WO2006094530A1 - Conjugues de proteines et de dipolymeres et leurs procedes de preparation - Google Patents

Conjugues de proteines et de dipolymeres et leurs procedes de preparation Download PDF

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WO2006094530A1
WO2006094530A1 PCT/EP2005/002632 EP2005002632W WO2006094530A1 WO 2006094530 A1 WO2006094530 A1 WO 2006094530A1 EP 2005002632 W EP2005002632 W EP 2005002632W WO 2006094530 A1 WO2006094530 A1 WO 2006094530A1
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Prior art keywords
protein
polymer
csf
peg
conjugate
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PCT/EP2005/002632
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English (en)
Inventor
Michael Bavand
Horst Blasey
Dietmar Lang
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Siegfried Ltd.
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Priority to PCT/EP2005/002632 priority Critical patent/WO2006094530A1/fr
Priority to PCT/EP2006/060671 priority patent/WO2006095029A2/fr
Priority to JP2008500216A priority patent/JP2008535793A/ja
Priority to EP06725029A priority patent/EP1869079A2/fr
Publication of WO2006094530A1 publication Critical patent/WO2006094530A1/fr

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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the present invention refers to di-polymer protein conjugates and processes for their preparation. Further, the present invention refers to the use of such di-polymer protein conjugate, especially di-pegylated protein conjugate, for the manufacture of a medicament for the treatment of disorders characterized by a reduced haematopoietic or immune function.
  • therapeutic proteins have been modified in order to increase their circulation half-life, e.g. by chemically or enzymatically coupling a polymer such as polyethylene glycol (PEG) to the protein.
  • PEG polyethylene glycol
  • the modification of a protein by attaching polyethylene glycol moiety thereto is also known as pegylation.
  • Proteins which were successfully modified by pegylation include erythropoietin (EPO), interferons such as interferon alpha, interferon beta and interferon gamma, granulocyte colony-stimulating factor (G-CSF or GCSF), interleukins such as IL-2 and IL-6, tumour necrosis factor (TNF), various cytokines and synthetic erythropoiesis protein (synthetic EPO).
  • EPO erythropoietin
  • interferons such as interferon alpha, interferon beta and interferon gamma
  • G-CSF or GCSF granulocyte colony-stimulating factor
  • interleukins such as IL-2 and IL-6
  • TNF tumour necrosis factor
  • various cytokines synthetic erythropoiesis protein
  • polyethylene glycol molecules are coupled to the protein via a functional group of the protein.
  • Each functional group in the amino acid chain of the protein which is nucleophilic, i.e. has electron donor ability, may react with a complementary group being attached to a PEG molecule.
  • nucleophilic groups include ⁇ -amino, ⁇ -amino, carboxyl, thiol, hydroxyl, imidazole, and guanidine groups.
  • the amino groups of a protein i.e. the alpha-amino group (N- terminus) and epsilon-amino groups of lysine residues, are used as the reactive or functional groups, which are involved in PEG coupling (see F.M. Veronese, Biomaterials 2001, 22: 405-417).
  • US 4,179,337 discloses PEG attachment to a protein via an acylation reaction resulting in an amide linkage between PEG and the protein.
  • pegylated proteins for biopharmaceutical applications preferably have a low number of PEG units per protein.
  • WO 96/11953 describes methods for attaching one polyethylene glycol molecule to the ⁇ -amino group (N-terminus) of a polypeptide under conditions of a reductive alkylation and at a pH sufficiently acidic to selectively activate the ⁇ -amino group.
  • di-pegylated proteins Compared to mono-pegylated proteins, di-pegylated proteins, however, have some significant advantages:
  • a more effective shielding of epitopes of the protein can be achieved by linkage of the protein with a second PEG molecule, so that proteolytic sensitivity and immunogenicity are reduced, while the stability of the PEG- protein conjugate is improved. This is particularly advantageous for proteinoids and non-recombinantly synthesised protein fragments that are joined via non-natural linkers, which may potentially increase immunogenicity.
  • WO 00/21574 describes di-pegylated protein muteins of leptin and G-CSF, wherein a cysteine mutation is introduced into the protein sequence.
  • the purpose of introducing a cysteine point mutation is to provide a second conjugation site, which complements pre-existing technology for PEG conjugation specifically to the N- terminus.
  • WO 01/51510 describes G-CSF mutein conjugates, wherein the G-CSF amino acid sequence differs from the amino sequence of human G-CSF in at least one specifically altered amino acid residue which comprises an attachment group for PEG.
  • WO 01/51510 describes a di-pegylated G-CSF mutant that is pegylated at specifically introduced amino groups by acylation.
  • Another object of the present invention provides polymer-protein conjugates with two polymer units per protein, in particular di-pegylated protein conjugates, which have a longer circulating half-time and a greater in vivo biological activity than the corresponding unconjugated protein.
  • a pharmaceutical preparation which comprises at least one, and preferably two di polymer-protein conjugates according to the present invention.
  • a process for the preparation of a polymer-protein conjugate comprises reacting a protein having at least two amino groups with a polymer reagent having a single aldehyde group in the presence of a reducing agent wherein the reaction time is chosen such that polymer- protein conjugates with two nitrogen atoms of amino groups of the protein being conjugated with a polymer unit via an amine linkage are preferably prepared.
  • the present invention relates to the use of a polymer-protein conjugate such as a polyethylene glycol-protein conjugate according to the present invention for the manufacture of a medicament for the treatment of a disorder characterized by reduced haematopoietic or immune function.
  • the disorder is usually neutropenia and/or leukaemia, which are induced by chemotherapy, radiotherapy and/or infections.
  • the invention is based on the finding that preferably two polymer units, in particular polyethylene glycol molecule, can be attached to a protein under conditions of a reductive alkylation by selecting a suitable pH, e.g. a pH about 7.
  • reaction time is a crucial parameter for the formation of polymer-protein conjugates with two polymer units per protein, in particular di-pegylated protein conjugates according to the present invention.
  • the formation of pegylated protein conjugates according to the invention occurs sequentially in a time-dependent order from mono-, di-, tri- to higher pegylated protein conjugates (see Figure 3).
  • the process yield could be significantly increased by introducing a recyclisation step in which non-desired reaction products are applied to a second polymer attachment process step such as a second pegylation reaction step yielding further di-pegylated protein conjugate and thus contributing to a higher overall process yield of di-pegylated protein conjugate.
  • a second polymer attachment process step such as a second pegylation reaction step yielding further di-pegylated protein conjugate and thus contributing to a higher overall process yield of di-pegylated protein conjugate.
  • the recyclisation step results in a prominent yield of di-pegylated protein conjugate with high quality, being comparable to the quality obtained in the first pegylation reaction.
  • This principle of recycling non-desired reaction products to obtain a higher yield of polymer-protein conjugates with two polymer units per protein is also applicable to other polymer protein conjugate forming reactions.
  • the method of protein modification according to the present invention allows one to preferably or predominantly attach polymer units at a protein at specific sites of the protein taking advantage of a different reactivity of amino groups of the same type (e.g. ⁇ -amino group of lysine residues) depending on the amino acid residues adjacent to the amino acid with the reactive amino group in the sequence of the protein.
  • the process according to the present invention is preferably performed at a pH, which as takes advantage of the pKa difference among e.g. different ⁇ -amino groups, different guanidino groups and/or different imidazole groups of the protein. This leads to a substantially homogenous preparation of di- polymer protein conjugates having the polymer predominantly attached to defined or pre-selectable or pre-selected or predetermined amino groups of the protein.
