WO2009141384A2 - Procédé de purification de polypeptides de facteur vii à l'aide de résines d'affinité comportant des ligands spécifiques - Google Patents

Procédé de purification de polypeptides de facteur vii à l'aide de résines d'affinité comportant des ligands spécifiques Download PDF

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WO2009141384A2
WO2009141384A2 PCT/EP2009/056149 EP2009056149W WO2009141384A2 WO 2009141384 A2 WO2009141384 A2 WO 2009141384A2 EP 2009056149 W EP2009056149 W EP 2009056149W WO 2009141384 A2 WO2009141384 A2 WO 2009141384A2
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fvii
ligand
drug substance
resin
factor vii
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PCT/EP2009/056149
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WO2009141384A3 (fr
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Phaedria Marie St. Hilaire
Roice Michael
Janus Krarup
Christine Bruun SCHIØDT
Line Naomi Lund
Ib Johannsen
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Novo Nordisk A/S
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Publication of WO2009141384A2 publication Critical patent/WO2009141384A2/fr
Publication of WO2009141384A3 publication Critical patent/WO2009141384A3/fr

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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
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    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
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Definitions

  • the present invention relates to a novel process for the purification of Factor VII polypeptides, e.g. recombinant Factor VII.
  • the process utilizes an affinity resin comprising a solid phase material having immobilized thereto one or more low- molecular weight synthetic ligands.
  • the affinity resins enable the separation of Factor VII polypeptide from closely related proteins, e.g. Protein S.
  • Therapeutic proteins are produced in living cells and must be purified from a complex mixture of proteins and other biological species before use in a patient. This separation can be very laborious and costly. A number of chromatography materials have been described for use in such separation.
  • Affinity chromatography enables selectively and reversibly adsorbing biological substances, such as proteins, to a complementary binding substance, such as an affinity ligand immobilised on a solid phase material, usually a porous, polymer matrix, packed in an affinity column.
  • a suitable ligand is covalently attached to the solid phase materials directly or by means of a linker.
  • a sample containing biological substances having affinity for the ligand can be brought into contact with the affinity ligand covalently immobilised to the solid phase material under suitable binding conditions which promote a specific binding between the ligand and the biological substance having an affinity for the ligand.
  • the affinity column can subsequently be washed with a buffer to remove unbound material, and in a further step, the biological substances having affinity for the ligand can be eluted and obtained in a purified or even in an isolated form.
  • the ligand should preferably exhibit specific and reversible binding characteristics to the biological substance, which it is the aim to purify or isolate.
  • US 6,498,236 Bl discloses affinity resins comprising synthetic affinity ligands for purification of immunoglobulins. An extensive list of possible aromatic and heteroaromatic groups are mentioned, among these 5-aminoisophtalic acid.
  • the invention furthermore relates to these novel affinity ligand-matrix conjugates and the preparation and use thereof in the purification of proteinaceous materials such as immunoglobulins, insulins, Factor VII, or human Growth Hormone or analogues, derivatives and fragments thereof and precursors.
  • US 5,633,350 discloses a method for the separation of vitamin K-dependent proteins from non-vitamin K-dependent accompanying proteins wherein the method is characterized in that at least anion exchange chromatography and optionally affinity chromatography is carried out as well.
  • the method is suitable especially for the purification of Factor II, VII, IX, X as well as Protein S, Protein C and Protein Z.
  • the optional affinity purification step is carried out with the use of an affinity resin comprising heparin affinity ligands.
  • US 2007/037966 discloses a process for the purification of a drug substance of a Factor VII polypeptide comprising contacting a drug substance with a hydrophobic interaction chromatography material under conditions which facilitate binding of a portion of said drug substance to said hydrophobic interaction chromatography material, wherein the hydrophobic interaction chromatography material comprises butyl and phenyl ligands.
  • Jurlander et al. Seminars in Thrombosis and Hemostasis (2001) discloses a process for purification of Factor VII involving an immunoaffinity chromatographic step.
  • Kelley et al. in Biotechnology and Bioengineering, Vol. 87, No. 3, August 5, 2004 describe a process for cGMP purification of Factor VIII and Factor Vlll-like molecules.
  • the biomolecule is purified in a multistep process wherein the immunoaffinity step is replaced by an affinity purification step using a peptide ligand (27 AA).
  • the bound Factor VIII was eluted by a buffer containing 50% ethylene glycol. This process is also described in United States Patent Application 20060193829.
  • US 2006/0051854 describes an immunoaffinity chromatographic step for purification of Factor VII employing an affinity resin comprising anti-FVIIa antibody ligands immobilized onto a sepharose base matrix. Washing of the column with a buffer comprising 2 M NaCI, 10 mM CaCI 2 and 10 mM histidine at pH 6.0 between loading and elution is described.
  • US 4,461,833 A describes a process for the purification of proteolytic procoagulant proteins from human and animal tissue by a multistep chromatographic process including purification on a benzamide affinity column.
  • recombinant Factor VII (rFVII) polypeptides are typically purified from the fermentation fluid by one of two different multi-step processes, either employing exclusively chromatography on conventional resins, or including affinity chromatography employing an affinity resin with protein ligands. Chromatography involving multiple steps on conventional resins suffers from one or more of the following drawbacks: reduced selectivity, low yield, high buffer consumption, long process time and high investments in process equipment.
  • Affinity resins with protein ligands result in increased selectivity, higher yield, lower buffer consumption, and reduced investment in process equipment.
  • affinity resins with protein ligands are very costly to manufacture, and inherently hold a risk that the purified Factor VII polypeptide be contaminated with protein fragments from the protein ligands or other biological matter from the production of the affinity resin with protein ligands.
  • the present invention solves the problem by providing affinity resins comprising novel synthetic affinity ligands that are selective for Factor VII polypeptides and that are not based on protein ligands.
  • the present inventors have developed a new process involving such synthetic ligands and have found that it is possible to reduce, or virtually eliminate, the presence of protein contaminants, such as plasma proteins or host cell proteins, and in particular to remove the contaminant Protein S, from a drug substance of a Factor VII polypeptide.
  • the present invention provides a process for the purification of a drug substance of a Factor VII polypeptide, using new affinity resins, cf. claims 1 and 3.
  • the present invention also provides novel affinity resins, cf. claims 12 and 14.
  • the present invention provides novel ligands (l)-(20), cf. claim 15. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 SDS-PAGE gel showing lane 1 : CCF, lane 2: FVII, lane 3 : Protein S and FVIIa, lane 4: Eluate from affinity resin with ligand 5, lane 5 : Eluate from affinity resin with ligand 20.
  • the present invention in particular provides a process for the purification of a drug substance of a Factor VII polypeptide.
  • drug substance used herein is intended to mean a solid mass as well as a liquid mass, e.g. a solution or suspension comprising the Factor VII polypeptide.
  • drug substance is in particular meant to refer to a "large” volume or mass, i.e. referring to volumes and masses known from large- scale and industrial-scale processes.
  • Vector VII polypeptide is defined further below.
  • the affinity resin is a solid phase material (see further below) having covalently immobilized thereto one or more ligands that have a high specificity towards the Factor VII polypeptide in question.
  • the term "ligand” means a molecule which can bind a target compound; in the present context a Factor VII polypeptide.
  • the ligands should bind to the Factor VII polypeptide in question at least in a substantially specific manner ("specific binding”).
  • the expression “one or more ligands” refers to the fact that the solid phase material may having more than one type of ligand immobilized thereto. This being said, immobilization of a single type of ligands (“a ligand”) will typically involve the immobilization of a plurality/multitude of species of identical ligands.
  • binding refers to the property of a ligand to bind to a Factor VII polypeptide ⁇ i.e. the binding partner), preferentially such that the relative mass of bound Factor VII polypeptide, is at least two-fold, such as 50-fold, for example 100-fold, such as 1000-fold, or more, greater than the relative mass of other bound species than the Factor VII polypeptide.
  • the current invention discloses affinity ligands and affinity resins wherein the ligands are specific binding partners of Factor VII polypeptide and can therefore be used for the purification of said polypeptide.
  • the affinity ligands are designed to bind in particular to the protease and EGF2 domains of Factor VII polypeptide.
  • An analysis of the desired ligand binding sites on the Factor VII polypeptide indicate that ligands comprising hydrophobic, polar groups and cationic groups will be especially well suited to bind specifically.
