecular weight of 40,000 to the N-terminus of hGH.

Example 5

PEG-ALD hGH with branched molecular weight 20,000

A PEG-aldehyde (PEG2-ALD) reactant (Shearwater Corp.) with a methoxy-branched chain molecular weight of 20,000 was prepared using the method described in Example 1 and the molar ratio of PEG to hGH based on amines of 0.1 to 0.5. Coupling to the N-terminus.

Example 6

SPA-PEG hGH with linear molecular weight 30,000

Example 6 shows how to prepare a substantially uniform hGH with one PEG attached using an N-hydroxysuccinimidyl (NHS) active ester. The hGH protein stock solution was dissolved at 10 mg / mL in 0.25 M HEPES buffer at pH 7.2 (optionally 8% acetonitrile may be added). SPA-PEG was then added to react the solution with methoxy-PEG-succinimidyl propionate (SPA-PEG) to obtain a relative molar ratio of PEG: hGH of 0.1 to 0.1-0.65. The reaction was carried out at 4 ° C. to room temperature for 5 minutes to 1 hour. The reaction was terminated by lowering the pH to 4.0 with 0.1 N acetic acid or adding 5 times molar excess of Tris HCl.

Example 7

SPA-PEG hGH with linear molecular weight 20,000

Using the method described in Example 6, the SPA-PEG reactant (Shearwater Corp.) with a linear molecular weight of 20,000 was coupled to the N-terminus of hGH.

Example 8

Biotin-SPA-PEG hGH with straight chain molecular weight 3,400

The biotin-SPA-PEG-CO ₂ -NHS reactant (Shearwater Corp.) of molecular weight 3,400 was coupled to hGH using the method described in Example 6.

Example 9

NHS-PEG hGH of Branched Chain Molecular Weight 10,000

PEG2-NHS (Shearwater Corp.) having a molecular weight of 10,000 was coupled to hGH using the method described in Example 6.

Example 10

NHS-PEG hGH with Branched Chain Molecular Weight 20,000

PEG2-NHS (Shearwater Corp.) with a molecular weight of 20,000 was coupled to hGH using the method described in Example 6.

Example 11

NHS-PEG hGH with Branched Chain Molecular Weight 40,000

Branched chain PEG2-NHS (Shearwater Corp.) having a molecular weight of 40,000 was coupled to hGH using the method described in Example 6.

Example 12

PEG-BTC-hGH with a linear molecular weight of 20,000

PEG-BTC (Shearwater Corp.) with a molecular weight of 20,000 was coupled to hGH using the method described in Example 6. Example 12 shows how a benzotriazole carbonate derivative of PEG produces a substantially uniform PEG-attached hGH.

Example 13

PEG-SS-hGH with a linear molecular weight of 5,000

Succinimidyl succinate-PEG (SS-PEG) (Shearwater Corp.) of molecular weight 5,000 was coupled to hGH using the method described in Example 6. Example 13 shows a method for producing PEG-attached hGH substantially uniformly using hydrolyzable bonds.

Example 14

PEG-CM-HBA-hGH with straight chain molecular weight 20,000

Carboxymethyl hydroxybutyric acid-PEG (CM-HBA-PEG) (Shearwater Corp.) having a molecular weight of 20,000 was coupled to hGH using the method described in Example 6. Example 14 shows a method for producing PEG-attached hGH substantially uniformly using hydrolyzable bonds.

Example 15

PEG-CM-HBA-hGH with straight chain 2-4x molecular weight 5,000

PEG-CM-HBA (Shearwater Corp.) having a molecular weight of 5,000 was coupled to hGH using the method described in Example 13.

Example 16

HZ-PEG-hGH with straight chain molecular weight 20,000

PEG-OCH ₂ CONHNH ₂

Example 16 shows a method for producing PEG-attached hGH substantially uniformly using methoxy-PEG-hydrazide, HZ-PEG (Shearwater Corp.) with a molecular weight of 20,000. The hGH protein stock solution was dissolved at 10 mg / mL in 10 mM MES at pH 4.0. Next, the solution was reacted with HZ-PEG by adding HZ-PEG mass to obtain a relative molar ratio of PEG: hGH of 0.1-5.0 to carboxyl groups. The reaction was catalyzed with carbodiimide (EDC, EOAC, EDEC) to give a final concentration of 2 mM to 4 mM. The reaction was carried out at 4 ° C. for 2 hours to overnight or at room temperature for 10 minutes to overnight. Purification by cation exchange terminated the reaction by removing unconjugated PEG and carbodiimide.