  • the polymer-protein conjugates according to the present invention are prepared by reductive alkylation.
  • the present invention preferably provides polymer-protein conjugates, wherein two nitrogen atoms of amino groups of the protein are each conjugated with a polymer unit via an amine linkage.
  • amino group includes primary and secondary amino groups and in particular NH- or NH 2 -groups in side-chains of amino acids such as NH 2 -groups in the side-chain of lysine, NH- or NH 2 -groups in the guanidino group of arginine or NH-groups in the imidazole side-chain of histidine.
  • the polymer unit preferably comprises at least one polymer moiety and a linker moiety, which is between at least the one polymer moiety and the amine linkage.
  • the linker moiety may be linear or branched. If the linker moiety is branched, a polymer unit may comprise more than one polymer moiety.
  • the linker moiety is preferably an aliphatic linker moiety. Suitable aliphatic linker moieties also include substituted alkyl diamines and triamines, lysine esters and malonic ester derivatives. The linker moieties are preferably non-planar, so that the polymer chains are not rigidly fixed. Preferably, the linker moiety includes a multiple-functionalized alkyl group containing up to 18, and more preferably from 1 to 10 carbon atoms. A hetero-atom such as nitrogen, oxygen or sulfur may also be included within the alkyl chain. The linker moiety may be branched, for example at a carbon or nitrogen atom. Examples for branched linker moieties and the resulting branched polymer units as well as methods for their preparation are described in WO 95/11924 and WO 03/049699, which are herein incorporated by reference.
  • the linker moiety comprises at least one methylene group attached to the nitrogen atom of the amine linkage, e.g. from 1 to 12, preferably from 1 to 5, more preferably from 1 to 3 and most preferably two methylene groups which are directly attached to the nitrogen atom of the amine linkage.
  • One preferred polymer-protein conjugate according to the present invention has the formula:
  • R is H, lower alkyl, aryl or any suitable protecting group
  • Polymer is a polymer which is suitable to be conjugated with proteins
  • m is an integer representing the number of methylene groups
  • P is a biologically active protein or proteinoid wherein two nitrogen atoms of amino groups of the protein or proteinoid are each conjugated with a polymer unit
  • L 1 is O, N, S and/or a branched or non- branched linker moiety which can be absent or present
  • L 2 is a branched or non- branched linker moiety which can be absent or present
  • y is a integer with the proviso that y is 1 in the absence of L 2 and y is at least 1 in the presence of L .
  • R is preferably a lower alkyl or an aryl such as benzyl.
  • the term lower alkyl includes lower alkyl groups containing e.g. from 1 to 7 carbon atoms, preferably from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, etc., with methyl being particularly preferred.
  • the linker L 1 which is part of the linkage between at least one polymer moiety and the suitable protecting group and/or the linker L 2 which is part of the linkage between the at least one polymer moiety, such as ethylene oxide residues of a PEG chain, and the nitrogen atom of the protein or proteinoid and, in particular, between the ethylene oxide residues of the PEG chain and the methylene groups, can independently be absent or present and are independently selected from a branched or non-branched linker moiety having any arbitrary structural formulae, such as the linker moieties described above, e.g. (-CO) 2 -CH-, -CO-O-, and/or -CO-NH-.
  • the number m of methylene groups forming a part of the linkage between polymer moiety, such as the ethylene oxide residues of a PEG chain, and the nitrogen atom of the protein or proteinoid, to which the polymer moiety such as a PEG reagent is attached to is in a range from 1 to 12, preferably from 1 to 5, more preferably from 1 to 3 and is most preferably 2.
  • the number y, in the presence of L 2 is in a range from 1 to 10, preferably from 1 to 5, more preferably from 1 to 3 and is most preferably 2.
  • the polymer moiety is usually a substantially non-antigenic or non-immunogenic polymer chain.
  • the polymer moieties used are preferably selected from among water-soluble polymer moieties. This has the advantage that the protein to which the water-soluble polymer moieties are attached or conjugated does not precipitate in an aqueous environment such as a physiological environment.
  • the polymer selected should further have a single reactive aldehyde, so that the degree of polymerization may be controlled as provided for in the present processes.
  • the polymer unit as well as the polymer moiety may be branched or unbranched.
  • the polymer will be pharmaceutically acceptable.
  • One skilled in the art will be able to select the desired polymer moiety based on considerations such as whether the polymer-protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis and other considerations.
  • the polymer moiety may be selected from the group consisting of polyalkylene glycol moieties, polysaccharide moieties such as dextran and its derivatives, polysaccharide and its derivatives, pyrrolidone moieties such as polyvinyl pyrolidone, cellulose moieties such as carboxymethyl cellulose, polyvinyl alcohol, poly-1 ,3-dioxolan, poly-1 ,3,6-trioxane, ethylene-maleic anhydride copolymer, polyaminoacid moieties and/or polyacrylamide moieties and/or other similar non-immunogenic polymer moieties (either homopolymers or random copolymers) and/or derivatives thereof.
  • Such polymers are also capable of being functionalized or activated for inclusion in the present invention.
  • polymer moiety is a polyalkylene glycol moiety.
  • polyalkylene glycol designates polyalkylene glycol radicals or polyalkylene glycol moieties, where the alkylene radical is a straight or branched chain radical.
  • polyalkylene glycol also comprises polyalkylene glycols formed from mixed alkylene glycols such as polymers containing a mixture of polyethylene and polypropylene radicals and polymers containing a mixture of polyisopropylene, polyethylene and polyisobutylene radicals.
  • a polyalkylene glycol moiety in the polymer protein conjugates according to the present invention is a polyethylene glycol moiety or polyethylene glycol residue formed by removal of the two terminal hydroxyl groups.
  • polyalkylene oxide derivatives such as methoxy polyethylene glycols (mPEG) or other suitable alkyl substituted polyalkylene oxide derivatives such as those containing mono- or bis-terminal Ci-C 4 groups.
  • Straight- chained non-antigenic polymers such as monomethyl polyethylene glycol homopolymers are preferred.
  • Alternative polyalkylene oxides such as other polyethylene glycol homopolymers, polyethylene glycol heteropolymers, other alkyl polyalkylene oxide block copolymers and copolymers of block copolymers of polyalkylene oxides are also useful.
  • An especially preferred polymer is polyethylene glycol according to the present invention resulting in PEG-protein conjugate of the formula:
  • PEG has the formula -(CH 2 -CH 2 -O) n with n representing the number of ethylene oxide residues in a polyethylene glycol unit. Further, it is preferred, that L 1 is O, L 2 is absent and y is 1.
  • the nitrogen atoms of the amino group to which a polymer unit may be attached according to the present invention is preferably selected from the group consisting of nitrogen atoms of the ⁇ -amino group, ⁇ -amino groups, guanidinium groups and imidazole groups. It is preferred, but not limited, to attach a polymer unit to the nitrogen atoms of a primary amino group.
  • the biologically active protein which can be conjugated in accordance with this invention, may be any of the conventional therapeutic proteins.
  • protein in the context of the present invention the term protein is understood to include proteins and proteinoids.
  • proteinoids are synthetic protein-like molecules which have the identical amino acid sequence as the corresponding native protein, but are chemically synthesised in the form of peptides which peptides are linked together to form the native protein-like structure with a resulting biological activity similar to the native protein. These proteinoids are potential protein therapeutics.