  • hydrophobic group is an organic group capable of binding to the surface of a bio-molecule mainly by hydrophobic interaction.
  • Hydrophobic groups are characterised by being essentially non-polar and uncharged at normal physiological conditions. Hydrophobic residues are repelled by aqueous solution so as to seek the inner positions in the conformation of a ligand when the ligand is in an aqueous medium. Also, hydrophobic residues will seek towards hydrophobic pockets or grooves of ligand binding partners when the ligand is associated with a binding partner under normal physiological conditions.
  • normal physiological condition means conditions that are typical inside a living organism or a cell. While it is recognized that some organs or organisms provide extreme conditions, the intra-organismal and intra-cellular environment normally varies around pH 7 ⁇ i.e., from pH 6.5 to pH 7.5), contains water as the predominant solvent, and exists at a temperature above O 0 C and below 5O 0 C.
  • Hydrophobic groups generally have a high content of carbon atoms.
  • Typical examples of hydrophobic groups are linear and branched alkanes, cyclic hydrocarbons, aromatic compounds, and combinations of linear and branched alkanes, cyclic hydrocarbons, and aromatic compounds.
  • substituted variants of such groups are considered as being hydrophobic as long as the relative content of carbon is above a certain limit.
  • the percentage of carbon atoms is not the only parameter, which influences the hydrophobicity.
  • the position and nature of other atoms play an important role.
  • an ether is typically more hydrophobic than an alcohol with the same number of carbon atoms and oxygen atoms
  • an ester is more hydrophobic than a di-ol with the same elemental composition.
  • a hydrophobic group as an organic group with 75% or more of its non-hydrogen atoms being carbon atoms, such as 80% or more, preferably 85% or more of its non-hydrogen atoms being carbon atoms.
  • the lower value, 75% applies to ethers and esters
  • the intermediate value, 80% applies to amides and secondary and tertiary amines
  • the upper value, 85% applies to alcohols and primary amines.
  • a polar group is an organic group capable of binding to the surface of a bio-molecule mainly by hydrogen bond formation.
  • Polar groups confer water solubility to the molecule.
  • polar groups comprise both groups with a permanent positive or negative charge such as a cationic or anionic group, as well as groups without a permanent charge.
  • Polar groups without a permanent charge usually contain electronegative atoms such as for example oxygen, sulphur and halogens, which cause unequal sharing of electrons between the nuclei of the electronegative atom and neighbouring atoms, such as e.g. hydrogen and carbon, and result in a molecule with opposing partial charges or a molecule with a dipole moment.
  • groups containing the atoms, oxygen, sulphur, nitrogen for example from groups such as carbonyls, secondary amines, anilines, primary and secondary amides, primary and secondary sulfonamides, phenols, alcohols, ethers, esters, nitriles and nitrogen containing heterocycles (i.e. a carbocyclic ring or ring system
  • the polar group without a permanent charge can be donated by an amino acid residue, such as serine, homoserine, threonine, homothreonine, citrulline, beta- citrulline, homocitrulline, beta-homocitrulline, hydroxyproline, tyrosine, homotyrosine, mono-, di-, tri- and tetra- hydroxy susbtituted phenylalanine, mono-, di-, tri- and tetra- alkoxy susbtituted phenylalanine, hydroxy and alkoxy substituted phenylalanine, cyano substituted phenylalanine, mono-, di-, tri- and tetra- amino susbtituted phenylalanine, asparagine, beta-asparagine, glutamine, beta-glutamine, homoglutamine, beta-homoglutamine.
  • an amino acid residue such as serine, homoserine, threonine, homo
  • anionic group Polar groups with a permanent negative charge in a particular pH range are termed "anionic group" for the purposes of this invention.
  • an "anionic group” is an organic group which has a negative charge in the pH range 3-10. Anionic groups are negatively charged due to abstraction of a proton in the pH range pH 5-10. Anionic groups of a ligand will seek cationic groups of ligand binding partners when the ligand is associated with a binding partner.
  • the anionic group is preferably selected from anionic groups comprising one or more negatively charged oxygens(s), one or more phosphorous atom(s) one or more nitrogen(s), and/or one or more sulphur atom(s).
  • Preferred anionic groups comprise a negatively charged oxygen from groups such as e.g. phosphate, sulphate, carboxylate, nitrate.
  • cationic group Polar groups with a permanent positive charge in a particular pH range are termed "cationic group" for the purposes of this invention.
  • a "cationic group” is an organic group which has a positive charge in the pH range 3-7.
  • Cationic groups are positively charged either due to a permanent positive charge or due to an association with an H ion at under normal physiological conditions, i.e. pH 3-7.
  • Cationic groups are attracted by aqueous solution so as to seek the surface positions in the conformation of a ligand when the ligand is in an aqueous medium under normal physiological conditions.
  • Cationic groups will seek towards anionic groups of ligand binding partners when the ligand is associated with a binding partner under normal physiological conditions.
  • the cationic group is preferably selected from cationic groups comprising one or more positively charged nitrogen(s), one or more phosphorous atom(s) and/or one or more sulphur atom(s).
  • Preferred cationic groups comprise a permanent positively charged nitrogen from groups such as e.g. alkyl ammonium, such as trimethyl ammonium, or triethylammonium, or dimethylammonium, or benzyldimethylammonium, or guanidinium, or a positively charged nitrogen from positively charged heterocycles, such as imidazolinium, piperidinium and pyrrolidinium. Guanidinium is particularly preferred.
  • cationic groups are mono- and disubstituted amines, such as monoalkyl amines, dialkyl amines, heterocyclic amines and aromatic amines which have a partial positive charged in aqueous solutions with pH in the range of 3-8, in particular in the range of 3-7.
  • the positively charged nitrogen can also be donated by an amino acid residue, such as lysine, arginine, histidine, ornithine, diaminobutyric acid, diaminopropionic acid, diaminopentanoic acid, diaminohexanoic acid, diaminopimelic acid, homoarginine, p-aminophenylalanine and 3-aminotyrosine.
  • Arginine is particularly preferred.
  • the desired ligands should include building blocks from the following group of amino acids leucine (Leu), isoleucine (He), 4-chloraniline (CA), phenylalanine (Phe), 5-aminoindane (AIND), 2,2-diphenylethylamine (DPEA), (aminomethyl)cyclohexane (CHMA), 1-ethyl propylamine (IEP), phenylcyclopentaneacetic acid (PCAA), 2-propylpentanoic acid (PPA), tryptophan (Trp), indole-3-carboxylic acid (3-ICA), threonine (Thr), tyrosine (Tyr), 3- hydroxy-4-methoxybenzylamine (HMOBA), 4-hydroxy-3-methoxybenzylamine (MOBHA), 4-aminophenol (4AP), 4-hydroxybenzoic acid (4HBA), glutamine (GIn), 4-aminobenzamide (ABA), 4-(2-aminoethylamine
  • amino acids with one to 5 carbon atoms more or less such as e.g. ornithine, diamino propanoic acid are homologs of lysine.
  • Related analogs of amino acids are isomers, i.e. one of two or more compounds that have the same chemical formula but different arrangements of the atoms within the molecules and that may have different physical/chemical properties such as for example regioisomers such as arginine and beta-arginine.
  • amino acids are isosteres that is one of two or more substances that exhibit similarity of some properties as a result of having the same number of total or valence electrons in the same arrangement and that consist of different atoms and not necessarily the same number of atoms such as for example tyrosine and 4-chlorobenzoic acid.
  • Related analogs of amino acids are compounds derivatives containing the same, similar or additional functional groups in an arrangement that is not isosteric, isomeric or homologic. Similar functional groups are similar by virtue of having similar physical or chemical properties.
  • Examples of compound derivatives are mono-, di-, tri- and tetra- hydroxy susbtituted phenylalanine, mono-, di-, tri- and tetra- alkoxy susbtituted phenylalanine, mono-, di-, tri- and tetra- alkyl susbtituted phenylalanine hydroxy and alkoxy substituted phenylalanine, cyano substituted phenylalanine, mono-, di-, tri- and tetra- amino susbtituted phenylalanine, 5-hydroxylysine, methyl lysine, adipic acid, gamma-carboxyglutamic acid, aromatic acids and aromatic amines, substituted aromatic acids and aromatic amines and the like.
