Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/61—Growth hormones [GH] (Somatotropin)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/27—Growth hormone [GH] (Somatotropin)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH

Definitions

• the present invention relates to a chemical modification of human Growth Hormone (hGH) and agonist variants thereof by which the chemical and/or physiological properties of hGH can be changed.
• the PEGylated hGH may have an increased plasma residency duration, decreased clearance rate, improved stability, decreased antigenicity, or a combination thereof .
• the present invention also relates to processes for the modification of hGH.
• the present invention relates to pharmaceutical compositions comprising the modified hGH.
• a further embodiment is the use of the modified hGH for the treatment of growth and development disorders .
• Human growth hormone is a protein comprising a single chain of 191 amino acids cross- linked by two disulphide bridges and the monomeric form has a molecular weight of 22 kDa. Human GH is secreted by the pituitary gland and which also can be produced by recombinant genetic engineering. hGH will cause growth in all bodily tissues that are capable of growth. Recombinant hGH has been commercially available for several years . Two types of therapeutically useful recombinant hGH preparations are present on the market: the authentic one, e.g. GenotropinTM, or NutropinTM and an analogue with an additional ethionine residue at the N-terminal end, e.g. SomatonormTM.
• the authentic one e.g. GenotropinTM, or NutropinTM
• an analogue with an additional ethionine residue at the N-terminal end e.g. SomatonormTM.
• hGH is used to stimulate linear growth in patients with hypo pituitary dwarfism also referred to as Growth Hormone Deficiency (GHD) or Turner's syndrome but other indications have also been suggested including long-term treatment of growth failure in children who were born short for gestational age (SGA) , for treatment of patients with Prader-Willi syndrome (PWS) , chronic renal insufficiency (CRI) , Aids wasting, and Aging.
• GDD hypo pituitary dwarfism
• SGA gestational age
• PWS Prader-Willi syndrome
• CRI chronic renal insufficiency
• Aids wasting Aids wasting
• Aging Aging.
• GH growth hormone
• the organ systems affected include the skeleton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys .
• Growth hormones exert their effect through interaction with specific receptors on the target cell's membrane.
• hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants or growth hormone (Nicoll, C. S., et al . (1986) Endocrine Reviews 7: 169) .
• hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W. , et al . [1987]
• hGH Human growth hormone
• hGH Human growth hormone
• hGH Human growth hormone
• GH Human growth hormone
• GH 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 potent anabolic agent, especially due to retention of nitrogen, phosphorus, potassium, and calcium.
• Treatment of hypophysecto ized rats with GH can restore at least a portion of the growth rate of the rats. Moore et al., Endocrinology 122:2920-2926 (1988).
• hypo pituitary (GH-deficient) subjects is accelerated linear growth of bone-growth- plate-cartilage resulting in increased stature. Kaplan, Growth Disorders in Children and Adolescents (Springfield, IL: Charles C. Thomas, 1964) .
• hGH causes a variety of physiological and metabolic effects in various animal models including linear bone growth, lactation, activation of macrophages, insulin-like and diabetogenic effects, and others (R. K. Chawla et al . , Annu . Rev. Med. 34:519 (1983); 0. G. P. Isaksson et al . , Annu . Rev. Physiol . 47, 483 (1985); C. K. Edwards et al . , Science 239, 769 (1988); M. 0. Thomer and M. L. Vance, J. Clin . Invest . 82:745 (1988); J. P. Hughes and H. G. Friesen, Ann . Rev. Physiol .
• water-soluble polymers such as copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, poly(vinyl alcohol) , poly(vinyl pyrrolidone) , poly (-1, 3-dioxolane) , poly(- 1, 3 , 6-trioxane) , ethylene/maleic anhydride copolymer, poly- amino acids (either homopolymers or random copolymers) .
• ADAGEN® a pegylated formulation of adenosine deaminase
• ONCASPAR® a pegylated L-asparaginase
• Pegylated superoxide dismutase has been in clinical trials for treating head injury.
• Pegylated - interferon U.S. 5,738,846, 5,382,657
• pegylated glucocerebrosidase and pegylated hemoglobin are reported to have been in preclinical testing.
• pegylated IL-6 EF 0 442 724, entitled, "Modified hIL-6, " which discloses poly (ethylene glycol) molecules added to IL-6.
• G-CSF granulocyte colony stimulating factor
• U.S. 5,824,784 Provided in U.S. 5,824,784 are methods for N-terminally modifying proteins or analogs thereof, and resultant compositions, including novel N-terminally chemically modified G-CSF compositions.
• U.S. 5,824,778 discloses chemically modified G-CSF.
• poly (ethylene glycol) For poly (ethylene glycol) , a variety of means have been used to attach the poly (ethylene glycol) molecules to the protein. Generally, pol (ethylene glycol) molecules are connected to the protein via a reactive group found on the protein.
• Amino groups such as those on lysine residues or at the N-terminus, are convenient for such attachment.
• Royer U.S. Pat. No. 4,002,531, above states that reductive alkylation was used for attachment of poly(ethylene glycol) molecules to an enzyme.
• US 5,298,643 and US 5,637,749 disclose PEG aryl imidates
• WO 93/00109 relates to a method for stimulating a mammal's or avian's GH responsive tissues comprising maintaining a continuous, effective plasma GH concentration for a period of 3 or more days .
• One way of achieving such plasma concentration is stated to be by use of GH coupled to a macromolecular substance such as PEG (polyethylene glycol) .
• the coupling to a macromolecular substance is stated to result in improved half-life.
• PEGylated human growth hormone has been reported in WO 93/00109 using PEG aldehyde-5000 and mPEG N-hydroxysuccinmidyl ester (mPEG-NHS-5000) .
• mPEG-NHS resulted in heterogeneous mixtures of multiply PEGylated forms of hGH.