  • Proteins which can be conjugated with two polymer units and especially be di- pegylated according to the present invention include non-mutated and mutated proteins such as but not limited to growth factors, antibodies, hormones, in particular therapeutically active proteins such as but not limited to erythropoietin, interferon alpha, interferon beta, interferon gamma, consensus interferon, G-CSF, GM-CSF, hemoglobin, interleukins such as interleukin-2 and interleukin-6, tumour necrosis factor, various cytokines as well as immuno-globulins such as IgG, IgE, IgM, IgA, IgD and/or structural and/or functional variants and/or fragments thereof as well as their proteinoids or synthetic protein-like forms such as synthetic erythropoiesis protein (synthetic EPO).
  • non-mutated and mutated proteins such as but not limited to growth factors, antibodies, hormones, in particular therapeutically active proteins such as but not limited
  • protein such as G-CSF useful in the practice of this invention may be of any form isolated from mammalian organisms, a product of prokaryotic or eukaryotic host expression of exogenous DNA sequences obtained by genomic or cDNA cloning or by DNA synthesis or alternatively a product of chemical synthetic procedures or by endogenous gene activation.
  • the protein can be of a natural or recombinant source obtained from tissue, mammalian- microbial cell cultures, plant cell cultures, transgenic animals, yeasts, fungi and/or transgenic plants.
  • Suitable prokaryotic hosts include various bacteria such as E. coli; suitable eukaryotic hosts include yeasts such as S.
  • the protein expression product may be glycosylated with mammalian, plant or other eukaryotic carbohydrates, or it may be non-glycosylated.
  • the G-CSF expression product may also include an initial methionine amino acid residue at position 1.
  • the present invention contemplates the use of any and all such forms of G-CSF, although recombinant G-CSF, especially E.c ⁇ /z-derived, is preferred.
  • Certain G-CSF analogues have been reported to be biologically functional, and these may also be conjugated according to the present invention. These G-CSF analogues may include those having amino acid additions, deletions and/or substitutions as compared to the G-CSF amino acid sequence according to SEQ ID No. 1.
  • the protein is one having the activity of G-CSF, including mutants of G- CSF, glycosylated G-CSF, non-glycosylated G-CSF and/or otherwise modified structural and/or functional variants of G-CSF.
  • the protein has the amino acid sequence of G-CSF identified in SEQ ID NO. 1 (see Figure 10) which corresponds to recombinant G-CSF produced in bacteria, having 174 amino acids and an extra N-terminal methionyl residue. Amino acid sequences of biological active G-CSF, which differ from SEQ ID NO. 1 in that they do not contain a methionyl residue at position 1 , are also preferred.
  • the two amino groups are preferably selected from the group consisting of the ⁇ -amino group (N-terminus) and ⁇ -amino groups of lysine residues of G-CSF.
  • N-terminus the ⁇ -amino group
  • ⁇ -amino groups of lysine residues of G-CSF preferably selected from the group consisting of the ⁇ -amino group (N-terminus) and ⁇ -amino groups of lysine residues of G-CSF.
  • WO 96/11953 it has been shown (i) that mono-pegylation occurred in descending order N-terminus > lysine 35 > lysine 41 » lysines 17, 24 (where pegylation was not significantly detectable) taking advantage of the different reactivity between the ⁇ - and ⁇ -amino groups of the protein based on pKa differences and (ii) that the biological activity of mono- pegylated G-CSF decreased in the order N-terminus >
  • the two amino groups of the protein, which are coupled to PEG are preferably selected from the group consisting of the N-terminal amino group, the ⁇ -amino group of lysine 17 and the ⁇ -amino group of lysine 35.
  • coupling of polymer groups such as PEG may occur at the ⁇ -amino group of lysine 24 and/or the ⁇ -amino group of lysine 41.
  • the residue numbers refer to the amino acid sequence of G-CSF identified in SEQ ID NO. 1 (see Figure 10).
  • the molecular weight of a polyethylene glycol moiety attached to a amino group is from 2 to 100 kDa, more preferably from 5 to 60 kDa and most preferably from 10 to 30 kDa.
  • the number n of ethylene oxide residues in a polyethylene glycol moiety is from about 40 to about 2270, more preferably from about 110 to about 1370 and most preferably from about 225 to about 680.
  • compositions of the above-described polymer protein conjugates are provided. Such pharmaceutical compositions may be for administration for injection or for oral, pulmonary, nasal or other forms of administration.
  • the present invention provides pharmaceutical compositions comprising effective amounts of di -polymer-protein conjugates of the present invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions includes diluents of various buffer content, such as Tris-HCl, acetate, phosphate, pH and ionic strength; additives such as detergents and solubilizing agents such as Tween 80, Polysorbate 80, antioxidants such as ascorbic acid and sodium metabisulfite, preservatives such as benzyl alcohol and bulking substances such as lactose or mannitol; incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
  • Such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the polymerprotein conjugates according to the present invention.
  • a pharmaceutical preparation according to the present invention comprises at least one di-polymer-protein conjugate according to the present invention. It may be advantageous to prepare a mixture of two di-polymer- protein conjugates in order to select the proportion of the two di-polymer-protein conjugates in the mixture. Furthermore, if desired, one may prepare a mixture of various proteins with various numbers of polymer moieties attached and combine this mixture with a di-polymer-protein conjugate according to the present invention which yields a mixture with a pre-determined proportion of di-polymer-protein conjugate according to the present invention.
  • the preparation according to the present invention comprises at least one di-polyethylene glycol-G-CSF conjugate, also referred to an isoform or di-pegylated G-CSF isoform, selected from the group consisting of G-CSF conjugated with polyethylene glycol moieties at the ⁇ -amino group (N-terminal) and the ⁇ -amino group of lysine 17, and G-CSF conjugated with polyethylene glycol moieties at the N-terminal amino group and the ⁇ -amino group of lysine 35.
  • Isoforms of di-pegylated protein conjugates may occur if there are more than two attachment sites for PEG, e.g. more than two nitrogen atoms of amino groups in the protein.
  • a defined di-pegylated G-CSF conjugate preparation may comprise two isoforms as described above, e.g. an isoform with polyethylene glycol moieties at the N-terminus and the ⁇ -amino group of lysine 17 (in the following also referred to as N-terminus + lysine 17), and an isoform with polyethylene glycol moieties at the N-terminus and the ⁇ -amino group of lysine 35 (in the following also referred to as N-terminus + lysine 35), in any defined mixed ratio, such as from 0.1 to 10, or from 0.5 to 2 or from 0.75 to 1.33 or about 1.
  • a defined product or preparation of the present invention consisting of a defined mixture of two di-PEG G-CSF isoforms as decribed above [(N-terminus + lysine 17) / (N-terminus + lysine 35) in a mixed ratio of e.g.
  • the present invention provides a mixed pharmaceutical preparation of PEG protein conjugates comprising at least one di-pegylated protein conjugate as described-above mixed with multi-pegylated protein conjugates wherein the proportion of said di-pegylated protein conjugate is pre-determined.
  • multi-pegylated protein conjugates means protein conjugates having at least one PEG moiety per protein.
  • a process for the preparation of a polymer-protein conjugate such as a polyethylene glycol-protein conjugate is provided which, under the conditions of a reductive alkylation, predominantly yields di-polymer-protein conjugates such as di-pegylated protein.