  • the ligand has at least one cationic group.
  • the ligand has at least one cationic group and at least one aromatic group. Even more preferably, the ligand has at least one cationic group and at least three aromatic groups.
  • aromatic groups are phenyl (a benzene ring), naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzooxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl,
  • each of Ri, R 2 and R 3 represents an organic moiety of a molecular weight of 15-1000 g/mol, the total molecular weight of said ligand being 200- 2000 g/mol; said ligand being immobilized to said solid phase material via one of said organic moieties R 1 , R 2 and R 3 .
  • the scaffold consisting of 5-aminoisophthalic acid to which the organic moieties R 1 , R 2 and R 3 are bound via amide bonds offers an interesting class of ligands which have excellent binding properties towards Factor VII polypeptides, and which further represents a specific binding to Factor VII polypeptides compared to other proteins present in, e.g., cell culture supernatants or plasma.
  • the ligand preferably has a molecular weight of more than 200 Da, such as more than 300 Da, for example more than 400 Da, such as more than 500 Da, for example more than 600 Da, such as more than 700 Da, for example a molecular weight of more than 800 Da.
  • the ligand preferably has a molecular weight of less than 5000 Da, such as less than 4000 Da, for example less than 3000 Da, such as less than 2500 Da, for example less than 2000 Da, such as less than 1500 Da, for example a molecular weight of less than 1000 Da.
  • organic moiety (and “organic moieties”) is intended to mean a molecular fragment comprising one or more carbon atoms and one or more hydrogen (H), oxygen (O), nitrogen (N), sulphur (S), bromine (Br), chlorine (Cl), fluorine (F), or phosphor (P) atoms colavently bonded.
  • Each of the organic moieties R 1 , R 2 and R 3 typically have the formula C x H y O z N k S
  • the preliminary results have suggested various preferences with respect to the organic moieties R 1 , R 2 and R 3 .
  • the R 2 in the ligand comprises a primary amine. More particularly, the ligand has the general formula (Ia)
  • R' 2 is a linker to the solid phase material.
  • R 2 in the ligand comprises a para-substituted phenyl group. More particularly, the ligand has the general formula (Ib)
  • R' 2 is a linker to the solid phase material.
  • this organic moiety in one variant comprises a para- substitutes chlorobenzene group.
  • R 1 comprises a di-phenyl- isopropyl group.
  • this organic moiety in one variant comprises a primary amine.
  • R 3 comprises a benzyl group, and in still another variant R 3 comprises a quinoline group, and in still another variant R 3 comprises an indole group.
  • especially preferred ligand are the ones selected from the group consisting of ligands (3)-(20)
  • especially preferred ligands are the ones selected from the group consisting of ligands (1) and (2)
  • the ligands described herein include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the description or depiction herein. Both racemic and diasteromeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric counterparts, and these are all within the scope of the invention.
  • Solid phase material
  • the affinity resin is a solid phase material substituted having immobilized thereto one or more synthetic ligands.
  • the solid phase material (sometimes referred to as "a matrix or polymer matrix”) may in principle be selected from a broad range of the materials conventionally use for chromatographic purposes and for peptides synthesis. Examples of such materials are described below.
  • the ligand is covalently immobilized to a solid phase material such as a porous, inorganic matrix or a polymer matrix, optionally in cross-linked and/or beaded form or in a monolithic porous entity.
  • a solid phase material such as a porous, inorganic matrix or a polymer matrix, optionally in cross-linked and/or beaded form or in a monolithic porous entity.
  • the pores of the polymer matrix are sufficiently wide for the target protein to diffuse through said pores and interact with the ligand on the inner surface of the pores.
  • an average pore diameter of 50- 200 nm is preferred, such as approx. 100 nm.
  • the beaded and optionally cross-linked polymer matrix in one embodiment comprises a plurality of hydrophilic moieties.
  • the hydrophilic moieties can be polymer chains which, when cross-linked, form the cross-linked polymer matrix. Examples include e.g. polyethylene glycol moieties, polyamine moieties, polyvinylamine moieties, and polyol moieties.
  • the core and/or the surface of a beaded polymer matrix comprises a polymeric material selected from the group consisting of polyvinyls, polyacrylates, polyacrylamides, polystyrenes, polyesters and polyamides.
  • the beaded polymer matrix can also be selected from the group consisting of PS, POEPS, POEPOP, SPOCC, PEGA, CLEAR, Expansin, Polyamide, Jandagel, PS- BDODMA, PS-HDODA, PS-TTEGDA, PS-TEGDA, GDMA-PMMA, PS-TRPGDA, ArgoGel, Argopore resins, ULTRAMINE, crosslinked LUPAMINE, high capacity PEGA, Silica, Fractogel, Sephadex, Sepharose, Glass beads, crosslinked polyacrylates, and derivatives of the aforementioned; in particular, the polymer matrix is selected from the group consisting of SPOCC, PEGA, HYDRA, POEPOP, PEG-polyacrylate copolymers, polyether-polyamine copolymers, and cross-linked polyethylene di-amines.
  • the core material of a bead is polymeric.
  • the core comprises or consists of hydrophilic polymeric material.
  • the core comprises or consists of hydrophobic polymeric material.
  • the surface of the beads comprises or consists of the same material as the core.
  • Resins useful for large-scale applications may be one of the above mentioned or other commercial resins such as SephadexTM, SepharoseTM, FractogelTM, CIMGELTM, Toyopearl, HEMATM, crosslinked agarose, and macroporous polystyrene or polyacrylate.
  • the matrix may also be of a mainly inorganic nature, such as macroporous glass or clay minerals, or combinations of resins and and inorganics, such as Ceramic HyperDTM or silica gel.
  • Polymer beads according to the invention can be prepared from a variety of polymerisable monomers, including styrenes, acrylates and unsaturated chlorides, esters, acetates, amides and alcohols, including, but not limited to, polystyrene (including high density polystyrene latexes such as brominated polystyrene), polymethylmethacrylate and other polyacrylic acids, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidenechloride and polydivinylbenzene.
  • polystyrene including high density polystyrene latexes such as brominated polystyrene
  • the beads are prepared from styrene monomers or PEG based macro-monomers.
  • the polymer is in preferred embodiments selected from the group consisting of polyethers, polyvinyls, polyacrylates, polymethacrylates, polyacylamides, polyurethanes, polyacrylamides, polystyrenes, polycarbonates, polyesters, polyamides, and combinations thereof.
  • Highly preferred surface and core moieties include cross-linked PEG moieties, polyamine moieties, polyvinylamine moieties, and polyol moieties.
  • a preferred hydrophobic polymer to be used for production of beads of the composition of the invention is PS-DVB (polystyrene divinylbenzene).
  • PS-DVB has been widely used for solid-phase peptide synthesis (SPPS), and has more recently demonstrated utility for the polymer-supported preparation of particular organic molecules (Adams et al. (1998) J.Org.Chem. 63 : 3706-3716). When prepared properly (Gr ⁇ tli et al. (2000) J.Combi.Chem.2: 108-119), PS-DVB solid phase materials display excellent properties for chemical synthesis such as high loading, reasonable swelling in organic solvents and physical stability.
  • the above-mentioned ligand is covalently immobilized to a solid phase material, possibly through a linker.
  • the ligand is covalently attached to a linker which is covalently attached to the polymer matrix.
  • General techniques for linking of affinity ligands to solid phase materials can be found in Hermanson, Krishna MaIMa and Smith, Immobilized Affinity Ligand Techniques", Academic Press, 1992.
  • Linkers are used for linking the ligand to a solid phase material such as e.g. a polymer matrix or an inorganic support.
  • the linker forms a strong and durable bond between the ligand and the solid phase material. This is particularly important, when the solid phase material of the present invention is to be used for repeated purification of Factor VII polypeptides.
  • linkers can be selectively cleavable. This can be useful when the solid phase material is to be used for analytical purposes.
  • linkers include carbohydrates and nucleic acids.
  • the linker residue L attached to the polymer matrix is cleavable by acids, bases, temperature, light, or by contact with a chemical reagent.
  • the linker attached to the polymer matrix can be (3- formylindol-l-yl)acetic acid, 2,4-dimethoxy-4'-hydroxy-benzophenone, HMPA, HMPB, HMPPA, Rink acid, Rink amide, Knorr linker, PAL linker, DCHD linker, Wang linker and Trityl linker.