• WO 93/00109 also discloses the use of mPEG-maleimide to PEGylate cysteine hGH variants.
• WO 99/03887 discloses a cysteine variant growth hormone that is PEGylated. Designated as BT-005, this conjugate is purported to be more effective at stimulating weight gain in growth hormone deficient rats and to have a longer half-life than hGH.
• WO 94/20069 prophetically discloses PEGylated hGH as part of a formulation for pulmonary delivery.
• US 4,179,337 discloses methods of PEGylating enzymes and hormones to obtain physiologically active non-immunogenic, water-soluble polypeptide conjugates.
• GH is mentioned as one example of a hormone to be PEGylated.
• EP 458064 A2 discloses PEGylation of introduced or naturally present cysteine residues in somatotropin .
• EP 458064 A2 further mentions the incorporation of two cysteine residues in a loop termed the omega loop stated to be located at residues 102-112 in wild type bovine somatotropin, more specifically EP 458064 A2 discloses the substitution of residues numbered 102 and 112 of bovine somatotropin from Ser to Cys and Tyr to Cys , respectively.
• WO 95/11987 suggests attachment of PEG to the thiol group of a cysteine residue being either present in the parent molecule or introduced by site directed mutagenesis.
• WO 95/11987 relates to PEGylation of protease nexxn-1, however PEGylation in general of hGH and other proteins is suggested as well.
• WO 99/03887 discloses, e.g., growth hormone modified by insertion of additional cysteine for serine residues_and attachment of PEG to the introduced cysteine residues .
• WO 00/42175 relates to a method for making proteins containing free cysteine residues for attachment of PEG.
• WO 00/42175 discloses the following muteins of hGH: T3C, S144C and T148C and the cysteine PEGylation thereof.
• WO 9711178 (as well as US 5849535, US 6004931, and US 6022711) relates to the use of GH variants as agonists or antagonists of hGH.
• WO 9711178 also discloses PEGylation of hGH, including lysine PEGylation and the introduction or replacement of lysine (e.g. K168A and K172R) .
• WO 9711178 also discloses the substitution G120K.
• a GH molecule with a longer circulation half-life would decrease the number of necessary administrations and potentially provide more optimal therapeutic hGH levels with concomitant enhanced therapeutic effect.
• the present invention provides chemically modified hGH conjugates having decreased heterogeneity, decreased clearance rate, increased plasma residency duration, improved solubility, increased stability, decreased antigenicity, or combinations thereof .
• the present invention relates to chemically modified hGH and agonist variants thereof, which have at least one improved chemical or physiological property selected from but not limited to decreased clearance rate, increased plasma residency duration, increased stability, improved solubility, and decreased antigenicity.
• the present invention has a number of aspects relating to chemically modifying hGH and agonist variants thereof as well as specific modifications using a variety of poly (ethylene glycol) moieties .
• the present invention also relates to methods of producing the chemically modified hGH and agonist variants thereof .
• the present invention also relates to compositions comprising the chemically modified hGH and agonist variants thereof.
• the modified hGH and agonist variants thereof of the present invention may be useful in the treatment of, but not limited to, dwarfism (GHD) , Adult GHD, Turner's syndrome, long-term treatment of growth failure in children who were born short for gestational age (SGA) , for treatment of patients with Prader-Willi syndrome (PWS) , chronic renal insufficiency (CRI) , Aids wasting, Aging, End-stage Renal Failure, and Cystic Fibrosis .
• GHD dwarfism
• SGA gestational age
• PWS Prader-Willi syndrome
• CRI chronic renal insufficiency
• Aids wasting Aging
• End-stage Renal Failure End-stage Renal Failure
• Cystic Fibrosis .
• Figure 1 is a reproduction of a reducing and non- reducing SDS-PAGE analysis of the products of the reaction of hGH and 2OK PEG-ALD and the anion exchange purified 20K PEG-ALD hGH.
• Lane 1. MW Protein standards; 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 linear PEG-ALD hGH-10 ug Lane 5. Blank; Lane 6. non-reduced hGH-10 ug; Lane 7. non-reduced 20 K linear PEG-ALD hGH reaction mix-10 ug; Lane 8. non-reduced anion exchange purified 20 K linear PEG-ALD hGH-10 ug; Lane 9. Blank; Lane 10. MW Protein standards.
• Figure 2 is a reproduction of a non-reducing SDS-PAGE analysis of various anion exchange purified pegylated hGH molecules.
• Lane 1. MW Protein standards; Lane 2. hGH-10 ug; Lane 2. 4-6 x 5K PEG-SPA hGH-10 ug; Lane 3. 20 K linear PEG-ALD hGH-10 ug; Lane 4. 20 K branched PEG-ALD hGH-10 ug Lane 5. 40 K branched PEG hGH-10 ug.
• Figure 3 shows reproductions of RP-HPLC elution profiles for trypsin digests of hGH, 40K Br PEG-ALD hGH and 4OK Br PEG-NHS hGH.
• PEG coupled primarily to the N-terminus of hGH results in a reduction in the N-terminal (TI) fragment peak with generation of a new PEGylated TI peak.
• Figure 4 compares the in vivo bioactivity of unPEGylated hGH dosed daily (0.3 mg/Kg/day) to ono- PEGylated hGH dosed subcutaneously(SC) once every six days (1.8 mg/Kg) by illustrating the weight gain in hypophysectomized rats during a period of 11 days.
• Figure 5 compares the in vivo bioactivity of unPEGylated hGH dosed SC daily (0.3 mg/Kg/day) to 4-6 x 5K PEG-SPA-hGH, mono-PEGylated 2OK branched PEG-ALD hGH, and mono-PEGylated 4OK branched PEG-ALD hGH each dosed SC once every six days (1.8 mg/Kg) by illustrating the weight gain in hypophysectomized rats during a period of 11 days .