  • this conversion of a polymer reagent such as a PEG reagent with protein to di-polymer- protein conjugates is achieved by selecting a reaction time which results in an enriched pool of di-polymer-protein conjugates. It is particularly preferred to provide a process for the predominant preparation of di-pegylated PEG-protein conjugates.
  • a selective or predominant or preferred conversion of polymer with protein to di-polymer-protein conjugates or a selective or predominant or preferred yield of di-polymer-protein conjugates or selectively or predominantly or preferably preparing polymer-protein conjugates having two nitrogen atoms of amino groups of the protein each conjugated with one polymer unit means that at least 20%, preferably at least 25%, more preferably at least 30% and most preferably at least 40% or at least 45% or even at least 50% or at least 60% of the products of the reaction mixture resulting from a process of the present invention are di-polymer-protein conjugates according to the present invention.
  • the percentage of di-polymer-protein conjugates in the reaction mixture is the amount of at least one di-polymer-protein conjugate in relation to the amount of all identified species or products in the reaction mixture based on cation exchange chromatography using a HPLC system and mass analysis.
  • a selective or predominant or preferred conversion of PEG with protein to di-pegylated PEG-protein conjugates or a selective or predominant or preferred yield of di-pegylated PEG-protein conjugates or selectively or predominantly or preferably preparing polyethylene glycol-protein conjugates having two nitrogen atoms of the amino groups of the protein each conjugated with one polyethylene glycol unit means that at least 20%, preferably at least 25%, more preferably at least 30% and most preferably at least 40% or at least 45% or even at least 50% or at least 60% of the products of the reaction mixture resulting from a process of the present invention are di-pegylated PEG-protein conjugates according to the present invention.
  • the percentage of di-pegylated PEG-protein conjugates in the reaction mixture is the amount of at least one di-pegylated PEG-protein conjugate in relation to the amount of all identified species or products in the reaction mixture based on cation exchange chromatography using a HPLC system and mass analysis (see also Table 1).
  • the reaction time of each of the two process cycles according to the present invention is from about 12 h to about 48 h, more preferably from 18 h to 32 h.
  • Other preferred reaction times are about 13 h, about 14 h, about 15 h, about 16 h, about 17h, about 19 h, about 20 h, about 21 h, about 22 h, about 23 h, about 24 h, about 25 h, about 26 h, about 27 h, about 29 h, about, 30 h, about 31 h and about 33h, about 34 h or about 35 h.
  • the reaction is performed at a protein concentration from 0.5 to 100 mg/ml, more preferably from 1 to 10 mg/ml and most preferably from 3 to 7 mg/ml.
  • the reaction is performed at a protein -to-polymer molar ratio of from 1 : 1 to 1 :400, and preferably from 1 :5 to 1 :30 and most preferably from 1 : 15 to 1 :30.
  • the reducing agent used in the reactive alkylation is preferably selected from, but not limited to, NaCNBH 4 or NaBH 4 .
  • the reaction be performed in the vicinity of neutrality, e.g. at a pH from 6 to 8, more preferably at a pH from 6.9 to 7.8, and most preferably at a pH from 7.2 to 7.5, especially if the protein is a protein having the biological activity of G-CSF.
  • a pH 4.0 to 10.0 or from 5.0 to 9.0 or from 5.5 to 8.5.
  • the selection of a specific pH may affect the reaction time, which is to be chosen such that polymer-protein conjugates with two nitrogen atoms of amino groups of the protein being conjugated with a polymer unit via an amine linkage are preferably prepared.
  • the reaction time which is preferably from about 12 h to about 48 h, may also be lower than 12 h, e.g. 1 h, 2 h, 4 h, 6 h, 8 h or 1O h.
  • the reaction may be performed in the presence of a buffer that, e.g., may be selected from a phosphate, acetate, HEPES, MES, and/or buffers.
  • a buffer that, e.g., may be selected from a phosphate, acetate, HEPES, MES, and/or buffers.
  • the reaction be performed at a temperature from 2°C to 50°C, more preferably at a temperature from 2 0 C to 8 0 C and most preferably about 4°C.
  • the selection of a specific temperature may affect the reaction time, which is to be chosen such that polymer-protein conjugates with two nitrogen atoms of amino groups of the protein being conjugated with a polymer unit via an amine linkage are preferably prepared.
  • the reaction time which is preferably from about 12 h to about 48 h, may also be lower than 12 h, e.g. 1 h, 2 h, 4 h, 6 h, 8 h or 10 h.
  • the PEG reagent used in the process according to the present invention is usually a reagent having the formula
  • R is H, a lower alkyl, aryl or any suitable protecting group
  • n is an integer representing the number of ethylene oxide residues in a polyethylene glycol moiety
  • m is an integer representing the number of methylene groups
  • L 1 is O, N, S and/or a branched or non-branched linker moiety which can be absent or present
  • L 2 is a branched or non-branched linker moiety which can be absent or present
  • y is a integer with the proviso that y is 1 in the absence of L 2 and y is at least 1 in the presence of L 2 .
  • R, m, n, L 1 , L 2 and y are defined as described above for the polymer- protein conjugates according to the present invention.
  • the polymer reagent is a PEG aldehyde, more preferably a PEG acetaldehyde, most preferably methoxy polyethylene glycol acetaldehyde (mPEG acetaldehyde).
  • the polyethylene glycol having a single aldehyde group, which is to be converted with the protein may be stored in the form of the corresponding acetal.
  • the PEG acetal precursor may be activated to the PEG aldehyde reagent at any time before the conversion with the protein to be conjugated, under the proviso that the activated PEG aldehyde is stable until it is conjugated with the protein according to process of the present invention.
  • a corresponding polyethylene glycol diethyl acetal may be activated.
  • a process for the long-term storage of a polyethylene glycol acetaldehyde such as methoxy polyethylene glycol acetaldehyde is provided, wherein the polyethylene glycol acetaldehyde is stored as a solid substance under an inert atmosphere such as nitrogen and at a temperature of about -2O 0 C or less.
  • a polyethylene glycol acetaldehyde such as methoxy polyethylene glycol acetaldehyde may be stored for at least 1 month, preferably at least 2 months, more preferably at least 3 months and most preferably at least 4 or 6 months.
  • the process further comprises a separation step or purification step leading to a pool of di-polymer-protein conjugates, preferably di-pegylated polyethylene glycol-protein conjugates, wherein two defined amino groups of the protein are each conjugated with one polymer unit, also referred to as the product pool, and to a pool which contains unreacted protein, unreacted polymer reagent, di-polymer-protein conjugate isoforms other than those of the product pool and/or polymer-protein conjugates with less or more than two amino groups of the protein conjugated with a polymer unit, also referred to as the rest pool.
  • a separation step or purification step leading to a pool of di-polymer-protein conjugates, preferably di-pegylated polyethylene glycol-protein conjugates, wherein two defined amino groups of the protein are each conjugated with one polymer unit, also referred to as the product pool, and to a pool which contains unreacted protein, unreacted polymer reagent, di-polymer-protein conjug
  • This separation step or purification step yielding a product pool and a rest pool is preferably performed by ion exchange chromatography.
  • the rest pool is again subjected to the process according to the present invention, e.g. instead of using solely unpegylated protein in the conversion with PEG reagent, the rest pool is reacted with a polyethylene glycol reagent having a single aldehyde group, e.g.