  • the ligand can be associated with the solid phase material through a linker having a length of preferably less than 50 A, such as a length of from 3 to 30 A, for example a length of from 3 to 20 A, such as a length of from 3 to 10 A.
  • the linker can be attached to a hydrophobic functional group or to a cationic functional group, or to a structural entity of the ligand joining a hydrophobic functional group and a cationic functional group.
  • the linker is attached to a cationic functional group.
  • the linker is attached to the affinity ligand via a carboxylic acid group, or an amino group, in particular via a carboxylic acid group.
  • the linker may also comprise a plurality of covalently linked subunits, e.g. such that the subunits are selected from identical and non-identical linker subunits.
  • the linker is flexible and comprises from 3 to preferably less than 50 identical or non-identical, covalently linked subunits.
  • the linker L is selected from the group consisting of glycine, alanine, 3-aminopropionic acid, 4-aminobutanoic acid, and HMBA.
  • the linker can also be selected from the group consisting of polydispersed polyethylene glycol; monodispersed polyethylene glycol, such as triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol; an amino acid; a dipeptide; a tripeptide; a tetrapeptide; a pentapeptide; a hexapeptide; a heptapeptide; octapeptide; a nonapeptide; a decapeptide, a polyalanine; a polyglycine, a polylysines, a polyarginine, including any combination thereof.
  • affinity resins Preparation of affinity resins
  • the affinity resins can in principle be prepared in two fundamentally different way, namely (i) by synthesizing the ligand in free from and subsequently immobilizing the ligand to the solid phase material directly or via a linker (see above), or (ii) by functionalising the solid phase material and thereafter sequentially synthesizing the ligand(s).
  • immobilization techniques are readily available in the art, e.g. in Hermanson et al. (see above).
  • techniques are also readily available, e.g. the techniques known in the art of solid phase peptide synthesis and derived techniques.
  • the drug substance of the Factor VII polypeptide is contacted with an affinity resin under conditions which facilitate binding of a portion of said drug substance to said affinity resin.
  • the aim is to facilitate binding of a relevant portion of said drug substance of the Factor VII polypeptide to said affinity resin.
  • portion in connection with step (a) is meant at least 30% ⁇ i.e. 30- 100%) of the mass of the Factor VII polypeptide present in the drug substance of the Factor VII polypeptide. It should be understood that it in most instances is desirable to bind far more than 30% of the mass of the Factor VII polypeptides, e.g. at least 50%, or at least 70%, or a predominant portion.
  • predominant portion is meant at least 90% of the mass of the Factor VII polypeptide present in the drug substance of the Factor VII polypeptide.
  • an even higher portion becomes bound to the affinity resin, e.g. at least 95% of the mass, or at least 98% of the mass, or at least 99% of the mass, or even substantially all of the mass of the Factor VII polypeptide present in the drug substance of the Factor VII polypeptide.
  • the drug substance of the Factor VII polypeptide typically originates from an industrial-scale production process, e.g. a cell culture, a microbial process, a cloned animal (e.g. cows, pigs, sheep, goats, and fish) or insect, or the like, in particular from a cell culture.
  • the drug substance of the Factor VII polypeptide may be derived from blood plasma, or the like.
  • the most common arrangement of the affinity resin is in a column format. Arrangement in a batch container is of course also possible.
  • the drug substance of the Factor VII polypeptide is typically obtained directly from cell culture fluid or from cell culture fluid with subsequent adjustment of pH, ionic strength, chelation of divalent metal ions, etc., if desirable or beneficial.
  • the drug substance of the Factor VII polypeptide is obtained directly from a preceding purification step, or from a preceding purification step with subsequent adjustment of pH, ionic strength, chelation of divalent metal ions, etc., if desirable or beneficial.
  • the contacting of the drug substance of the Factor VII polypeptide is typically conducted according to conventional protocols, i.e. the concentration, temperature, ionic strength, etc. of the drug substance may be as usual, and the affinity resin may be washed and equilibrated before application as usual.
  • the load of Factor VII polypeptide is typically at least 250 mg per litre of affinity resin, such as in the range of 0.25-10.0 g, e.g. 2.0-5.0 g, Factor VII polypeptide per litre of affinity resin in wet form, and the drug substance is typically loaded at a flow of 1-50 column volumes per hour.
  • the pH of the drug substance before and upon application to the affinity resin appears to play a relevant role for the formation of contaminants, e.g. in the form of dimers and degradation products of the Factor VII polypeptide.
  • the drug substance is in liquid form and has a pH in the range of 3.0-10.0, such as in the range of 3.0-7.0, or 6.5-10.0, upon application to the affinity resin.
  • the drug substance has a pH of in the range of 4.0-7.0, or in the range of 7.0-9.0, or in the range of 4.5-8.5.
  • a preferred pH range would be 5.0-6.5.
  • the content of calcium ions may play a role in connection with the stability of the Factor VII polypeptide.
  • the drug substance in step (a) has a concentration of calcium ions of at least 5 mM, such as in the range of 5-100 mM. In such instances, a preferred pH range would be 5.0-9.5.
  • the drug substance applied in step (a) has 0-1600 mM NaCI, 0-20 mM CaCI 2 and 0-20 mM His added at pH 5.0-7.0, when contacted with the affinity resin according to step (a).
  • the conductivity is at least 40 mS/cm, such as at least 50 mS/cm, such as at least 100 mS/cm such at lest 200 mS/cm.
  • the temperature of the drug substance is typically 0-30 0 C, such as around 2- 25 0 C.
  • the temperature of the affinity resin with the bound Factor VII polypeptide is typically 0-30 0 C, such as around 2-25 0 C, e.g. kept within a specified range by using a cooling jacket and solutions of controlled temperature.
  • a washing step (b) is typically conducted in order to remove proteins which are bound unspecific to the affinity resin.
  • the remaining (bound) fraction of the Factor VII polypeptide on the affinity resin will have a much lower abundance of contaminants.
  • This washing step (b) is preferably conducted with a washing buffer having a pH in the range of 2.0-6.9.
  • the washing buffer has a pH in the range of 3.0-10.0, such as in the range of 3.0-7.0, or 6.5-10.0, upon application to the affinity resin.
  • the washing buffer has a pH of in the range of 4.0-7.0, or in the range of 7.0-9.0, or in the range of 4.5-8.5.
  • the washing buffer in step (b) typically has a concentration of calcium ions of at least 1 mM, such as in the range of 1-100 mM.
  • the washing step (b) is typically conducted at a flow of 1-50 column volumes per hour.
  • the washing buffer is typically an aqueous solution comprising a buffering agent, typically a buffering agent comprising at least one component selected from the groups consisting of acids and salts of MES, PIPES, ACES, BES, TES, HEPES, TRIS, histidine, imidazole, glycine, glycylglycine, glycinamide, phosphoric acid, acetic acid (e.g. sodium acetate), lactic acid, glutaric acid, citric acid, tartaric acid, malic acid, maleic acid, and succinic acid.
  • the buffering agent may comprise a mixture of two or more components, wherein the mixture is able to provide a pH value in the specified range.
  • step (b) involves at least one washing buffer comprising 0-10 mM EDTA and 0-20 mM His at pH 5-7.
  • a first washing buffer, a second washing buffer, and a third washing buffer are sequentially applied, said first washing buffer comprising 0- 4,000 mM NaCI, 0-2 mM CaCI 2 , 0-0.2% Tween-20, and 0-20 mM His at pH 5-7, said second washing buffer comprising 0-10 mM CaCI 2 , and 0-20 mM His at pH 5-7, and said third washing buffer comprising 0-10 mM EDTA and 0-20 mM His at pH 5-7.
  • washing step (b) may be conducted by using one, two or several different washing buffers, or by the application of a gradient washing buffer.
  • washing step and the elution step need not to be discrete steps, but may be combined, in particular if a gradient elution buffer is utilised in the elution step.
  • the affinity resin containing bound drug substance is eluted with an elution buffer, and a purified drug substance of the Factor VII polypeptide is collected as an eluate.
  • the type of elution is not particularly critical, thus, it is, e.g., possible to elute with an elution buffer comprising a stepwise decreasing gradient of salts, elute with a linear decreasing gradient of the salts (or a gradient-hold-gradient profile, or other variants), or to use a pH gradient, or to use a temperature gradient, or a combination of the before-mentioned.