• Figure 6 compares the in vivo bioactivity of unPEGylated hGH dosed SC daily (0.3 mg/Kg/day) to 4-6 x 5K PEG-CMHBA-hGH, mono-PEGylated 2OK linear ALD, ono-PEGylated 3 OK linear ALD, mono-PEGylated 2OK branched PEG-ALD hGH, and mono-PEGylated 4OK branched PEG-ALD hGH each dosed SC once every six days (1.8 mg/Kg) by illustrating the increase in tibial bone growth in hypophysectomized rats during a period of 11 days .
• Figure 7 compares the in vivo bioactivity of a single 1.8 mg/Kg SC dose of unPEGylated hGH, mono-PEGylated 5K linear PEG-ALD hGH, mono-PEGylated 20K linear PEG- ALD hGH, mono-PEGylated 2OK branched PEG-ALD hGH, mono-PEGylated 20K linear PEG-Hydrazide hGH, mono- PEGylated 3OK linear PEG-ALD hGH, mono-PEGylated 4OK branched PEG-ALD hGH, 4-6 x 5K PEG SPA hGH, 4-6 x 5K PEG-CMHBA hGH by illustrating the increase in plasma IGF-1 levels in hypophysectomized rats during a period of 9 days .
• hGH and agonist variants thereof are members of a family of recombinant proteins, described in US 4,658,021 and US 5,633,352. Their recombinant production and methods of use are detailed in US 4,342,832, 4,601,980; US 4,898,830; US 5,424,199; and US 5,795,745.
• hGH or agonist variant thereof which is produced by host cells such as E. coli and animal cells transformed or transfected by using recombinant genetic techniques, may be used in the present invention. Additional hGH variants are described in U.S. Ser. No. 07/715,300 filed Jun.14, 1991 and Ser. No. 07/743,614 filed Aug. 9, 1991, and WO 92/09690 published Jun. 11, 1992. Among them, hGH or agonist variant thereof, which is produced by the transformed E. coli , is particularly preferable. Such hGH or agonist variant thereof may be obtained in large quantities with high purity and homogeneity. For example, the above hGH or agonist variant thereof may be prepared according to a method disclosed in US
• the term "substantially has the following amino acid sequence” means that the above amino acid sequence may include one or more amino-acid changes (deletion, addition, insertion or replacement) as long as such changes will not cause any disadvantageous non-similarity in function to hGH or agonist variant thereof. It is more preferable to use the hGH or agonist variant thereof substantially having an amino acid sequence, in which at least one lysine, aspartic acid, glutamic acid, unpaired cysteine residue, a free N-terminal ⁇ -amino group or a free C-terminal carboxyl group, is included.
• poly (ethylene glycol) is covalently bound through amino acid residues of hGH or agonist variant thereof.
• 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, which may be used to create PEG-hGH conjugates or PEG-hGH agonist variant conjugates (for reviews see Roberts M.J. et al . , Adv. Drug Del . Rev. 54:459-476, 2002), Harris J.M. et al .
• the amino acid residue may be any reactive one(s) having, for example, free amino, carboxyl, sulfhydryl (thiol) , hydroxyl, guanidinyl, or imidizoyl groups, to which a terminal reactive group of an activated poly (ethylene glycol) may be bound.
• the amino acid residues having the free amino groups may include lysine residues and/or N-terminal amino acid residue, those having a free carboxyl group may include aspartic acid, glutamic acid and/or C-terminal amino acid residues, those having a free sulfhydryl (thiol) such as cysteine, those having a free hydroxyl such as serine or tyrosine, those having a free guanidinyl such as arginine, and those having a free imidizoyl such as histidine.
• thiol such as cysteine
• cysteine those having a free hydroxyl such as serine or tyrosine
• guanidinyl such as arginine
• free imidizoyl such as histidine.
• oxime chemistries (Lemieux & Bertozzi Tib Tech 16:506-513, 1998) are used to target N-terminal serine residues.
• the poly(ethylene glycol) used in the present invention is not restricted to any particular form or molecular weight range.
• the poly (ethylene glycol) molecular weight may between 500 and 100,000. Normally, a molecular weight of 500-60,000 is used and preferably of from 1,000-40,000. More preferable, the molecular weight is greater than 5,000 to about 40,000.
• the poly (ethylene glycol) is a branched PEG having more than one PEG moiety attached.
• Preferred examples of branched PEGs are described in U.S. 5,932,462; U.S. 5,342,940; U.S. 5,643,575; U.S. 5,919,455; U.S. 6,113,906; U.S. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5:283-288 (1994); and Ya asaki et al . ,
• each poly (ethylene glycol) of the branched PEG is 5,000-20,000.
• Poly (alkylene oxide) s are bound to hGH or agonist variant thereof via a terminal reactive group, which may or may not leave a linking moiety (spacer) between the PEG and the protein.
• a terminal reactive group which may or may not leave a linking moiety (spacer) between the PEG and the protein.
• polymers such as poly (alkylene oxide) are converted into activated forms, as such term is known to those of ordinary skill in the art.
• the reactive group for example, is a terminal reactive group, which mediates a bond between chemical moieties on the protein, such as amino, carboxyl or thiol groups, and poly (ethylene glycol) .
• one or both of the terminal polymer hydroxyl end-groups, i.e.
• alpha and omega terminal hydroxyl groups are converted into reactive functional groups, which allows covalent conjugation.
• This process is frequently referred to as “activation” and the poly (ethylene glycol) product having the reactive group is hereinafter referred to as “an activated poly (ethylene glycol)".
• Polymers containing both and ⁇ linking groups are referred to as “bis- activated poly (alkylene oxides)” and are referred to as “bifunctional” .
• Polymers containing the same reactive group on ⁇ and ⁇ terminal hydroxyls are sometimes referred to as “homobifunctional” or “homobis-activated” .