  • reaction time is chosen such that polyethylene glycol-protein conjugates having two nitrogen atoms of amino groups of the protein each conjugated with one polyethylene glycol unit are selectively prepared.
  • di-pegylated PEG-protein conjugates such as di-pegylated PEG-protein conjugates according to the present invention.
  • di-pegylated PEG-protein conjugate isoforms having PEG moieties attached to the nitrogen atoms of amino groups at defined positions of the amino acid chain of the protein may be obtained.
  • a di-pegylated protein conjugate broadly includes any protein, which is conjugated with two polyethylene glycol units, wherein both of the polyethylene glycol units are each attached to one amino group of the protein.
  • the di-pegylated protein conjugates of the present invention are preferably obtained by reacting a polyethylene acetaldehyde molecule with the protein under conditions of a reductive alkylation which selectively leads to a di-pegylated PEG protein conjugate having a C 2 linker between the amine group resulting from the amino group of the protein and the chain of ethylene oxide residues of the polyethylene glycol unit.
  • G-CSF is exemplified as the protein and PEG is exemplified as the polymer moiety.
  • PEG polymer moiety
  • One aspect of the present invention relates to a method for preparing di-pegylated protein conjugates by attaching polyethylene glycol aldehyde moieties to the ⁇ - and ⁇ -amino groups of proteins under the conditions of a reductive alkylation.
  • a first step in this method can optionally be the provision of the corresponding polyethylene glycol reagent, which is to be converted with the protein.
  • the polyethylene glycol reagent is a polyethylene glycol acetaldehyde, more preferred a methoxy polyethylene glycol acetaldehyde.
  • US 5,252,714 describes that methoxy PEG acetaldehyde is unstable in aqueous solution due to aldol decompositions and oxidation.
  • the process for providing the polyethylene glycol reagent comprises the activation of methoxy PEG di-ethyl acetal which is a stable storage form of the methoxy PEG acetaldehyde, into methoxy PEG acetaldehyde.
  • the PEG di-ethyl acetal precursor may be activated into PEG acetaldehyde, preferably methoxy PEG acetaldehyde, at some hours, e.g. 12 hours or 24 hours or preferably some weeks, e.g. one or two weeks or even some months, e.g. two, three or four months or more before the reaction with the protein, preferably G-CSF.
  • parameters have been identified which influence the yield and/or the reaction kinetics of the di-pegylation process including the recyclisation step according to the present invention. These parameters include protein concentration, protein -to-PEG molar ratio, pH, the reducing agent used in the reductive alkylation, temperature, incubation time, co-solvent, stirring speed and method, ionic strength of the buffer.
  • WO 96/11953 teaches that a sufficiently acidic pH selectively leads to mono- pegylation of ⁇ -amino groups in view of the pKa difference between ⁇ - and ⁇ - amino groups.
  • WO 03/049699 describes that pegylation at a pH in the range from 5.5 to 7.5 only occurred at ⁇ -amino groups which results in mono-pegylated protein.
  • the pegylation reaction according to the present invention is preferably performed in the vicinity of neutrality, e.g. at pH 6.5 to 7.5.
  • one crucial parameter for the degree of pegylation of proteins such as G-CSF is the reaction time.
  • the preferred reaction time is chosen such that at least 20% of the reaction mixture comprises di-pegylated PEG protein conjugates, i.e. polyethylene glycol-protein conjugates, having two amino groups of the protein each conjugated with one polyethylene glycol unit.
  • the reaction time for the di-pegylation reaction is chosen such that at least 25, at least 30, at least 40 or at least 50% of the reaction mixture comprises di-pegylated protein such as di-pegylated G-CSF.
  • the yield of mono-pegylated species reaches a maximum and starts to decline (data not shown) while di- and tri-PEG G-CSF conjugates are increasingly formed.
  • the di-PEG G-CSF conjugate yield reaches and maintains its maximum for several hours before starting to decrease, while the tri-PEG G-CSF conjugate is increasingly formed.
  • tri- and higher pegylated conjugates do not reach their yield maxima.
  • the selection of the defined time frame for the reaction time of a process according to the present invention is essential for the production of specific isoforms or species of pegylated proteins such as G-CSF at high yield.
  • the process according to the present invention leads to a product mixture of differently pegylated forms, which is highly enriched in di-pegylated protein conjugate, i.e. preferably at least 20%, or more preferably at least 30% of the reaction mixture comprises di-pegylated protein conjugates.
  • the reaction mixture also contains unreacted protein, unreacted polymer and otherwise pegylated protein conjugates.
  • the process according to the present invention further comprise a purification step in order to separate the di-pegylated species from undesired substances, e.g. by chromatography, preferably by an ion exchange chromatography step, more preferably by a cation exchange chromatography step (C-IEX).
  • the di-pegylated protein conjugates may be separated from unreacted protein, unreacted PEG reagent and/or polyethylene glycol conjugates which have more or less than two polyethylene glycol units per protein.
  • di-pegylated protein conjugate isoforms may be separated which differ in their PEG attachment sites form the desired di-pegylated PEG protein conjugate products.
  • the pool comprising the di-pegylated protein conjugate products is also designated as the product pool, whereas the pool comprising the undesired compounds is also designated as the rest pool, or product- depleted eluate.
  • the overall yield of di- pegylated protein conjugate could be further increased by recyclisation the product- depleted eluate resulting from a purification step such as the C-IEX step, in another pegylation process according to the present invention.
  • the eluate of the chromatography column e.g. a C-IEX column
  • the polyethylene glycol aldehyde reagent preferably an acetaldehyde such as methoxy polyethylene glycol acetaldehyde.
  • the di-pegylated protein conjugates according to the present invention show a high biological activity, which was demonstrated in a bioassay for di-pegylated G-CSF according to the present invention.
  • the conjugate according to the present invention showed haematopoietic biological properties of naturally occurring G-CSF.
  • in vivo data revealed that the application of di-pegylated G-CSF according to the present invention results in an increase in neutrophil and white blood cell counts and further revealed a sustained duration of biological activity, which provides a practical benefit for the patient by allowing fewer G-CSF injections per course of treatment compared to repeated injection of unconjugated G-CSF (Neupogen®).
  • R-O-(CH 2 -CH 2 -O) n VCH 2 -CHO is preferably used for the conjugation of proteins, wherein R is H or lower alkyl such as methyl; n is an integer representing the number of ethylene oxide residues in a polyethylene glycol moiety, preferably from 225 to 680; m is an integer representing the number of methylene groups; L 1 is absent; L 2 is absent; and y is 1.
  • R is H or lower alkyl such as methyl
  • n is an integer representing the number of ethylene oxide residues in a polyethylene glycol moiety, preferably from 225 to 680;
  • m is an integer representing the number of methylene groups; L 1 is absent; L 2 is absent; and y is 1.
  • the skilled person is well aware of protocols and conditions for producing these compounds and other polymer reagents according to the present invention (see, e.g. EP 0 154 316; US 5,990,237; Chamov et al., Bioconju
  • the process of the present invention is started by converting a precursor compound of the polyethylene glycol reagent, such as methoxy polyethylene glycol acetal, to the activated polyethylene glycol acetaldehyde such as methoxy polyethylene glycol aldehyde.
  • This activation is usually performed in an aqueous solution, e.g. for up to 3h, at an acidic pH, e.g. pH 2, which may be achieved by adding any organic and/or inorganic acid.