  • the conductivity of the final elution buffer is preferably lower than the conductivity of the composition comprising the drug substance in step (a).
  • the elution buffer in step (c) typically has a pH as in step (a) and (b).
  • the elution buffer in step (c) has a concentration of calcium ions of at least 1 mM, such as in the range of 1-100 mM.
  • the elution buffer comprises 0-1 M Arginine, 0-0.4 M NaCI, and 0-10 mM CaCI 2 at pH 5.5-7.5.
  • a first elution buffer and a second elution buffer are sequentially used, said first elution buffer comprising 0-30 mM NaCI, 0-100 mM CaCI 2 , 0-100 mM His, 0-0.04% Tween-20 and 0-60% ethylene glycol at pH 5 - 7, and said second elution buffer comprising 0-1 M Arginine, 0-0.4 M NaCI and 0-10 mM CaCI 2 at pH 5.5-7.5.
  • the elution step (c) is typically conducted at a flow of 1-50 column volumes per hour.
  • purified drug substance means that the resulting drug substance, i.e. the drug substance collected in step (c), has a lower content of other proteins than the drug substance applied in step (a).
  • purification refers to the process wherein a purified drug substance can be obtained, i.e. the process of the present invention.
  • the process of the present invention is capable reducing the content of other proteins with at least 50%, however more preferably, and also realistically, the reduction is at least 60%, such as at least 70% or even at least 80% or at least 85%.
  • the affinity resin can be regenerated for the purpose of subsequent use by a sequence of steps.
  • washing step and the elution step need not to be discrete steps, but may be combined, in particular if a gradient elution buffer is utilised in the elution step.
  • the process of the present invention is particularly feasible for "industrial-scale” (or “large-scale”) drug substances of a Factor VII polypeptide.
  • “industrial-scale” is typically meant methods wherein the volume of liquid Factor VII polypeptide compositions is at least 10 L, such as at least 50 L, e.g. at least 500 L, or at least 5000 L, or where the weight of the product is at least 1 g (dry matter), such as at least 10 g, e.g. at least 50 g, e.g. 1-1000 g.
  • a process for the purification of a drug substance of a Factor VII polypeptide comprising the steps of:
  • affinity resin is a solid phase material having covalently immobilized thereto one or more ligands selected from ligands (l)-(20).
  • Vector VII polypeptide or “FVII polypeptide” means any protein comprising the amino acid sequence 1-406 of wild-type human
  • Factor Vila i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950
  • variants thereof as well as Factor VII-related polypeptides, Factor VII derivatives and Factor VII conjugates.
  • FVII variants, Factor VII-related polypeptides, Factor VII derivatives and Factor VII conjugates exhibiting substantially the same or improved biological activity relative to wild-type human Factor Vila.
  • Such variants, derivatives or conjugates of Factor VII may exhibit different properties relative to human Factor VII, including stability, phospholipid binding, altered specific activity, and the like.
  • Factor VII is intended to encompass Factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor Vila. Typically, Factor VII is cleaved between residues 152 and 153 to yield Factor Vila, i.e. the activated form of Factor VII.
  • Factor VII is also intended to encompass a mixture of zymogen and activated Factor VII polypeptides.
  • Fractor VII or “Factor Vila” within the above definition also includes natural allelic variations that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment.
  • wild type human FVIIa is a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950.
  • Factor VII-related polypeptides encompasses polypeptides, including variants, in which the Factor Vila biological activity has been substantially modified, such as reduced, relative to the activity of wild-type Factor Vila.
  • These polypeptides include, without limitation, Factor VII or Factor Vila into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.
  • Fractor VII derivative is intended to designate a FVII polypeptide exhibiting substantially the same or improved biological activity relative to wild-type Factor VII, in which one or more of the amino acids of the parent peptide have been genetically and/or chemically and/or enzymatically modified, e.g. by alkylation, glycosylation, PEGylation, acylation, ester formation or amide formation or the like. This includes but is not limited to PEGylated human Factor Vila, cysteine-PEGylated human Factor Vila and variants thereof.
  • Non-limiting examples of Factor VII derivatives includes GlycoPegylated FVII derivatives as disclosed in WO 03/31464 and US Patent applications US 20040043446, US 20040063911, US 20040142856, US 20040137557, US 20040132640, WO2007022512, and US 20070105755 (Neose Technologies, Inc.); FVII conjugates as disclosed in WO 01/04287, US patent application 20030165996, WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, US patent application 20030211094 (University of Minnesota).
  • improved biological activity refers to FVII polypeptides with i) substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila or ii) to FVII polypeptides with substantially the same or increased TF binding activity compared to recombinant wild type human Factor Vila or iii) to FVII polypeptides with substantially the same or increased half life in blood plasma compared to recombinant wild type human Factor Vila.
  • PEGylated human Factor Vila means human Factor Vila, having a PEG molecule conjugated to a human Factor Vila polypeptide. It is to be understood, that the PEG molecule may be attached to any part of the Factor Vila polypeptide including any amino acid residue or carbohydrate moiety of the Factor Vila polypeptide.
  • cyste-PEGylated human Factor Vila means Factor Vila having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced in human Factor Vila.
  • Factor VII variant is intended to designate a FVII polypeptide exhibiting substantially the same or better bioactivity than wild-type Factor VII, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type Factor VII, and are polypeptides having an amino acid sequence that differs from the sequence of wild-type Factor VII by insertion, deletion, or substitution of one or more amino acids.
  • Non-limiting examples of Factor VII variants having substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased proteolytic stability as disclosed in U.S. Patent No. 5,580,560; Factor Vila that has been proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng.
  • Non-limiting examples of FVII variants having increased biological activity compared to wild-type FVIIa include FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, WO 03/027147, WO 04/029090, WO 05/075635, and European patent application with application number 05108713.8 (Novo Nordisk A/S), WO 02/38162 (Scripps Research Institute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).
  • variants of Factor VII include, without limitation, PlOQ-FVII, K32E- FVII, P10Q/K32E-FVII, L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337
  • K316Q/L305V/V158T/E296V/K337A-FVII K316Q/L305V/V158D/K337A/M298Q- FVII, K316Q/L305V/V158D/E296V/K337A -FVII, K316Q/L305V/V158D/E296V/M298Q/K337A-FVII, K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII, F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII, F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII, F
  • F374Y/L305V/E296V/M298Q/S314E-FVII F374Y/V158D/E296V/M298Q/K337A- FVII, F374Y/V158D/E296V/M298Q/S314E-FVII, F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E- FVII, F374Y/V158D/E296V/K337A/S314E-FVII,
  • F374Y/L305V/V158D/E296V/M298Q-FVII F374Y/L305V/V158D/M298Q/K337A- FVII, F374Y/L305V/V158D/E296V/K337A-FVII, F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E- FVII, F374Y/V158T/E296V/M298Q/K337A-FVII,
  • FVII having substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn; FVII having substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys; and FVII having substitutions, additions or deletions in the amino acid sequence from 1531Ie to 223Arg.
  • substitution variants in a Factor VII polypeptide include, without limitation substitutions in positions PlO, K32, L305, M306, D309, L305, L305, F374, V158, M298, V158, E296, K337, M298, M298, S336, S314, K316, K316, F374, S52, S60, R152, S344, T106, K143, N145, V253, R290, A292, G291, R315, V317, and substitutions, additions or deletions in the amino acid sequence from T233 to N240 or from R304 to C329; or from 1153 to R223, or combinations thereof, in particular variants such as PlOQ, K32E, L305V, M306D, D309S, L305I, L305T, F374P, V158T, M298Q, V158D, E296V, K337A, M298Q, M298K, S336G, S314E, K316H, K316
  • the biological activity of Factor Vila in blood clotting derives from its ability to (i) bind to Tissue Factor (TF) and (ii) catalyze the proteolytic cleavage of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively).
  • biological activity of Factor VII polypeptides may be quantified by measuring the ability of a preparation to promote blood clotting, cf. Assay 4 described herein. In this assay, biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "Factor VII units" by comparison with a pooled human serum standard containing 1 unit/mL Factor VII activity.