• Polymers containing different reactive groups on ⁇ and ⁇ terminal hydroxyls are sometimes referred to as “heterobifunctional” (see for example WO 01/26692) or “heterobis-activated” .
• Polymers containing a single reactive group are referred to as “mono-activated” polyalkylene oxides or “mono-functional” .
• Other substantially non-antigenic polymers are similarly “activated” or “functionalized” .
• the activated polymers are thus suitable for mediating a bond between chemical moieties on the protein, such as a- or ⁇ -amino, carboxyl or thiol groups, and poly (ethylene glycol).
• Bis-activated polymers can react in this manner with two protein molecules or one protein molecule and a reactive small molecule in another embodiment to effectively form protein polymers or protein-small molecule conjugates through cross linkages.
• Functional groups capable of reacting with either the amino terminal ⁇ -amino group or ⁇ -amino groups of lysines found on the hGH or agonist variant thereof include: N-hydroxysuccinimidyl esters, carbonates such as the p-nitrophenyl , or succinimidyl (US 5,808,096, 5,612,460, US 5,324,844, US 5,5,122,614); carbonyl imidazole; azlactones (US 5,321,095, US 5,567,422); cyclic imide thiones (US 5,405,877, 5,349,001); isocyanates or isothiocyanates (Greenwald R.B., J " . Org. Chem.
• Functional groups capable of reacting with carboxylic acid groups, reactive carbonyl groups and oxidized carbohydrate moieties on hGH or agonist variant thereof include; primary amines; and hydrazine and hydrazide functional groups such as the acyl hydrazides, carbazates, semicarbamates, thiocarbazates, etc (WO 01/70685) .
• Mercapto groups if available on the hGH or agonist variant thereof, can also be used as attachment sites for suitably activated polymers with reactive groups such as thiols; maleimides, sulfones, and phenyl glyoxals; see, for example, U.S. Pat. No. 5,093,531, the disclosure of which is hereby incorporated by reference.
• Other nucleophiles capable of reacting with an electrophilic center include, but are not limited to, for example, hydroxyl, amino, carboxyl, thiol, active ethylene and the like.
• polymers including lipophilic and hydrophilic moieties disclosed in US 5,359,030 and US 5,681,811; US 5,438,040; and US 5,359,030.
• halogenated PEGs are disclosed on WO 98/32466 that can react with amino, thiol groups, and aromatic hydroxy groups, which directly covalently attach the PEG to the protein.
• secondary amine or amide linkages are formed using the N- terminal -amino group or ⁇ -amino groups of lysine of hGH or agonist variant thereof and the activated PEG.
• a secondary amine linkage is formed between the N- terminal primary ⁇ - or ⁇ -amino group of hGH or agonist variant thereof and single or branched chain PEG aldehyde by reduction with a suitable reducing agent such as NaCNBH 3 , NaBH 3 , Pyridine Borane etc. as described in Chamow et al . , Bioconjugate Chem. 5: 133- 140 (1994) and US Pat. No 5,824,784.
• a suitable reducing agent such as NaCNBH 3 , NaBH 3 , Pyridine Borane etc.
• 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 97%, and most preferably at least 98% of the pol (ethylene glycol) is on the amino terminal ⁇ -amino group.
• polymers activated with amide-forming linkers such as succinimidyl esters, cyclic imide thiones, or the like are used to effect the linkage between the hGH or agonist variant thereof and polymer, see for example, U.S. Pat. No. 5,349,001; U.S. Pat. No. 5,405,877; and Greenwald, et al . , Cri t . Rev. Ther. Drug Carrier Syst . 17:101-161, 2000, which are incorporated herein by reference.
• amide-forming linkers such as succinimidyl esters, cyclic imide thiones, or the like
• One preferred activated poly(ethylene glycol) which may be bound to the free amino groups of hGH or agonist variant thereof includes single or branched chain N-hydroxysuccinylimide poly (ethylene glycol) may be prepared by activating succinic acid esters of poly (ethylene glycol) with N- hydroxysuccinylimide .
• Other preferred embodiments of the invention include using other activated polymers to form covalent linkages of the polymer with the hGH or agonist variant thereof via ⁇ -amino or other groups .
• isocyanate or isothiocyanate forms of terminally activated polymers can be used to form urea or thiourea-based linkages with the lysine amino groups (Greenwald R.B., J. Org. Chem. , 60:331-336, 1995) .
• carbamate (urethane) linkages are formed with protein amino groups as described in U.S. Pat. Nos. 5,122,614, 5,324,844, and 5,612,640, which are hereby incorporated by reference.
• Examples include N- succinimidyl carbonate, para-nitrophenyl carbonate, and carbonyl imidazole activated polymers .
• a benzotriazole carbonate derivative of PEG is linked to amino groups on hGH or agonist variant thereof .
• Another aspect of the invention represents a prodrug or sustained release form of hGH or agonist variant thereof, comprised of a water soluble polymer, such as poly (ethylene glycol) , attached to an hGH or agonist variant thereof molecule by a functional linker that can predictably break down by enzymatic or pH directed hydrolysis to release free hGH or agonist variant thereof or other hGH or agonist variant thereof derivative.
• the prodrug can also be a "double prodrug" (Bundgaard in Advanced Drug Delivery Reviews 3:39-65, 1989) involving the use of a cascade latentiation.
• the hydrolytic reaction involves an initial rate-limiting (slow) enzymatic or pH directed step and a second step involving a rapid non-enzymatic hydrolysis that occurs only after the first has taken place.
• a releasable polymer provides protein conjugates, which are impermanent and could act as a reservoir, that continually discharge hGH or agonist variant thereof .
• Such functional linkers are described in US 5,614,549; US 5,840,900; US 5,880,131; US 5,965,119; us 5,965,565; US 6,011,042; US 6,153,655; US 6,180,095 Bl; US 6,413,507; Greenwald R.B. et al . , J. Med. Chem.