  • an acidic pH e.g. pH 2
  • the conversion from polyethylene glycol acetal to polyethylene glycol aldehyde is tracked by analysing the components by 1 H-NMR spectroscopy.
  • any natural or recombinant protein or protein variant or protein mutant as obtained from any prokaryotic or eukaryotic cell may be used.
  • Human proteins are preferred.
  • any protein having the activity of the wild-type form, including variants, muteins, structurally or functionally equivalent protein as well as synthetic protein-like forms may be used.
  • a protein having the activity of G-CSF is used in the process according to the present invention.
  • G-CSF is stored in a suitable buffer, e.g. 10 mM Na acetate buffer at pH 4.0 at low concentration, e.g. about 1 mg/ml.
  • the pegylation reaction according to the present invention is commonly performed in a suitable incubator, containing the protein such as G-CSF, a physiological buffer system, and activated polyethylene glycol aldehyde such as methoxy polyethylene glycol acetaldehyde, and a reducing agent. Further, it is preferred that the reaction mixture is gently stirred at low temperature. The reaction may be stopped by acidification of the reaction mixture. Reaction buffer
  • Typical reaction buffers for the pegylation reaction according to the present invention include phosphate, citrate/phosphate, cacodylate, carbonate, HEPES, MES, BES, MOPS and/or other suitable buffers.
  • the concentration at which the buffers are used may depend on the amount of protein used. Typical concentrations range from 50 to 200 mM buffer such as phosphate buffer. However, the different ionic strengths do not affect the di- pegylated product yield.
  • the concentration of protein used for the pegylation process according to the present invention depends on the physicochemical properties of the protein, such as solubility and aggregation, as well as on process economics.
  • the concentration of protein in the reaction mixture is preferably in the range from 1 to 20 mg/ml, more preferably from about 2 or 2.4 to about 5.7 or 6 mg/ml and most preferred at about 3.2 mg/ml.
  • di-pegylation of proteins according to the present invention may be performed in a wide range of protein to polyethylene glycol reagent molar ratios.
  • the molar ratios of polyethylene glycol aldehyde, such as methoxy polyethylene glycol acetaldehyde to protein of about 5 to 400 are preferred. More preferably, the molar ratio, especially for the di-pegylation of G-CSF, is 10 to 50, most preferably 15 to 30.
  • Di-pegylation of proteins according to the present invention may be performed in a wide range of polyethylene glycol reagent molecular weights.
  • Preferred molecular weights of the polyethylene glycol aldehyde reagents are in the range from 10 to 30 kDa. Higher molecular weights may lead to lower di-pegylation yields and reaction mixtures may then show an increase in viscosity.
  • the chemical structure of the polyethylene glycol aldehyde reagent is not essential and thus not limited to be linear, but may also be branched.
  • the reaction temperature for the process according to the present invention will be in a range from about 2°C to about 50°C, more preferably between 2°C and 20°C.
  • the process according to the present invention is performed at lower temperature to minimize non-specific modification such as degradation, aggregation of protein by chemical or physical processes.
  • the most preferred reaction temperatures for the process according to the present invention are in the range from about 2°C to about 8 0 C, e.g. at about 4°C.
  • EP 0 154 316 states that the reducing agent should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation.
  • Preferred reducing agents may be selected from the group consisting of sodium borohydride and sodium cyanoborohydride and should be added in excess to the protein concentration used. It was found that a low molar excess of a reducing agent, such as sodium cyanoborohydride, i.e. a less than 117-fold molar excess, decelerates the formation of di-pegylated G-CSF, but reached the same maximum di-pegylated protein conjugate yield compared to high concentrations of reducing agents such as a molar excess of up to 180-fold molar excess. Above a molar excess of sodium cyanoborohydride in the range of a 120-fold molar excess, the kinetic formation of di-pegylated protein conjugate does not depend on the concentration of reducing agent used.
  • the reaction mixture is usually subjected to a purification step, preferably by subjecting the reaction mixture to a cation exchange chromatography.
  • Cation exchange chromatography may be performed with all common and/or commercially available cation exchange matrices.
  • Typical ion exchange resins that may be employed for the purpose of the present invention comprise SP-5PW (Tosohaas Biosciences, Germany), Source S (Amersham Biosciences, Germany), Fractogel SO 3 650 (Merck, Germany), SP Sepharose HP (Amersham Biosciences, Germany) and SP FF Sepharose (Amersham Biosciences, Germany).
  • the purification step is performed at a temperature from 2°C to ambient temperature, preferably at room temperature.
  • any buffer may be used which is typically used for ion exchange chromatography, comprising phosphate, sodium acetate, TRIS/HCl, HEPES or other suitable buffers.
  • the buffers are chosen depending on the protein being analysed and are adjusted to pH values from 3.5 to 8.5, preferably from 4 to 6.
  • the dilution, washing, and equilibration buffer contains 15 mM sodium acetate at pH 4.2.
  • the elution buffer may be of the same composition as the equilibration buffer and may contain about 1 M NaCl.
  • the reaction mixture may be diluted, e.g. 3-fold with equilibration buffer in order to reduce the conductivity to approximately 6 mS/cm. This provides conditions for the complete binding of the protein conjugate according to the present invention to the ion exchange column.
  • Purification by C-IEX typically includes the following steps:
  • linear flow rates of about 68 cm/h may be used for all chromatography steps except sample loading which may be done at 17 cm/h.
  • the first pool also designated as product pool I
  • the second pool also designated as rest pool
  • the rest pool may also contain traces of di-pegylated isoforms of the product pool but essentially does not comprise di-pegylated isoforms of the product pool.
  • This rest pool is preferably recycled in another pegylation reaction, which is performed as described above for the process according to the present invention.
  • the rest pool is usually applied to ultrafiltration / diafiltration in order to concentrate the modified protein and to exchange the buffer for a buffer suited to perform a pegylation reaction according to the process of the present invention.
  • the rest pool is then subjected to a pegylation reaction as described above for the process of the present invention, optionally followed by cation exchange purification as described above.
  • the product pool II resulting from this re-pegylation step, optionally purified, may then be combined with the product pool I (see Figure 1).
  • G-CSF is used in these examples.
  • Other proteins may also be conjugated to PEG by the methods exemplified.
  • 150 mg 12 kDa mPEG acetal was dissolved in 80 raM phosphoric acid and hydrolyzed for 3h at 50°C. After cooling down on ice to approximately 2-8°C the solution was neutralized to pH 7 by drop wise addition of sodium bicarbonate solution (5%). The mixture was saturated with NaCl and three times extracted with 2 ml methylene chloride. The three collected organic phases were combined, dried by adding disodium sulphate and the volume was reduced to 1.5 ml (nitrogen stream).
  • mPEG acetaldehyde was precipitated by adding 30 ml ice-cold diethyl ether. The precipitate was isolated by vacuum filtration (G4 funnel), dried at room temperature under vacuum and transferred in gas-tight tubes, which were flushed with nitrogen gas before sealing them and stored at -20°C.
  • the mPEG acetal was usually activated freshly for each pegylation reaction, or was at maximum stored over one night.
  • mPEG aldehyde appears to be stable for at least 125 days when stored light-protected, in sealed tubing, under N 2 atmosphere and at -2O 0 C (see Figure 2).