Example 17

PEG-attached species

In Examples 1 and 4 modified hGH with two or more PEGs attached were also obtained, and anion exchange chromatography was used to isolate from the species with one PEG attached. Modified hGH with two or more PEGs attached was also isolated from species with one PEG attached using cation exchange chromatography. Modified hGH with two or more PEGs attached was also obtained in Examples 2, 3, 5-13 and purified in a similar manner to Examples 1 and 4.

Example 18

Purification of hGH with PEG attached

PEG-attached hGH species were purified at least 95% from the reaction mixture using a single ion exchange chromatography step (SEC analysis).

Anion Exchange Chromatography

PEG hGH species were purified at least 95% from the reaction mixture using a single anion exchange chromatography step (SEC analysis). Anion exchange chromatography was used to purify hGH with one PEG attached from unmodified hGH and hGH species with multiple PEG attachments. A typical 20K aldehyde hGH reaction mixture (5-100 mg protein) mentioned above was Q-Sepharose Hitrap column (1 or 5 mL) equilibrated with 25 mM HEPES (buffer A) at pH 7.3 (Amershan Pharmacia Biotech, Piscataway). , NJ) or Q-Sepharose high flow column (26/20, 70 mL bed volume) (Amershan Pharmacia Biotech, Piscataway, NJ). The reaction mixture was diluted 5-10 times with Buffer A and loaded onto the column at a flow rate of 2.5 mL / min. The column was washed with 8 volumes of column buffer A. Subsequently, various hGH species were eluted with a buffer A of 80-100 times the volume of this column in a column with a NaCl linear gradient of 0-100 mM. Eluent was observed at absorbance of 280 nm (A ₂₈₀ ) and 5 mL fractions collected. Fractions were collected to the extent of PEG attachment, for example 1, 2, 3, etc. (as assessed in Example 15). The pooled fractions were concentrated to 0.5-5 mg / mL in a Centrirep YM10 concentrator (Amicon, Technology Corporation, Northborough, Mass.). The protein concentration of the collected fractions was measured by A ₂₈₀ using an extinction coefficient of 0.78. The total yield of purified mono 20K PEG-aldehyde hGH obtained in this process was 25-30%.

<Cation Exchange Chromatography>

Cation exchange chromatography was performed on an SP Sepharose high performance column (Pharmacia XK 26/20, 70 mL bed volume) equilibrated with 10 mM sodium acetate (Buffer B) at pH 4.0. The reaction mixture was diluted 10-fold with Buffer B and loaded onto the column at a flow rate of 5 mL / min. The column was washed with column 5 volumes of buffer B and then with column 5 volumes of 12% buffer C (10 mM acetate pH 4.5, 1M NaCl). PEG-hGH species were then eluted at 20 times this column volume in a column with a buffer C linear gradient of 12 to 27%. The eluate was observed at 280 nm and 10 mL fractions collected. Fractions were collected according to the degree of PEG attachment (1, 2, 3, etc.), exchanged in 10 mM acetate buffer at pH 4.5, and concentrated to 1-5 mg / mL in a stirred cell with Amicon YM10 membrane. I was. The protein concentration of the collected fractions was measured by A ₂₈₀ using an extinction coefficient of 0.78. The total yield of purified mono PEG-attached hGH obtained in this process was 10-50%.

Example 19

<Biochemical Characteristics>

The purified PEG-attached hGH fractions were characterized by reduced and non-reduced SDS-PAGE, modified and unmodified size exclusion chromatography, analytical anion exchange chromatography, N-terminal sequencing, hydrophobic interaction chromatography and reversed phase HPLC.

<Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)>

Unmodified SEC-HPLC

Unmodified SEC-HPLC was used to evaluate the reaction of methoxy-PEG with hGH, anion exchange purification pool, and final product with various binding chemistries, sizes, linkers, and geometric configurations. Analytical undenatured SEC-HPLC using a Tosohaas G4000PWXL column, 7.8 mm x 30 cm (Tosohaas Amersham Bioscience, Piscataway, NJ) or Superdex 200 (Amersham Bioscience, Piscataway, NJ) It was performed in 20 mM phosphate, 150 mM NaCl, pH 7.2 at a flow rate of minutes. PEGylation significantly increased the hydrodynamic volume of the protein and consequently reduced the settling time. New species along with unmodified hGH were also observed in the PEG aldehyde hGH reaction mixture. These PEG attached and unattached species were separated in Q-Sepharose chromatography. The resulting purified mono PEG-aldehyde hGH species were eluted in turn, showing a single peak in undenatured SEC (> 95% purity). The Q-Sepharose chromatography step effectively separated free PEG, hGH, hGH species with multiple PEG attachments from hGH with one PEG attachment. Unmodified SEC-HPLC showed that the effective size of the various PEG attached hGH was much larger than their respective theoretical molecular weights (Table 1).

Size Exclusion Chromatography (SEC) (Theory) Molecular weight Size (SEC) hGH 22,000 21,000 4-6x5K PEG-SPA GH 47,000 128,000 2-4x5K PEG-CMHBA (NHS) GH 37,000 71,000 20K PEG-ALD GH 42,000 120,000 20K branched PEG-ALD GH 42,000 114,000 20K PEG-CMHBA (NHS) GH 42,000 115,000 20K PEG-hydrazide GH 42,000 125,000 2x20K PEG-ALD GH 62,000 250,000 30K PEG-ALD GH 52,000 231,000 30K PEG-SPA GH 52,000 183,000 2x30K PEG-SPA GH 82,000 569,000 40K Branched PEG-ALD GH 62,000 330,000 40K Branched PEG-NHS GH 62,000 253,000

Denatured SEC-HPLC

Various reactions of methoxy-PEG with hGH, anion exchange purification and final purified product were evaluated using denaturing SEC-HPLC Analytical denaturation SEC-HPLC was 100 mM phosphate pH 6.8, 0.1% SDS at 0.8 mL / min flow rate. In a Tosohaas 3000SWXL column 7.8 mm × 30 cm (Tosohaas Pharmacia Biotech, Piscataway, NJ). PEG attachment increased the hydrodynamic volume of the protein and caused the migration to earlier retention times. A new species was observed in the 20K PEG aldehyde hGH reaction mixture with unmodified hGH. The PEG attached and non PEG attached species were separated by Q-Sepharose chromatography and the resultant single 20K PEG aldehyde hGH was later eluted with a single peak in denatured SEC (> 95% purity). The Q-Sepharose chromatography step effectively removed free PEG, hGH and multiple PEG attached hGH species from a single PEG attached hGH.

SDS PAGE / PVDF transition

SDS-PAGE was used to assess the reaction of various PEG reagents with hGH and the purified final product. The above example technology was shown using single 20K straight and branched 20K and 40K PEG aldehydes and 4 × 6 5K SPA PEG. (Figures 1 & 2). SDS-PAGE was run on 1-20 mm thick 10-20% Tris Tricine gel (Invitrogen, Carlsbad, Calif.) Under reducing and non-reducing conditions and Novex Colloidal Coomassie ^™ G-250 Staining was performed using a staining kit (Invitrogen, Carlsbad, Calif.). Purified single PEG-aldehyde hGH species shifted to one major band in SDS-PAGE. The bands were blotted onto PVDF membranes for later N-terminal sequence identification.

Analytical Anion Exchange HPLC

Analytical anion exchange HPLC was used to evaluate various mPEG and hGH reactions, anion exchange purified fractions and final purified products. Analytical anion exchange HPLC was performed using a Tosohas Q5PW or DEAE-PW anion exchange column, 7.5 mm × 75 mm (Tosohaas Pharmacia Biotech, Piscataway, NJ) at 50 mM Tris pH 8.6 at 1 mL / min flow rate. Samples were eluted using a linear gradient of 5-200 mM NaCl.