• Conjugation reactions referred to as pegylation reactions, were historically carried out in solution with molar excess of polymer and without regard to where the polymer will attach to the protein. Such general techniques, however, have typically been proven inadequate for conjugating bioactive proteins to non-antigenic polymers while retaining sufficient bioactivity.
• One way to maintain the hGH or agonist variant thereof bioactivity is to substantially avoid the conjugation of those hGH or agonist variant thereof reactive groups associated with the receptor binding site(s) in the polymer coupling process.
• Another aspect of the present invention is to provide a process of conjugating poly (ethylene glycol) to hGH or agonist variant thereof maintaining high levels of retained activity.
• the chemical modification through a covalent bond may be performed under any suitable condition generally adopted in a reaction of a biologically active substance with the activated poly (ethylene glycol) .
• the conjugation reaction is carried out under relatively mild conditions to avoid inactivating the hGH or agonist variant thereof. Mild conditions include maintaining the pH of the reaction solution in the range of 3 to 10 and the reaction temperatures within the range of from about 0°-37°C.
• suitable buffers pH 3 to 10
• suitable buffers including phosphate, MES, citrate, acetate, succinate or HEPES, for 1-48 hrs at 4°-37°C.
• the activated poly (ethylene glycol) may be used in about 0.05-100 times, preferably about 0.01 -2.5 times, the molar amount of the number of free amino groups of hGH or agonist variant thereof.
• the above modification is preferably carried out in pH from about 3.5 to about 5.5, for example, the modification with poly (oxyethylenediamine) is carried out in the presence of carbodiimide (pH 3.5-5) for 1-24 hrs at 4° -37°C.
• the activated poly (ethylene glycol) may be used in 0.05-300 times the molar amount of the number of free carboxyl groups of hGH or agonist variant thereof .
• the upper limit for the amount of polymer included in the conjugation reactions exceeds about 1:1 to the extent that it is possible to react the activated polymer and hGH or agonist variant thereof without forming a substantial amount of high molecular weight species, i.e. more than about 20% of the conjugates containing more than about one strand of polymer per molecule of hGH or agonist variant thereof.
• ratios of up to about 6 : 1 can be employed to form significant amounts of the desired conjugates which can thereafter be isolated from any high molecular weight species .
• bifunctionally activated PEG derivatives may be used to generate polymeric hGH or agonist variant thereof- PEG molecules in which multiple hGH or agonist variant thereof molecules are crosslinked via PEG.
• the reaction conditions described herein can result in significant amounts of unmodified hGH or agonist variant thereof, the unmodified hGH or agonist variant thereof can be readily recycled into future batches for additional conjugation reactions.
• the processes of the present invention generate surprisingly very little, i.e. less than about 30% and more preferably, less than about 10%, of high molecular weight species and species containing more than one polymer strand per hGH or agonist variant thereof .
• reaction conditions are to be contrasted with those typically used for polymeric conjugation reactions wherein the activated polymer is present in several-fold molar excesses with respect to the target.
• the polymer is present in amounts of from about 0.1/amino group to about 50 equivalents per equivalent of hGH or agonist variant thereof.
• the polymer is present in amounts of from about 1 to about 10 equivalents per equivalent of hGH or agonist variant thereof.
• the conjugation reactions of the present invention initially provide a reaction mixture or pool containing mono- and di-PEG-hGH conjugates, unreacted hGH, unreacted polymer, and usually less than about 20% high molecular weight species.
• the high molecular weight species include conjugates containing more .than one polymer strand and/or polymerized PEG-hGH or agonist variant thereof species. After the unreacted species and high molecular weight species have been removed, compositions containing primarily mono- and di-polymer-hGH or agonist variant thereof conjugates are recovered. Given the fact that the conjugates for the most part include a single polymer strand, the conjugates are substantially homogeneous. These modified hGH or agonist variant thereof have at least about 0.1% of the in vitro biological activity associated with the native or unmodified hGH or agonist variant thereof as measured using standard FDC-P1 cell proliferation assays, (Clark et al .
• the modified hGH or agonist variant thereof have about 25% of the in vitro biological activity, more preferably, the modified hGH or agonist variant thereof have about 50% of the in vitro biological activity, more preferably, the modified hGH or agonist variant thereof have about 75% of the in vitro biological activity, and most preferably the modified hGH or agonist variant thereof have equivalent or improved in vi tro biological activity.
• the processes of the present invention preferably include rather limited ratios of polymer to hGH or agonist variant thereof.
• the hGH or agonist variant thereof conjugates have been found to be predominantly limited to species containing only one strand of polymer.
• the attachment of the polymer to the hGH or agonist variant thereof reactive groups is substantially less random than when higher molar excesses of polymer linker are used.
• the unmodified hGH or agonist variant thereof present in the reaction pool, after the conjugation reaction has been quenched, can be recycled into future reactions using ion exchange or size exclusion chromatography or similar separation techniques.
• a poly (ethylene glycol) -modified hGH or agonist variant thereof may be purified from a reaction mixture by conventional methods which are used for purification of proteins, such as dialysis, salting-out, ultrafiltration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC) , gel chromatography and electrophoresis. Ion- exchange chromatography is particularly effective in removing unreacted poly (ethylene glycol) and hGH or agonist variant thereof.
• the mono- and di-polymer-hGH or agonist variant thereof species are isolated from the reaction mixture to remove high molecular weight species, and unmodified hGH or agonist variant thereof.
• Separation is effected by placing the mixed species in a buffer solution containing from about 0.5-10 mg/mL of the hGH or agonist variant thereof-polymer conjugates.
• Suitable solutions have a pH from about 4 to about 8.