  • the diluted G-CSF sample was concentrated by ultrafiltration. Depending on the final sample concentration, G-CSF was concentrated to approximately 4-8 mg/ml using an Amicon cell (50 ml) or centricon tubes (2 ml) equipped with YM 10 membranes of 10 kDa molecular weight cut off.
  • an absorbance of 0.86 ⁇ 0.015 at 280 nm corresponds to 1 mg/ml protein.
  • the same molar extinction coefficient was applied.
  • the species distribution (mono-, di-, tri-, tetra-) in the PEG G-CSF reaction mixture was determined by C-IEX HPLC. Therefore, the stopped reaction mixture was diluted for three times with 15 mM acetate buffer (pH 4.2) and passed through a column packed with SP-5PW (75 x 7.5 mm, 10 ⁇ m particle size, 3.3 ml bed volume, manufactured by Tosohaas Bioscience), mounted on a Dionex system or Shimadzu HPLC system.
  • the column was equilibrated in 15 mM NaAc, pH 4.2. The flow rate was 0.4 ml/min and 200 ⁇ g of protein were typically injected. The column was run at room temperature; however, the samples were maintained at 5 0 C in the auto-injector.
  • each pegylated G-CSF conjugated could be allocated to the retention time of an elution peak.
  • Mass of the single pegylated G-CSF species determined by MALDI-TOF agreed with the preliminary mass estimation based on the amino acid sequence and PEG moiety (see Table 1).
  • Table 1 Comparison of calculated and experimental determined molecular weight of different pegylated G-CSF species.
  • the analytic C-IEX allowed the separation of one mono-PEG G-CSF, three di-PEG G-CSF isoforms, four tri-PEG G-CSF isoforms, and one tetra-PEG G-CSF in dependence of the retention time.
  • the average isoform composition in the PEG G-CSF reaction mixture at 30 h incubation was determined (see Table 2).
  • Table 2 Relative percentage of differently pegylated G-CSF species in five PEG G-CSF syntheses at a 30 h reaction time with a single coupling cycle.
  • the rest pool of the preparative C-IEX chromatography (see Figure 4) containing all species including traces of product species according to the present invention, was collected, de-salted and concentrated by ultrafiltration/diafiltration and subjected again to the pegylation reaction (see Example 3), except that PEG modified G-CSF (3.2 mg/ml) was used.
  • PEG modified G-CSF 3.2 mg/ml
  • C-IEX HPLC Analytical cation exchange high performance liquid chromatography
  • Table 4 Allocation of elution peaks from the preparative C-IEX run with corresponding retention time and allocated PEG G-CSF species (isoforms).
  • di-pegylated G-CSF For di-pegylated G-CSF, three out often isoforms could be identified. Two isoforms, Di and Di' PEG G-CSF, eluted in major single peaks. The third minor isoform co- eluates with a tri-PEG G-CSF isoform.
  • di-PEG G-CSF product which comprises two isoforms, di- (peak 2) and di'- (peak 3).
  • PEG attachment sites for these isoforms have been identified to be located at the N- terminus, lysine 17 and lysine 35 by using a combination of MALDI-MS/ nano-LC ESI-MS/MS and Edman sequencing (see product characterisation).
  • the defined di-PEG G-CSF product contained at least 90% di-PEG G-CSF species, comprising di and di 1 isoforms in an approximately 0.76 (di) to 1 (di 1 ) ratio (see Table 5):
  • the isolated di-PEG G-CSF product consists of two major (di and di 1 ) di-PEG G- CSF isoforms and represent the major active di-pegylated product species.
  • composition and purity of the final di-PEG G-CSF product were analysed by cation exchange and size exclusion chromatography.
  • C-IEX HPLC to control the composition of final di-PEG G-CSF product
  • Buffer A 15 mM NaAc, pH 4,2
  • Buffer B 15 mM NaAc, pH 4,2; 1.000 mM NaCl
  • the isomer composition of the di-PEG G-CSF product was analysed by C-IEX HPLC (see Figure 5) and the composition was within the defined product specification (see Table 6).
  • Table 6 Averaged Di-PEG G-CSF Product definition optimized for the total process.
  • Buffer A 10O mM Pi; pH 6.9
  • the final product consists of > 90% di- pegylated G-CSF, comprising di and di' PEG G-CSF isoforms in a defined mixed ratio.
  • the peaks between 11 and 13 min retention time were not further analysed, but could potentially be higher molecular aggregates (-1.5 %, see Table 7).
  • Table 7 PEG G-CSF isoform distribution in the final di-PEG G-CSF product analysed by SEC analysis.
  • the final di-PEG G-CSF product (sample B) and native unmodified G-CSF (sample A) were analysed by circular dichroism spectroscopy.
  • CD spectra were recorded from 180-260 nm wavelengths using a Jasco J-720 (Japan) spectropolarimeter.
  • di-pegylated G-CSF isoforms (di- and di 1 PEG G-CSF) were analysed by peptid mapping.
  • non-pegylated G-CSF was treated identically.
  • the procedure comprised the steps (i) denaturation of the PEG G-CSF isoforms and capping of the free SH groups by reductive alkylation, (ii) chymotrypsin digestion of the denatured PEG G-CSF isoforms, (iii) rpHPLC peptide separation and (iv) sequence analysis by combination of electrospray tandem mass analysis and MALDI-MS/N-terminal sequencing. (i) Carboxymethylation of SH groups
  • Chymotrypsin digestion Dried samples of di-PEG G-CSF isoforms were reconstituted to a concentration of lmg in 1 ml 0.1 M ammonium hydrogen carbonat buffer, pH 7.8 for digestion. The isoforms were digested with chymotrypsin (enzyme to substrate ratio per weight of 1 :100) at 37 0 C for 3 h.
  • Protein digests were injected onto a Vydac C4 column (4.6 x 250 mm, 5 m particle size, 300 A pore size) and peptides were mapped by HPLC using a linear gradient of acetonitrile in 0.1% TFA (1% acetonitrile increase/min over 90 min). The resulting peptides were collected for peptide sequencing by a combination of nano-LC ESI MS/MS and N-terminal sequencing.
  • peptide mass sequencing with nano-LC ESI-MS/MS identified the sequence from amino acid 1 to 13: M-T-P-L-G-P-A-S-S-L-P-Q-S-F (MW 1470 m/z) having the PEG attachment to the ⁇ -amino group of the N-terminal methionine.
  • N-terminal sequence analysis determined the sequence from amino acid 14 to 40: LLKCLEQ VRKIQGDGAALQEKLC AT Y having the PEG attachment to the ⁇ -amino group of lysine 17.
  • N-terminal sequence analysis determined the sequence from amino acid 14 to 40: LLKCLEQVRKIQGDGAALQEKLCATY having the PEG attachment to the ⁇ - amino group of lysine 35.
  • the Di-PEG G-CSF isoform is di-pegylated at the N-terminus and lysine 17 and the Di'-PEG G-CSF isoform is di-pegylated at the N-terminus and lysine 35. d) Bioactivity
  • the di-PEG G-CSF product was tested in vivo, to show the improved activity versus unmodified G-CSF (Neupogen®) and to compare its biological activity with commercially available mono-pegylated G-CSF (Neulasta®).
  • the in vivo study was an open randomized, parallel-grouped single dose biocomparability study of Neupogen® and Neulasta® with our di-PEG G-CSF product. It was carried out by subcutaneously injecting 0.1 mg/kg to male rats. Six animals were subjected to bleeding per group per time point. Serum samples were subject to a complete blood count on the same day that the samples were collected. The average white blood cell counts and neutrophil counts were calculated.