  • Factor Vila biological activity may be quantified by (i) measuring the ability of Factor Vila or a Factor VII-related polypeptide to produce activated Factor X (Factor Xa) in a system comprising TF embedded in a lipid membrane and Factor X. (Persson et al., J. Biol.
  • Factor VII variants having substantially the same or improved biological activity relative to wild-type Factor Vila encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75% and most preferably at least about 90% of the specific activity of Factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay (Assay 4), proteolysis assay (Assay 2), or TF binding assay as described above.
  • a clotting assay Assay 4
  • proteolysis assay Assay 2
  • TF binding assay as described above.
  • Factor VII variants having substantially reduced biological activity relative to wild-type Factor Vila are those that exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the specific activity of wild-type Factor Vila that has been produced in the same cell type when tested in one or more of a clotting assay (Assay 4), proteolysis assay (Assay 2), or TF binding assay as described above.
  • Factor VII variants having a substantially modified biological activity relative to wild-type Factor VII include, without limitation, Factor VII variants that exhibit TF-independent Factor X proteolytic activity and those that bind TF but do not cleave Factor X.
  • affinity resins described herein are novel as such.
  • the present invention also provides novel affinity resins comprising a solid phase material having covalently immobilized there to one or more ligands, i.e. the ligands described hereinabove.
  • the present invention provides an affinity resin comprising a solid phase material having covalently immobilized thereto one or more ligands of the general formula (I),
  • each of Ri, R 2 and R 3 represents an organic moiety of a molecular weight of 15-1000 g/mol, the total molecular weight of said ligand being 200- 2000 g/mol; said ligand being immobilized to said solid phase material via one of said organic moieties R 1 , R 2 and R 3 .
  • each of the organic moieties Ri, R 2 and R 3 has the formula C x H y O z N k S
  • each of Ri, R 2 and R 3 represents an organic moiety of a molecular weight of 50-500 g/mol, wherein the total molecular weight of the ligand is 250-1500 g/mol, such as 300-1200 g/mol, e.g. 350-1000 g/mol.
  • the ligand is as specified hereinabove.
  • the currently most interesting ligands are those of the formula (l)-(20), such as those of the formula (l)-(2) or those of the formula (3)-(20).
  • R represents the solid phase material of said affinity resins.
  • the novel affinity resins are particularly useful in the purification and/or isolation of biomolecules, such as proteins, in particular Factor VII polypeptides.
  • the affinity ligands are specific binding partners for Factor VII polypeptides and can isolate said polypeptide from closely related proteins such as e.g. protein S.
  • the ligand is immobilized to the surface of a sensor or an array plate (the "solid phase material") and is used to detect and/or quantify Factor VII polypeptides in a biological sample.
  • biological sample includes natural samples or samples obtained from industrial processes, e.g. recombinant processes, and include "body fluid", i.e. any liquid substance extracted, excreted, or secreted from an organism or tissue of an organism.
  • body fluid i.e. any liquid substance extracted, excreted, or secreted from an organism or tissue of an organism.
  • a body fluid need not necessarily contain cells.
  • Body fluids of relevance to the present invention include, but are not limited to, whole blood, serum, urine, plasma, cerebral spinal fluid, tears, milk, sinovial fluid, and amniotic fluid.
  • a plurality of ligands are immobilized to the surface of an array plate (the "solid phase material") and arranged in a plurality of spots, with each spot representing one ligand.
  • an array plate the "solid phase material”
  • Such a functionalized array can be used to detect the presence of Factor VII polypeptides in a solution.
  • Such an array can be used for diagnostic applications to detect the presence of Factor VII polypeptides in a biological sample.
  • a plurality of ligands are immobilized to the binding surface of a cantilever sensor (the "solid phase material") for detection and optionally quantification of Factor VII polypeptides.
  • a plurality of affinity ligands can be immobilized to a plurality of cantilevers with each cantilever representing one ligand.
  • Such a functionalized array can be used to detect the presence of various antibodies in a solution.
  • Such a multi-sensor can be used for diagnostic applications to detect the presence of certain Factor VII polypeptides in a biological sample.
  • the invention further provides an affinity ligand selected from the group consisting of ligands (l)-(20), such as those of the formula (l)-(2) or (3)-(20).
  • affinity ligands selected from the group consisting of ligands (l)-(20), such as those of the formula (l)-(2) or (3)-(20).
  • Preferred examples hereof are those of the formula (1) and (2) :
  • Embodiment 1 A process for the purification of a drug substance of a Factor VII polypeptide, said process comprising the steps of:
  • affinity resin eluting said affinity resin containing bound drug substance with an elution buffer, and collecting a purified drug substance of the Factor VII polypeptide as an eluate; wherein said affinity resin is a solid phase material having covalently immobilized thereto one or more ligands of the general formula (I),
  • each of Ri, R 2 and R 3 represents an organic moiety of a molecular weight of 15-1000 g/mol, the total molecular weight of said ligand being 200- 2000 g/mol;
  • said ligand being immobilized to said solid phase material via one of said organic moieties Ri, R 2 and R 3 .
  • Embodiment 2 The process according to Embodiment 1, wherein each of the organic moieties Ri, R 2 and R 3 has the formula C x HyO z N
  • Embodiment 3 A process for the purification of a drug substance of a Factor VII polypeptide, said process comprising the steps of:
  • affinity resin is a solid phase material having covalently immobilized thereto one or more ligands
  • Embodiment 4 The process according to Embodiment 3, wherein the hydrophobic, polar and positively charged groups include one or more of leucine (Leu), isoleucine (He), 4-chloraniline (CA), phenylalanine (Phe), 5-aminoindane (AIND), 2,2-diphenylethylamine (DPEA), (aminomethyl)cyclohexane (CHMA), 1- ethyl propylamine (IEP), phenylcyclopentaneacetic acid (PCAA), 2- propylpentanoic acid (PPA), tryptophan (Trp), indole-3-carboxylic acid (3-ICA), threonine (Thr), tyrosine (Tyr), 3-hydroxy-4-methoxybenzylamine (HMOBA), 4- hydroxy-3-methoxybenzylamine (MOBHA), 4-aminophenol (4AP), 4- hydroxybenzoic acid (4HBA), glutamine (GIn), 4-amin
  • Embodiment 5 The process according to any one of the preceding Embodiments, wherein the ligand has at least one cationic group.
  • Embodiment 6 The process according to Embodiment 5, wherein the ligand has at least one cationic group and at least one aromatic group.
  • Embodiment 7 The process according to Embodiment 6, wherein the ligand has at least one cationic group and at least three aromatic groups.
  • Embodiment 8 The process according to any one of the Embodiments 1-2 and 5-7, wherein the R 2 in the ligand comprises a primary amine.
  • Embodiment 9 The process according to Embodiment 8, wherein the ligand has the general formula (Ia)
  • R' 2 is a linker to the solid phase material.
  • Embodiment 10 The process according to any one of Embodiments 1-2 and 5-7, wherein R 2 in the ligand comprises a para-substituted phenyl group.
  • Embodiment 11 The process according to Embodiment 10, wherein the ligand has the general formula (Ib)
  • R' 2 is a linker to the solid phase material.
  • Embodiment 12 The process according to any one of Embodiments 1-2 and 5-7, wherein R 1 comprises a para-substituted chlorobenzene group.
  • Embodiment 13 The process according to any one of Embodiments 1-2 and 5-7, wherein R 1 comprises a di-phenyl-isopropyl group.
  • Embodiment 14 The process according to any one of Embodiments 1-2 and 5- 13, wherein R 3 comprises a primary amine.
  • Embodiment 15 The process according to any one of Embodiments 1-2 and 5- 13, wherein R 3 comprises a benzyl group.
  • Embodiment 16 The process according to any one of Embodiments 1-2 and 5- 13, wherein R 3 comprises a quinoline group.
  • Embodiment 17 The process according to any one of Embodiments 1-2 and 5- 13, wherein R 3 comprises an indole group.
  • Embodiment 18 The process according to Embodiment 1, wherein the ligand is one selected from the group consisting of ligands (3)-(20)
  • Embodiment 19 The process according to Embodiment 3, wherein the ligand is selected from the group consisting of ligands (1) and (2) :
  • Embodiment 20 The process according to any one of the preceding Embodiments, wherein, in step (a), the drug substance has 0-1600 mM NaCI, 0- 20 mM CaCI 2 and 0-20 mM His added at pH 5.0-7.0, when contacted with the affinity resin according to step (a).