• the solutions preferably contain one or more buffer salts selected from KCl, NaCl, K 2 HP0 4 , KH 2 P0 4 , Na 2 HP0 , NaH 2 P0 4 , NaHC0 3 , NaB0 4 , CH 3 C0 2 H, and NaOH.
• the hGH or agonist variant thereof polymer conjugate solution may first have to undergo buffer exchange/ultrafiltration to remove any unreacted polymer.
• the PEG- hGH or agonist variant thereof conjugate solution can be ultrafiltered across a low molecular weight cut-off (10,000 to 30,000 Dalton) membrane to remove most unwanted materials such as unreacted polymer, surfactants, if present, or the like.
• the fractionation of the conjugates into a pool containing the desired species is preferably carried out using an ion exchange chromatography medium.
• Such media are capable of selectively binding PEG-hGH or agonist variant thereof conjugates via differences in charge, which vary in a somewhat predictable fashion.
• the surface charge of hGH or agonist variant thereof is determined by the number of available charged groups on the surface of the protein. These charged groups typically serve as the point of potential attachment of poly (alkylene oxide) polymers. Therefore, hGH or agonist variant thereof conjugates will have a different charge from the other species to allow selective isolation.
• Strongly polar anion or cation exchange resins such as quaternary amine or sulfopropyl resins, respectively, are used for the method of the present invention. Ion exchange resins are especially preferred.
• a non-limiting list of included commercially available cation exchange resins suitable for use with the present invention are SP-hitrap®, SP Sepharose HP® and SP Sepharose® fast flow. Other suitable cation exchange resins e.g. S and CM resins can also be used.
• a non-limiting list of anion exchange resins, including commercially available anion exchange resins, suitable for use with the present invention are Q-hitrap®, Q Sepharose HP®, and Q sepharose® fast flow. Other suitable anion exchange resins, e. g. DEAE resins, can also be used.
• the anion or cation exchange resin is preferably packed in a column and equilibrated by conventional means .
• a buffer having the same pH and osmolality as the polymer conjugated hGH or agonist variant thereof solution is used.
• the elution buffer preferably contains one or more salts selected from KCl, NaCl, K 2 HP0 4 , KH 2 P0 4 , Na 2 HP0 , NaH 2 P0 4 , NaHC0 3 , NaB0 4/ and (NH ) 2 C0 3 .
• the conjugate-containing solution is then adsorbed onto the column with unreacted polymer and some high molecular weight species not being retained.
• a gradient flow of an elution buffer with increasing salt concentrations is applied to the column to elute the desired fraction of polyalkylene oxide-conjugated hGH or agonist variant thereof.
• the eluted pooled fractions are preferably limited to uniform polymer conjugates after the cation or anion exchange separation step. Any unconjugated hGH or agonist variant thereof species can then be back washed from the column by conventional techniques. If desired, mono and multiply pegylated hGH or agonist variant thereof species can be further separated from each other via additional ion exchange chromatography or size exclusion chromatography.
• the temperature range for elution is between about 4°C and about 25°C.
• elution is carried out at a temperature of from about 4°C to about 22°C.
• the elution of the PEG-hGH or agonist variant thereof fraction is detected by UV absorbance at 280 nm. Fraction collection may be achieved through simple time elution profiles.
• a surfactant can be used in the processes of conjugating the poly (ethylene glycol) polymer with the hGH or agonist variant thereof moiety.
• Suitable surfactants include ionic-type agents such as sodium dodecyl sulfate (SDS) .
• SDS sodium dodecyl sulfate
• Other ionic surfactants such as lithium dodecyl sulfate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decane sulfonic acid, etc. can also be used.
• Non-ionic surfactants can also be used.
• materials such as poly (oxyethylene) sorbitans (Tweens) , poly (oxyethylene) ethers (Tritons) can be used.
• the only limitations on the surfactants used in the processes of the invention are that they are used under conditions and at concentrations that do not cause substantial irreversible denaturation of the hGH or agonist variant thereof and do not completely inhibit polymer conjugation.
• the surfactants are present in the reaction mixtures in amounts from about 0.01-0.5%; preferably from 0.05-0.5%; and most preferably from about 0.075-0.25%. Mixtures of the surfactants are also contemplated. It is thought that the surfactants provide a temporary, reversible protecting system during the polymer conjugation process. Surfactants have been shown to be effective in selectively discouraging polymer aggregates while allowing lysine-based or amino terminal-based conjugation to proceed.
• the present poly (ethylene glycol) -modified hGH or agonist variant thereof has a more enduring pharmacological effect, which may be possibly attributed to its prolonged half-life in vivo. Furthermore,' the present poly (ethylene glycol) - modified hGH or agonist variant thereof may be useful for the treatment of hypo pituitary dwarfism (GHD) , Adult Growth Hormone Deficiency, Turner's syndrome, growth failure in children who were born short for gestational age (SGA) , Prader-Willi syndrome (PWS) , chronic renal insufficiency (CRI) , Aids wasting, and Aging .
• GDD hypo pituitary dwarfism
• SGA gestational age
• PWS Prader-Willi syndrome
• CRI chronic renal insufficiency
• Aids wasting Aids wasting
• Aging Aging .
• the present poly (ethylene glycol) -modified hGH or agonist variant thereof may be formulated into pharmaceuticals containing also a pharmaceutically acceptable diluent, an agent for preparing an isotonic solution, a pH-conditioner and the like in order to administer them into a patient.
• the above pharmaceuticals may be administered subcutaneously, intramuscularly, intravenously, pulmonary, intradermally, or orally, depending on a purpose of treatment.
• a dose may be also based on the kind and condition of the disorder of a patient to be treated, being normally between 0.1 mg and 5 mg by injection and between 0.1 mg and 50 mg in an oral administration for an adult
• the polymeric substances included are also preferably water-soluble at room temperature.