Reversed Phase HPLC (RP-HPLC)

The PEG-GH reaction mixture and the purified PEG attached product were analyzed by RP HPLC to identify hGH species, single and multiple PEG attached hGH species, oxidized hGH forms and different positions (e.g., N-terminus). PEG hGH isoform with single PEG linked to lysine ε-amino group) was monitored. RP-HPLC was performed using a Zorbax SB-CN 150 or 250 mm × 4.6 mm (3.5 mm or 5 mm) reversed phase HPLC column. The experiment was conducted at ambient temperature with typically 10 mg of protein per sample loaded. Buffer A is 0.1% trifluoroacetic acid in water and Buffer B is 0.1% trifluoroacetic acid in acetonitrile. The gradient causing a ½% increase in B per minute was as follows:

N-terminal sequence and peptide mapping

NH2-terminal protein sequences were determined using automated Edman degradation chemistry. Applied Biosystems Model 494 Procise Sequencer (Perkin Elmer, Wellesley, Mass.) Was used for digestion. Perkin Elmer / Brownlee 2.1 mm For RP-HPLC analysis on-line using an Applied Biosystems Model 140C PTH analyzer fitted using the same PTH-C18 column as above. Each PTH-AA derivative was identified. Solutions of 20K straight and 20 and 40K branched PEG-ALD hGH protein bands or purified 20K straight and branched chain 20 and 40K PEG-ALD hGH transferred to PVDF membranes were sequenced. It was observed that purified 20K straight chain PEG-hGH, which yields a major signal (about 88% yield), has the sequence predicted for hGH except when no N-terminal amino acid is present. The results are predicted for proteins with PEG attached to the N-terminus via aldehyde chemistry. The residues of the first cycle could not be recovered because of the attached PEG moiety. Less important signals (about 12% yield) had the correct N-terminal amino acid sequence. Given that the peak collected from the RP-HPCL is 100% PEG attached, the data means that about 88% of the PEG modifications are at the N-terminus and the remainder are obviously linked to some possible lysine residues.

Trypsin digestion was performed at a concentration of 1 mg / mL and typically 50 ug material per digest was used. Trypsin was added so that the trypsin to PEG-hGH ratio was 1: 30 (w / w). Tris buffer was present at 30 mM, pH 7.5. Samples were incubated at room temperature for 16 ± 0.5 hours. The reaction was stopped by adding 50 μl of 1N HC1 per mL of digestion solution. Samples were diluted to a final concentration of 0.25 mg / ml in 6.25% acetonitrile before placing the samples in an automated sampler. Acetonitrile was added first (to 19.8% acetonitrile), mixed slowly, and then water was added to the final volume (4 times the starting volume). The additional degradation solution can be removed and stored for one week at -20 ° C.

The Waters Alliance 2695 HPLC system is used for analysis, but other systems should yield similar results. The column used was an Astec C-4 polymeric 25 cm × 4.6 mm column with 5 μm particles. The experiment was carried out at ambient temperature, typically loading 50 μg of protein per sample. Buffer A is 0.1% trifluoroacetic acid in water and Buffer B is 0.85% trifluoroacetic acid in acetonitrile. The gradient was as follows:

The column was heated to 40 ° C. using a heat jacket. Data was collected at 210 and 300 nm to detect peaks using a Waters 996 PDA detector. The chromatogram extracted at 214 nm was used for sample analysis to determine the extent of N-terminal PEG attachment (loss of T-1 fragment) and is shown in Table 2.

Example 20

Pharmacokinetic Studies

Efficacy study in pituitary gland rats

Female Sprague Dawley rats from which the pituitary gland was removed from the Harlan Lab were preselected for growth rate over a period of 7 to 10 days. Thereafter, growth studies were run for 11 days. Rats were divided into 6-8 groups. Group 1 consists of rats subcutaneously administered with vehicle daily or on days 0 and 6. Group 2 received subcutaneous GH (0.3 mg / kg / dose) daily. Group 3 received subcutaneous GH at day 0 and day 6 (1.8 mg / kg / dose). Group 4 received subcutaneous PEG-GH at day 0 and day 6 (1.8 mg / kg / dose). Rats whose pituitary glands were removed were weighed more than two days apart during the study to monitor weight gain. The weight gain (mean +/- SEM) for 20K PEG-ALD hGH, 20K and 40K branched PEG-ALD hGH, and 4-6 × 5PEG-SPA hGH administration once a week was similar to the daily dose of hGH. (Tables 3 & 4). Table 3 summarizes the total weight gain (mean +/- SEM) at 11 days for the administration of various PEG-attached hGH molecules once a week compared to daily hGH administration.