  • Embodiment 21 The process according to any one of the preceding Embodiments, wherein, in step (b), at least one washing buffer comprising 0-10 mM EDTA and 0-20 mM His at pH 5-7 is used.
  • Embodiment 22 The process according to any one of the preceding Embodiments, wherein, in step (b), a first washing buffer, a second washing buffer, and a third washing buffer are sequentially applied, said first washing buffer comprising 0-4,000 mM NaCI, 0-2 mM CaCI 2 , 0-0.2% Tween-20, and 0-20 mM His at pH 5-7, said second washing buffer comprising 0-10 mM CaCI 2 , and 0-20 mM His at pH 5-7, and said third washing buffer comprising 0-10 mM EDTA and 0-20 mM His at pH 5-7.
  • Embodiment 23 The process according to any one of the preceding Embodiments, wherein, in step (c), the elution buffer comprises 0-1 M Arginine, 0-0.4 M NaCI, and 0-10 mM CaCI 2 at pH 5.5-7.5.
  • Embodiment 24 The process according to any one of the preceding Embodiments, wherein, in step (c), a first elution buffer and a second elution buffer, are sequentially used, said first elution buffer comprising 0-30 mM NaCI, 0-100 mM CaCI 2 , 0-100 mM His, 0-0.04% Tween-20 and 0-60% ethylene glycol at pH 5 - 7, and said second elution buffer comprising 0-1 M Arginine, 0-0.4 M NaCI and 0-10 mM CaCI 2 at pH 5.5-7.5.
  • Embodiment 25 The process according to any one of the preceding Embodiments, wherein the Factor VII polypeptide is human Factor VII, and wherein the drug substance is human blood plasma.
  • Embodiment 26 The process according to any one of the preceding Embodiments, wherein the Factor VII polypeptide is recombinant Factor VII, and wherein the drug substance is a cell culture supernatant.
  • Embodiment 27 The process according to any one of the preceding Embodiments, wherein the Factor VII polypeptide is recombinant Factor VII, and wherein the drug substance is an eluate from previous chromatographic step.
  • Embodiment 28 The process according to any one of Embodiments 26-27, wherein the Factor VII polypeptide is a PEGylated Factor VII polypeptide.
  • Embodiment 29 An affinity resin comprising a solid phase material having covalently immobilized thereto one or more ligands of the general formula (I),
  • each of Ri, R 2 and R 3 represents an organic moiety of a molecular weight of 15-1000 g/mol, the total molecular weight of said ligand being 200- 2000 g/mol;
  • Embodiment 30 The affinity resin according to Embodiment 29, wherein each of the organic moieties R 1 , R 2 and R 3 has the formula wherein 0 ⁇ x ⁇ 15, 0 ⁇ y ⁇ 2x+ l, O ⁇ z ⁇ x, O ⁇ k ⁇ x, O ⁇ l ⁇ x, 0 ⁇ m ⁇ 3x, 0 ⁇ n ⁇ 3x, 0 ⁇ p ⁇ 3x, O ⁇ q ⁇ x, and 15 ⁇ 12x+y+ 16z+14k+32l+80m-l-35n-l- 19p-l-31q ⁇ IOOO, and wherein one of the organic moieties Ri, R 2 and R 3 comprises a link to the solid phase material.
  • Embodiment 31 The affinity resin according to any one of the Embodiments 29- 30, wherein the ligand is as specified in any one of the Embodiments 5-18.
  • Embodiment 32 An affinity resin selected from the group consisting of affinity resins (Rl) and (R2) :
  • R represents the solid phase material of said affinity resins.
  • Embodiment 33 An affinity ligand selected from the group consisting of ligands (l)-(20) defined herein, in particular ligands (1) and (2) :
  • the ligands were synthesized on amino functional resin. First the resin was glycinated, then the ligands were coupled to the glycine derivatised resin. The glycine derivatised base resin was suspended in ample DMF for 30 min.
  • the resin was transferred to a fritted syringe.
  • the first building block (protected natural/unnatural amino acid; 3 equiv) dissolved in DMF and N-ethylmorpholine (4 equiv) and TBTU (2.88 equiv) was added.
  • the reaction mixture was kept for 5 min and added to the swollen resin in DMF for 3 h.
  • the resin was washed with DMF (10 x) and the negative Kaiser test indicated the quantitative reaction of the first building block.
  • the unmasked carboxyl group on the first building block was activated by adding diisopropylethylamine (4 equiv) and TBTU (2.88 equiv) in DMF.
  • the reaction mixture was kept for 30 min and the solvent was drained off.
  • the second building block (aliphatic/cyclic/aromatic amines) in DMF was added to the resin and the reaction mixture was kept for 3 h at room temperature.
  • the resin was washed with DMF (1Ox).
  • the amino group protection on the first building block was removed and the third building block (amino acids or aliphatic/cyclic/aromatic carboxylic acid) along with diisopropylethylamine (4 equiv) and TBTU (2.88 equiv) in DMF were added.
  • the resin was washed with DMF (10 x), EtOH/DMF (10 x) and DCM (10 x).
  • the protective groups were removed by TFA/scavenger cocktail for 3 h at room temperature.
  • the resin washed thoroughly with DCM, DMF, EtOH and water.
  • Fractogel EMD-amino resin (12 ml, 1.9 mmol, supplied by Merck KGaA) was washed with DMF(IO x) and suspended in DMF for 1 h.
  • Fmoc-Gly-OH (1.7 g, 3 equiv, 5.7 mmol), TBTU (1.76 g, 2.88 equiv, 5.47 mmol) and NEM (967 ⁇ l, 4 equiv, 7.6 mmol) in DMF (10 ml) was added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with DMF (10 x).
  • a negative Kaiser test indicated the absence of free primary amino group on resin.
  • the Fmoc-group was cleaved off with 20% Piperidine in DMF (30 min) and the resin was washed with DMF (10 x) and the Kaiser test was positive.
  • Fmoc-Aminoisophthalic acid (Fmoc- AIPA-OH, 2.3 g, 3 equiv, 5.7 mmol) activated with TBTU (1.76 g, 2.88 equiv, 5.47 mmol) and DIPEA (1.33 ml, 4 equiv, 7.6 mmol) and added to the resin.
  • the reaction mixture stirred for overnight at room temperature and washed with DMF (10 x).
  • Fractogel EMD-amino resin (12 ml, 1.9 mmol) washed with DMF(IO x) and suspended in DMF for 1 h.
  • Fmoc-Gly-OH (1.7 g, 3 equiv, 5.7 mmol)
  • TBTU (1.76 g, 2.88 equiv, 5.47 mmol
  • NEM 967 ⁇ l, 4 equiv, 7.6 mmol
  • the Fmoc-group was cleaved off with 20% Piperidine in DMF (30 min) and the resin was washed with DMF (10 x) and the Kaiser test was positive.
  • Fmoc-(L)Tyr(tBu)-OH (2.6 g, 3 equiv, 5.7 mmol) activated with TBTU (1.76 g, 2.88 equiv, 5.47 mmol) and NEM (967 ⁇ l, 4 equiv, 7.6 mmol) and added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with DMF (10 x).
  • Fmoc-group was cleaved off with 20% Piperidine in DMF (30 min) and the resin was washed with DMF (10 x).
  • Fmoc-Aminoisophthalic acid (Fmoc-AIPA-OH, 2.3 g, 3 equiv, 5.7 mmol) activated with TBTU (1.76 g, 2.88 equiv, 5.47 mmol) and DIPEA (1.33 ml, 4 equiv, 7.6 mmol) and added to the resin.
  • the reaction mixture stirred for overnight at room temperature and washed with DMF (10 x).
  • Boc-(L)Thr(tBu)-OH (1.57 g, 3 equiv, 5.7 mmol) activated with TBTU (1.76 g, 2.88 equiv, 5.47 mmol) and NEM (967 ⁇ l, 4 equiv, 7.6 mmol) and added to the resin.
  • the reaction mixture stirred for overnight at room temperature and washed with DMF (10 x) and DCM (10 x). All the protecting groups were removed by TFA/water/TIS (93 : 5 : 2) cocktail for 2 h and the resin washed thoroughly with DCM, DMF EtOH and water.