• a non- limiting list of such polymers include poly (alkylene oxide) homopolymers such as poly (ethylene glycol) or poly (propylene glycols), pol (oxyethylenated polyols) , copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
• effectively non-antigenic materials such as dextran, poly(vinyl pyrrolidones) , poly (acrylamides) , poly (vinyl alcohols), carbohydrate-based polymers, and the like can be used. Indeed, the activation of ⁇ - and ⁇ -terminal groups of these polymeric substances can be effected in fashions similar to that used to convert poly (alkylene oxides) and thus will be apparent to those of ordinary skill. Those of ordinary skill in the art will realize that the foregoing list is merely illustrative and that all polymer materials having the qualities described herein are contemplated. For purposes of the present invention, "effectively non- antigenic" means all materials understood in the art as being nontoxic and not eliciting an appreciable immunogenic response in mammals .
• the hGH is that of SEQ ID NO:l. It is understood that other members of the hGH or agonist variant thereof family of polypeptides could also be pegylated in a similar manner as exemplified in the subsequent examples .
• This example demonstrates a method for generation of substantially homogeneous preparations of N-terminally monopegylated hGH by reductive alkylation.
• Methoxy- linear PEG-propionaldehyde reagent of approximately 20,000 MW (Shearwater Corp.) was selectively coupled via reductive animation to the N-terminus of hGH by taking advantage of the difference in the relative pK a value of the primary amine at the N-terminus versus pK a values of primary amines at the ⁇ -amino position of lysine residues.
• hGH protein dissolved at 10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical, St.
• Methoxy-linear 30,000 MW PEG-propionaldehyde reagent Shearwater Corp. was coupled to the N-terminus of hGH using the procedure described for Example 1.
• Methoxy-linear 5,000 MW PEG-propionaldehyde reagent (Fluka) was coupled to the N-terminus of hGH using the procedure described for Example 1.
• PEG2-ALD PEG-aldehyde
• Branched chain 20 000 MW PEG-ALD hGH Methoxy-branched 20,000 MW PEG-aldehyde (PEG2-ALD) reagent (Shearwater Corp.) was coupled to the N- terminus of hGH using the procedure described for Example 1 using PEG to hGH molar ratios of 0.1-0.5 per amine.
• PEG2-ALD PEG-aldehyde
• This example demonstrates a method for generation of substantially homogeneous preparations of mono- pegylated hGH using N-hydroxysuccinimidyl (NHS) active esters.
• hGH protein stock solution was dissolved at 10 mg/mL in 0.25 M HEPES buffer, pH 7.2 (optionally 8% acetonitrile may also be added) .
• the solution was then reacted with Methoxy-PEG-succinimidyl propionate (SPA-PEG) by addition of SPA-PEG to yield a relative PEG:hGH molar ratio of 0.1-0.65 per amine. Reactions were carried out at 4°C to RT for from 5 minutes to 1 hour. Reactions were stopped by lowering the pH to 4.0 with 0.1 N acetic acid or by adding a 5X molar excess of Tris HC1.
• SPA-PEG Methoxy-PEG-succinimidyl propionate
• Example 6 20,000 MW PEG-BTC (Shearwater Corp.)- is coupled to hGH using the procedure described for Example 6.
• This example demonstrates a method for generation of substantially homogeneous preparations of pegylated hGH using benzotriazole carbonate derivatives of PEG.
• PEG Shearwater Corp.
• CM- HBA-PEG carboxymethyl hydroxybutyric acid-PEG
• This example demonstrates a method for generation of substantially homogeneous preparations of pegylated hGH using 20,000 MW methoxy-PEG-hydrazide, HZ-PEG (Shearwater Corp.) .
• hGH protein stock solution was dissolved at 10 mg/mL in 10 mM MES, pH 4.0. The solution was then reacted with HZ-PEG by addition of solid to yield a relative PEG: hGH molar ratio of 0.1- 5.0 per carboxyl group.
• Reactions were catalyzed with carbodiimide (EDC, EOAC, EDEC) at a final concentration of 2 mM to 4 mM. Reactions were carried out at 4°C for 2 hours to overnight or room temperature from 10 minutes to overnight. Reactions were stopped by Removing the unconjugated PEG and the carbodiimide by purification on cation exchange.
• EDC carbodiimide
• Modified hGHs having two or more PEGs (multi- pegylated) attached were also obtained from Examples 1 and 4 and were separated from the mono-pegylated species using anion exchange chromatography. Modified hGHs having two or more PEGs (multi-pegylated) attached are also separated from mono-PEGylated species using cation exchange chromatography. Modified hGHs having two or more PEGs (multi- pegylated) attached are also obtained in examples 2,3,5-13 and are purified in similar fashion to examples 1 and 4.
• Pegylated hGH species were purified from the reaction mixture to >95% (SEC analysis) using a single ion exchange chromatography step
• the PEG hGH species were purified from the reaction mixture to >95% (SEC analysis) using a single anion exchange chromatography step. Mono-pegylated hGH was purified from unmodified hGH and multi-pegylated hGH species using anion exchange chromatography.
• a typical 20K aldehyde hGH reaction mixture (5-100 mg protein) , as described above, was purified on a Q- Sepharose Hitrap column (1 or 5 mL) (Amersham Pharmacia Biotech, Piscataway, NJ) or Q-Sepharose fast flow column (26/20, 70 mL bed volume) (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in 25 mM HEPES, pH 7.3 (Buffer A) .
• the reaction mixture was diluted 5-10X with buffer A and loaded onto the column at a flow rate of 2.5 mL/min. The column was washed with 8 column volumes of buffer A.
• Cation exchange chromatography is carried out on an SP Sepharose high performance column (Pharmacia XK 26/20, 70 ml bed volume) equilibrated in 10 mM sodium acetate pH 4.0 (Buffer B) .