At the completion of each growth study, animals were sacrificed and bone (thigh) length analyzed. FIG. 6 shows changes in tibia length (mean +/- SEM) at 11 days in response to administration of various PEG-GH conjugates on days 0 and 6 or daily administration of hGH.

IGF-1 Levels in Pituitary Removal Rats

The experiment was run as in the weight gain study but blood samples were taken on days 0, 1, 2, 3, 4, 5 and animals were sacrificed on day 9. IGF-1 levels were determined by ELISA. FIG. 7 compares the increase in serum IGF-1 levels (mean +/- SEM) after daily hGH or single dose of hGH or PEG-attached hGH in pituitary gland rats on day 0 and day 6 Doing.

Pharmacokinetic Studies

Pharmacokinetic studies were conducted in normal, Sprague-Dawley male rats, mice and cynomolgus monkeys. Six rats and up to 60 mice per group were used to inject either 1.8 mg / kg of a single subcutaneous bolus or a single iv dose of 1.0 mg / kg GH or PEG-GH in rats and mice. In cynomolgus monkeys, 0.18 mg / kg GH or PEG-GH was administered in both a single subcutaneous bolus and iv using 2-4 monkeys per group. Blood samples were appropriately taken over 1 to 5 days for evaluation of the relevant PK parameters (Table 4). (t _½ ) = final half-life, (Cl) = elimination, (T _max ) = time to maximum concentration, V _ss = volume distribution (normal) at steady state and (C _max ) = GH maximum concentration and PEG-GH Blood levels were monitored for each sample using an immunoassay test.

hGH immune assay

HGH and PEG-attached hGH in rat, mouse and cynomolgus monkey plasma using hGH AutoDELFIA kit fluorescence immunoassay assay (PerkinElmer Life Sciences), using appropriate PEG hGH to generate a standard curve Protein concentration levels were determined.

SEQUENCE LISTING <110> Pharmacia Corporation Finn, Rory Liao, Wei Siegel, Ned <120> CHEMICALLY-MODIFIED HUMAN GROWTH HORMONE CONJUGATES <130> 03582/1 / PCT <150> US 60/331907 <151> 2001-11-20 <160> 1 <170> PatentIn version 3.1 <210> 1 <211> 191 <212> PRT <213> homo sapiens <400> 1 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asp Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asp Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asp Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asp Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Ser Leu Arg Ser Val 85 90 95 Phe Ala Asp Ser Leu Val Tyr Gly Ala Ser Asp Ser Asp Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asp Ser His Asp Asp Asp Ala Leu Leu Lys Asp Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190

Claims (79)