  • Di-PEG G-CSF product according to the present invention yielded higher WBC and neutrophil counts and longer sustained WBC and neutrophil response compared to unmodified G-CSF (Neupogen®) (see Figure 8 and Figure 9).
  • Figure 1 Overview of all process steps in the production of di-PEG G-CSF product according to the present invention.
  • Figure 2 Maximum yield of all di-PEG G-CSF conjugate isoforms and kinetic of formation as function of time-dependent low temperature storage of activated mPEG aldehyde used for the pegylation reaction.
  • Figure 3 Sequential formation of recombinant G-CSF conjugates with one to four strands of 12 kDa mPEG aldehyde in time-dependent manner. Maximum di-PEG G- CSF yield at 30 h incubation.
  • Figure 4 A typical preparative C-IEX elution profile for the separation of di-PEG G-CSF from the pegylation reaction mixture (protein load: 6.1 mg pegylated G-CSF species; column: SP-5PW C-IEX column (Tosohaas, 20 ⁇ m, 10.6 ml bed volume), Buffer A: 15mM NaAc, pH 4.2; Buffer B: 1 M NaCl in 15 mM NaAc, pH 4.2; linear gradient); E- eluat, RP - rest pool.
  • protein load 6.1 mg pegylated G-CSF species
  • column SP-5PW C-IEX column (Tosohaas, 20 ⁇ m, 10.6 ml bed volume)
  • Buffer A 15mM NaAc, pH 4.2
  • Buffer B 1 M NaCl in 15 mM NaAc, pH 4.2
  • linear gradient E- eluat, RP - rest pool.
  • Figure 5 C-IEX HPLC separation of Di-PEG G-CSF product. Chromatogramm recorded at 214 nm.
  • Figure 6 SEC separation of di-PEG G-CSF product. Chromatogramm recorded at 214 nm. Peak at 23 min corresponds to salt.
  • Figure 7 CD spectrum of non-modified G-CSF (black), and di-PEG G-CSF product (red).
  • Figure 8 In vivo bioactivity of di-PEG G-CSF conjugate in comparison to unmodified rhG-CSF and mono-pegylated rhG-CSF illustrated by the average WBC after single subcutaneous injection (PBS - phosphate buffered saline).
  • Figure 9 In vivo bioactivity of di-PEG G-CSF conjugate in comparison to unmodified rhG-CSF and mono-pegylated rhG-CSF illustrated by the average neutrophil counts after single subcutaneous injection (PBS - phosphate buffered saline).
  • Figure 10 SEQ ID No. 1 (amino acid sequence of G-CSF including methionine at position 1).

Abstract

L‘invention concerne les conjugués de protéines et de polymères ainsi que leurs procédés de préparation. Selon cette invention, deux atomes d’azote de groupes amino de la protéine sont conjugués chacun avec une unité polymère par une liaison amine.
PCT/EP2005/002632 2005-03-11 2005-03-11 Conjugues de proteines et de dipolymeres et leurs procedes de preparation WO2006094530A1 (fr)

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PCT/EP2005/002632 WO2006094530A1 (fr) 2005-03-11 2005-03-11 Conjugues de proteines et de dipolymeres et leurs procedes de preparation
PCT/EP2006/060671 WO2006095029A2 (fr) 2005-03-11 2006-03-13 Conjugues di-polymere-proteine et procedes de preparation de ceux-ci
JP2008500216A JP2008535793A (ja) 2005-03-11 2006-03-13 ジポリマー・タンパク質コンジュゲートおよびその調製方法
EP06725029A EP1869079A2 (fr) 2005-03-11 2006-03-13 Conjugues di-polymere-proteine et procedes de preparation de ceux-ci

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US10195274B2 (en) 2015-05-28 2019-02-05 Armo Biosciences Inc. Method of modulating a chimeric antigen receptor t cell immune response by administering IL-10
US10209261B2 (en) 2013-06-17 2019-02-19 Armo Biosciences Inc. Method for assessing protein identity and stability
US10293043B2 (en) 2014-06-02 2019-05-21 Armo Biosciences, Inc. Methods of lowering serum cholesterol
US10357545B2 (en) 2013-04-18 2019-07-23 Armo Biosciences, Inc. Methods of using interleukin-10 for treating solid tumors
US10398761B2 (en) 2015-08-25 2019-09-03 Armo Biosciences, Inc. Methods of using combinations of PEG-IL-10 and IL-15 for treating cancers
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US10618970B2 (en) 2015-02-03 2020-04-14 Armo Biosciences, Inc. Method of treating cancer with IL-10 and antibodies that induce ADCC
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EP1834963A1 (fr) * 2006-03-13 2007-09-19 Siegfried Ltd. Conjugues de proteines et de dipolymeres et leurs procedes de preparation
WO2007104768A2 (fr) * 2006-03-13 2007-09-20 Siegfried Ltd. Conjugués de protéines di-polymères et leurs procédés d'élaboration
WO2007104768A3 (fr) * 2006-03-13 2007-11-08 Siegfried Ltd Conjugués de protéines di-polymères et leurs procédés d'élaboration
US10568968B2 (en) 2006-09-28 2020-02-25 Merck Sharp & Dohme Ltd. Methods for treatment of cancer with therapeutic combinations comprising PEG-IL-10
CN101627056B (zh) * 2007-12-29 2013-05-15 厦门伯赛基因转录技术有限公司 Y型聚乙二醇修饰的g-csf及其制备方法和应用
EP2236521A4 (fr) * 2007-12-29 2013-07-10 Biosteed Gene Expression Tech Co Ltd G-csf modifié par du polyéthylène glycol de type y, méthode de préparation et son utilisation
US8530417B2 (en) 2007-12-29 2013-09-10 Biosteed Gene Expression Tech Co. Ltd. Y-shaped polyethylene glycol modified G-CSF, the preparation and use thereof
AU2007363326B2 (en) * 2007-12-29 2014-01-30 Biosteed Gene Expression Tech. Co., Ltd. Y-type polyethylene glycol modified G-CSF and preparation method and use thereof
WO2009086656A1 (fr) * 2007-12-29 2009-07-16 Biosteed Gene Expression Tech. Co., Ltd. G-csf modifié par du polyéthylène glycol de type y, méthode de préparation et son utilisation
EP2236521A1 (fr) * 2007-12-29 2010-10-06 Biosteed Gene Expression Tech. CO., LTD. G-csf modifié par du polyéthylène glycol de type y, méthode de préparation et son utilisation
EP2313457A2 (fr) * 2008-07-31 2011-04-27 Pharmaessentia Corp. Conjugués peptide-polymère
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US20170202923A1 (en) * 2008-12-17 2017-07-20 Merck Sharp & Dohme Corp. Mono- and Di-Peg IL-10 Production; and Uses
US10639353B2 (en) * 2008-12-17 2020-05-05 Merck Sharp & Dohme Corp Mono- and di-PEG IL-10 production; and uses
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US11413332B2 (en) 2013-11-11 2022-08-16 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders
US10293043B2 (en) 2014-06-02 2019-05-21 Armo Biosciences, Inc. Methods of lowering serum cholesterol
US10143726B2 (en) 2014-10-22 2018-12-04 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders
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