• the reaction mixture is diluted 10X with buffer B and loaded onto the column at a flow rate of 5 mL/min.
• the column is washed with 5 column volumes of buffer B, followed by 5 column volumes of 12% buffer C (10 mM acetate pH 4.5, 1 M NaCl) .
• the PEG-hGH species is eluted from the column with a linear gradient of 12 to 27% buffer C in 20 column volumes.
• the eluant is monitored at 280 nm and 10 mL fractions are collected. Fractions are pooled according to extent of pegylation (mono, di, tri etc.), exchanged into 10 mM acetate pH 4.5 buffer and concentrated to 1-5 mg/mL in a stirred cell fitted with an Amicon YM10 membrane. Protein concentration of pool is determined by A280 nm using an extinction coefficient of 0.78. Total yield of monopegylated hGH from this process is 10 to 50%.
• the purified pegylated hGH pools were characterized by reducing and non-reducing SDS-PAGE, non-denaturing and denaturing Size Exclusion Chromatography, analytical
• Non-denaturing SEC-HPLC The reaction of Methoxy-PEG of various attachment chemistries, sizes, linkers, and geometries with hGH, anion exchange purification pools and final purified products were assessed using non-denaturing SEC-HPLC.
• Analytical non-denaturing SEC-HPLC was carried out using a Tosohaas G4000PWXL column, 7.8 mm X 30 cm, (Tosohaas Amersham Bioscience, Piscataway, NJ) or Superdex 200 (Amersham Bioscience, Piscataway, NJ) in 20 mM Phosphate pH 7.2, 150 mM NaCl at a flow rate of 0.5 L/minute.
• PEGylation greatly increases the hydrodynamic volume of the protein resulting in a shift to an earlier retention time.
• New species were observed in the PEG aldehyde hGH reaction mixtures along with unmodified hGH. These PEGylated and non- PEGylated species were separated on Q-Sepharose chromatography, and the resultant purified mono PEG- Aldehyde hGH species were subsequently shown to elute as a single peak on non-denaturing SEC (> 95% purity) .
• the Q-Sepharose chromatography step effectively removed free PEG, hGH, and multi PEGylated hGH species from the mono-Pegylated hGH.
• Non-denaturing SEC-HPLC demonstrated that the effective size of the various PEGylated-hGH was much greater than their respective theoretical molecular weights (Table 1)
• SDS-PAGE was used to assess the reaction of the various PEG reagents with hGH and the purified final products . Examples of this technique are shown with mono 2OK linear and branched 2OK and 4OK PEG aldehyde, and 4X6 5K SPA PEG. ( Figures 1 & 2) .
• SDS-PAGE was carried out on 1 mm thick 10-20% Tris tricine gels (Invitrogen, Carlsbad, CA) under reducing and non- reducing conditions and stained using a Novex Colloidal CoomassieTM G-250 staining kit (Invitrogen, Carlsbad, CA) . Purified mono PEG-aldehyde hGH species migrate as one major band on SDS-PAGE. Bands were blotted onto PVDF membrane for subsequent N-terminal sequence identification.
• Analytical anion exchange HPLC was used to assess the reaction of various mPEGs with hGH, anion exchange purification fractions and final purified products.
• Analytical anion exchange HPLC was carried out using a Tosohaas Q5PW or DEAE-PW anion exchange column, 7.5 mm x 75 mm (Tosohaas Pharmacia Biotech, Piscataway, NJ) in 50 mM Tris ph 8.6 at a flow rate of 1 mL/min. Samples were eluted with a linear gradient of 5-200 mM NaCl.
• PEG-GH reaction mixtures and purified PEGylated products were analyzed by RP HPLC to elucidate hGH species, mono and multiply PEGylated hGH species, and, to monitor oxidized hGH forms, as well as, PEG hGH isoforms having a single PEG linked at different sites (e.g. N-terminus vs Lysine ⁇ -amino groups).
• RP-HPLC was carried out utilizing a Zorbax SB-CN 150 or 250 mm x 4.6 mm (3.5 mm or 5 mm) reversed phase HPLC column. Experiments were conducted at ambient temperature on a typical load of 10 mg of protein per sample.
• Buffer A is 0.1% triflouroacetic acid in water; Buffer B is 0.1% trifluoroacetic acid in acetonitrile.
• the gradient, which results in a V2% increase in B per minute, is as follows: Step Time Flow %A %B Step
• Tryptic digests were performed at a concentration of 1 mg/mL and, typically, 50 ug of material is used per digest. Trypsin was added such 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. Reactions were quenched by the addition of 50 ⁇ L of IN HCl per mL of digestion solution. Samples were diluted, prior to placing the samples in the autosampler, to a final concentration of 0.25 mg/ml in 6.25 % acetonitrile . Acetonitrile is added first (to 19.8% acetonitrile), mixed gently, and then water is added to final volume (four times the starting volume) . Extra digestion solution may be removed and stored for up to 1 week at -20°C.
• Rats were divided into groups of six to eight. Group 1 consisted of rats given either daily or day 0 and day 6 subcutaneous dose(s) of vehicle. Group 2 were given daily subcutaneous doses of GH (0.3 mg/kg/dose) . Group 3 were given subcutaneous doses of GH on day 0 and day 6(1.8 mg/kg/dose). Group 4 were given subcutaneous doses of PEG-GHs on day 0,6 (1.8 mg/kg/dose) .
• FIG. 6 shows the change in tibial bone length (average +/- SEM) at day 11 in response to various PEG-GH conjugates dosed on day 0 and day 6 or hGH dosed daily.
• hGH Immunoassay hGH and pegylated hGH protein concentration levels in rat, mouse, and cynomolgus monkey plasma were determined using the hGH AutoDELFIA kit fluorescence immunoassay (PerkinElmer Life Sciences) , using the appropriate PEG hGH to generate standard curve .

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