A conjugate comprising one or more water soluble polymers covalently attached to one or more amino acid residues of a bioactive human growth hormone (hGH) polypeptide or agonist variant thereof. The conjugate of claim 1, wherein the hGH polypeptide comprises the amino acid sequence of SEQ ID NO: 1. The conjugate of claim 1 or 2, wherein said polymer is a poly (ethylene oxide) molecule. 4. The conjugate of claim 3, wherein said poly (ethylene oxide) molecule is a poly (ethylene glycol) molecule. The conjugate of claim 4 wherein the poly (ethylene glycol) is attached to an amino acid residue having free amino group (s), carboxy group (s) or sulfhydryl group (s). The conjugate of claim 5, wherein the conjugate is formed using activated poly (ethylene glycol). 7. The conjugate of claim 6, wherein said activated poly (ethylene glycol) comprises a functional group. 8. The conjugate of claim 7, wherein said attachment is at an amino acid having free amino groups. The conjugate according to claim 8, wherein said functional group is selected from the group consisting of carbonate, carbonyl imidazole, active ester of carboxylic acid, azlactone, cyclic imide thione, isocyanate or isothiocyanate, imidate and aldehyde gate. 10. The conjugate of claim 9, wherein said functional group is a carbonate or carbonyl imidazole. The conjugate of claim 10, wherein the activated poly (ethylene glycol) is selected from the group consisting of: The conjugate of claim 11 having the structure [Where R is a human growth hormone polypeptide] The conjugate of claim 12, wherein the human growth hormone polypeptide comprises the amino acid of SEQ ID NO: 1. 10. The conjugate of claim 9, wherein said functional group is a cyclic imide thione. 15. The conjugate of claim 14, wherein said activated poly (ethylene glycol) is selected from the group: [Where L is -O-, -NH-, -OCH _2- , -NH-CO (CH ₂ ) _n , -NH-CO (CH ₂ ) _n O-, -CO-NH (CH ₂ ) _n- , -S-, -CO-NH (CH ₂ ) _n O-, -O (CH ₂ ) _n O-, -O (CH ₂ ) _n- , -SCH ₂ CH ₂ -and -NH (CH ₂ ) _n Selected from the group consisting of 10. The conjugate of claim 9, wherein said functional group is azlactone. The conjugate of claim 16 wherein said activated poly (ethylene glycol) is selected from the group consisting of: [Wherein R ¹ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, carbocyclic and heterocyclic aromatic rings, α, β-unsaturated alkyl; R ² and R ³ are independently selected from the group consisting of hydrogen, alkyl, aryl and alkylaryl] 10. The conjugate of claim 9, wherein said functional group is an isocyanate or isothiocyanate. 19. The conjugate of claim 18, wherein said activated poly (ethylene glycol) is selected from the group consisting of mPEG-N = C = O and mPEG-N = C = S. 10. The conjugate of claim 9, wherein said functional group is an aldehyde or an aldehyde hydrate. The conjugate of claim 20, wherein the activated poly (ethylene glycol) is selected from the group consisting of: 10. The conjugate of claim 9, wherein said functional group is an active ester of carboxylic acid. The conjugate of claim 22, wherein said activated poly (ethylene glycol) is selected from the group consisting of: The conjugate of claim 23, wherein the conjugate has a structure selected from the group consisting of: [Where R is a human growth hormone polypeptide] The conjugate of claim 24, wherein said human growth hormone polypeptide comprises the amino acid of SEQ ID NO: 1. 10. The conjugate of claim 9, wherein said activated poly (ethylene glycol) is selected from the group consisting of: mPEG-O-SO ₂ -CH ₂ CF ₃ and The conjugate of claim 26 having a structure selected from the group consisting of: PEG-NH-R and [Where R is a human growth hormone polypeptide] The conjugate of claim 27, wherein said growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO: 1. 29. The conjugate of claim 28, wherein said functional group is an imidate. 10. The conjugate of claim 9, wherein said activated poly (ethylene glycol) is selected from the group consisting of: [Wherein m is 0 to 20] Wherein R ¹ is alkyl, phenyl, phenylalkyl and cycloalkyl] 10. The conjugate of claim 9, wherein said free amino group is an amino terminal a-amino group. 32. The conjugate of claim 31, wherein said amino terminal α-amino group is phenylalanine. The conjugate of claim 8, wherein said attachment occurs at an amino acid having a free carboxyl group. 34. The conjugate of claim 33, wherein said functional group is selected from the group consisting of primary amine, hydrazine and hydrazide functional groups. 35. The conjugate of claim 34, wherein said functional group is selected from the group consisting of The conjugate of claim 8, wherein said attachment occurs at an amino acid having a free sulfhydryl group. 37. The conjugate of claim 36, wherein said functional group is selected from the group consisting of thiols, maleimides, vinyl sulfones, and phenyl glyoxal. 38. The conjugate of claim 37, wherein said functional group is selected from the group consisting of The conjugate of claim 38 having a structure selected from the group consisting of: [Where R is a human growth hormone polypeptide] 40. The conjugate of claim 39, wherein said human growth hormone polypeptide comprises an amino acid of SEQ ID NO: 1. The conjugate of claim 8, wherein said poly (ethylene glycol) has a molecular weight of about 0.5 kDa to about 100 kDa. 42. The conjugate of claim 41, wherein said poly (ethylene glycol) has a molecular weight of about 5 kDa to about 40 kDa. 9. The conjugate of claim 8, wherein said poly (ethylene glycol) is a branched chain polymer. 44. The conjugate of claim 43, wherein said branched chain polymer is selected from the group consisting of: Human growth hormone-PEG having the structure [Where R is a human growth hormone polypeptide] 46. The conjugate of claim 45, wherein said human growth hormone polypeptide comprises an amino acid of SEQ ID NO: 1. 47. The conjugate of claim 46, wherein at least 80% of said polypeptide glycol is conjugated to amino-terminal phenylalanine. 47. The conjugate of claim 46, wherein at least 90% of said polypeptide glycol is conjugated to amino-terminal phenylalanine. 49. The conjugate of claim 47 or 48, wherein each mPEG has a molecular weight of about 20 kDa. Human growth hormone-PEG conjugate having the formula [Where R is a human growth hormone polypeptide] 51. The conjugate of claim 50, wherein said human growth hormone polypeptide comprises an amino acid of SEQ ID NO: 1. 53. The conjugate of claim 51, wherein at least 80% of said polypeptide glycol is conjugated to amino-terminal phenylalanine. 53. The conjugate of claim 51, wherein at least 90% of said polypeptide glycol is conjugated to amino-terminal phenylalanine. The conjugate of claim 52 or 53, wherein each mPEG has a molecular weight of about 20 kDa. Human growth hormone-PEG having the structure mPEG-OCH ₂ CH ₂ CH ₂ NH-R [Where R is a human growth hormone polypeptide] The conjugate of claim 55, wherein said human growth hormone polypeptide comprises the amino acid of SEQ ID NO: 1. 57. The conjugate of claim 56, wherein at least 80% of said polypeptide glycol is conjugated to amino-terminal phenylalanine. 59. The conjugate of claim 56, wherein at least 90% of said polypeptide glycol is conjugated to amino-terminal phenylalanine. 59. The conjugate of claim 57 or 58, wherein each mPEG has a molecular weight of about 20 kDa. 9. The conjugate of claim 8, wherein said poly (ethylene glycol) is a bifunctional polymer. 9. The conjugate of claim 8, wherein said poly (ethylene glycol) is a prodrug. A composition comprising the hGH of claim 1 and a pharmaceutically acceptable carrier. A method of treating a patient with a growth or development disorder comprising administering a therapeutically effective amount of the hGH conjugate of claim 1 to a patient with a growth or development disorder. The method of claim 63, wherein the growth or development disorder is growth hormone deficiency (GHD). 64. The method of claim 63, wherein said growth or development disorder is Turner's syndrome. 64. The method of claim 63, wherein said growth or development disorder is chronic renal failure. The method of claim 63, wherein the growth or development disorder is small for gestational age (SGA). 49. A composition comprising the hGH of claim 48 and a pharmaceutically acceptable carrier. A method of treating a patient with a growth or development disorder comprising administering a therapeutically effective amount of the hGH conjugate of claim 48 to a patient with a growth or development disorder. 70. The method of claim 69, wherein the growth or development disorder is growth hormone deficiency (GHD). The method of claim 69, wherein the growth or development disorder is Turner syndrome. 70. The method of claim 69, wherein said growth or development disorder is chronic renal failure. The method of claim 69, wherein the growth or development disorder is low birth weight baby (SGA). A composition comprising the hGH of claim 50 or 55 and a pharmaceutically acceptable carrier. A method of treating a patient with a growth or development disorder comprising administering a therapeutically effective amount of the hGH conjugate of claim 1 to a patient with a growth or development disorder. 76. The method of claim 75, wherein said growth or development disorder is growth hormone deficiency (GHD). 76. The method of claim 75, wherein said growth or development disorder is Turner syndrome. 76. The method of claim 75, wherein the growth or development disorder is chronic renal failure. 76. The method of claim 75, wherein said growth or development disorder is low birth weight baby (SGA).