  • CCF cell culture fluid
  • the column was washed sequentially with Buffer A (2 M NaCI, 1 mM CaCI 2 , 0.1% Tween-20, 10 mM His, pH 6; 5 CV), Buffer B (5 mM CaCI 2 , 10 mM His, pH 6; 5 CV), and Buffer C (5 mM EDTA, 10 mM His, pH 6; 5 CV).
  • Buffer A 2 M NaCI, 1 mM CaCI 2 , 0.1% Tween-20, 10 mM His, pH 6; 5 CV
  • Buffer B 5 mM CaCI 2 , 10 mM His, pH 6; 5 CV
  • Buffer C 5 mM EDTA, 10 mM His, pH 6; 5 CV.
  • Bound protein was eluted by application of 2 buffers: Buffer D (15 mM NaCI, 50 mM CaCI 2 , 50 mM His, 0.02% Tween-20, 30% ethylene glycol, pH 6; 1 CV) and Buffer F (0.5 M Arginine,
  • the purity of the eluates was determined by SDS-PAGE gels run on a Novex system using Cambrex Pager gels. Gels were run according to the manufacturer's protocol. The resin was effective in purifying FVII from CCF (see Fig 1). This can be seen from the SDS-PAGE gel in Figure 1 showing: lane 1 : CCF, lane 2: FVII, lane 3 : Protein S and FVIIa, lane 4: Eluate from affinity resin with ligand 5, lane 5 : Eluate from affinity resin with ligand 20.
  • PL-PEGA resin (3 g, 0.4 mmol/g, 150-300 ⁇ m) washed with NMP (10 x).
  • the HMBA linker (548 mg, 3.6 mmol, 3 equiv) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and NEM (0.61 ml, 4.8 mmol, 4 equiv) in NMP was added to the resin and kept in a shaker for 3 h at room temperature.
  • the resin washed with NMP (10 x), DCM (10 x) and lyophilized.
  • Boc-(L)Pap(Fmoc)-OH (2.87 g, 3 equiv, 5.7 mmol) activated with TBTU (1.76 g, 2.88 equiv, 5.47 mmol) and NEM (967 ⁇ l, 4 equiv, 7.6 mmol) and added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with DMF (10 x).
  • the Fmoc-group was cleaved off with 20% Piperidine in DMF (30 min) and the resin was washed with DMF (10 x).
  • Fmoc-Aminoisophthalic acid (Fmoc-AIPA-OH, 2.3 g, 3 equiv, 5.7 mmol) activated with TBTU (1.76 g, 2.88 equiv, 5.47 mmol) and DIPEA (1.33 ml, 4 equiv, 7.6 mmol) and added to the resin.
  • the reaction mixture stirred for overnight at room temperature and washed with DMF (10 x).
  • Ligand 5 was synthesized (see below), purified, and dissolved in DMF. 1 mole of TBTU per mole of ligand and 0.5 mole of NEM per mole of ligand were added to the solution. The resulting mixture was added to amino functional FragtoGel. Two moles of ligand per mole of resin bound amine was used. The mixture was shaken for five days. Then resulting affinity resin was drained and washed with 10 resin volumes of di-methylformamide, then with 10 resin volumes of methanol, and finally with 10 resin volumes of demineralized water.
  • PL-PEGA resin (3 g, 0.4 mmol/g, 150-300 ⁇ m) washed with NMP (10 x).
  • the HMBA linker (548 mg, 3.6 mmol, 3 equiv) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and NEM (0.61 ml, 4.8 mmol, 4 equiv) in NMP was added to the resin and kept in a shaker for 3 h at room temperature.
  • the resin washed with NMP (10 x), DCM (10 x) and lyophilized.
  • Fmoc-Gly-OH (1.07 g, 3.6 mmol, 3 equiv) dissolved in DCM and add MeIm (0.57 ml, 7.2 mmol, 6 equiv) followed by MSNT (1.06 g, 3.6 mmol, 3 equiv).
  • MeIm 0.57 ml, 7.2 mmol, 6 equiv
  • MSNT 1.06 g, 3.6 mmol, 3 equiv
  • the activated Fmoc-Gly was added to the resin and kept in a shaker for 2 h at room temperature.
  • the resin washed with DCM (10 x) and NMP (10 x).
  • the Fmoc protection was cleaved off with 20% piperidine/NMP (25 ml, 20 min).
  • Fmoc-(L)Tyr(tBu)-OH (1.66 g, 3.6 mmol) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and NEM (0.61 ml, 4.8 mmol, 4 equiv) in NMP and added to the resin.
  • the reaction mixture stirred for 3 h at room temperature and washed with NMP (10 x).
  • the Fmoc-group was cleaved off with 20% Piperidine in DMF (20 min) and the resin was washed with NMP (10 x).
  • Fmoc- Aminoisophthalic acid (Fmoc-AIPA-OH, 1.45 g, 3 equiv, 3.6 mmol) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and DIPEA (0.81 ml, 4 equiv, 4.8 mmol) and added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with NMP (10 x).
  • the Ligand was cleaved off from the resin by treating with 0.1 M NaOH for 2 h at room temperature.
  • the resin was filtered off and the filtrate was neutralized with 0.1 M HCI.
  • the solvent was evaporated under pressure and the residue was lyophilized.
  • Fractogel EMD-amino resin (3 ml, 0.18 mmol) washed with NMP(IO x) and suspended in NMP for 1 h.
  • the side-chain protected ligand 20 (0.39 g, 3 equiv, 0.54 mmol), HATU (0.21 g, 3 equiv, 0.54 mmol), HOAt (0.073 g, 3 equiv, 0.54 mmol) and DIPEA (125 ⁇ l, 4 equiv, 0.72 mmol) in NMP (5 ml) was added to the resin.
  • the reaction mixture stirred for 5 h at 6O 0 C.
  • the resin was filtered off and washed with NMP (1Ox), 75% NMP/EtOH (5x), 50% NMP/EtOH (5x), 25% NMP/EtOH (5x), EtOH (5x), 75% EtOH/water (5x), 50% EtOH/water (5x), 25% EtOH/water (5x), water (5x), and 20% EtOH/water (5x).
  • PL-PEGA resin (3 g, 0.4 mmol/g, 150-300 ⁇ m) washed with NMP (10 x).
  • the HMBA linker (548 mg, 3.6 mmol, 3 equiv) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and NEM (0.61 ml, 4.8 mmol, 4 equiv) in NMP was added to the resin and kept in a shaker for 3 h at room temperature.
  • the resin washed with NMP (10 x), DCM (10 x) and lyophilized.
  • Boc-(L)Pap(Fmoc)-OH (1.8 g, 3 equiv, 3.6 mmol) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and NEM (0.61 ml, 4.8 mmol, 4 equiv) in NMP and added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with NMP (10 x).
  • the Fmoc-group was cleaved off with 20% Piperidine in NMP (30 min) and the resin was washed with NMP (10 x).
  • Fmoc-Aminoisophthalic acid (Fmoc-AIPA-OH, 1.45 g, 3 equiv, 3.6 mmol) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and DIPEA (0.81 ml, 4 equiv, 4.8 mmol) and added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with NMP (10 x).
  • Fmoc-(L)l_ys(Boc)-OH (1.67 g, 3 equiv, 3.6 mmol) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and DIPEA (0.81 ml, 4 equiv, 4.8 mmol) and added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with NMP (10 x).
  • the Fmoc-group was cleaved off with 20% Piperidine in NMP (30 min) and the resin was washed with NMP (10 x).
  • the Ligand was cleaved off from the resin by treating with 0.1 M NaOH for 2 h at room temperature.
  • the resin was filtered off and the filtrate was neutralized with 0.1 M HCI.
  • the solvent was evaporated under pressure and the residue was lyophilized.
  • Fractogel EMD-amino resin (3 ml, 0.18 mmol) washed with NMP (10 x) and suspended in NMP for 1 h.
  • the side-chain protected ligand 5 (0.46 g, 3 equiv, 0.54 mmol), HATU (0.21 g, 3 equiv, 0.54 mmol), HOAt (0.073 g, 3 equiv, 0.54 mmol) and DIPEA (125 ⁇ l, 4 equiv, 0.72 mmol) in NMP (5 ml) was added to the resin.
  • the reaction mixture stirred for 5 h at 6O 0 C.
  • PL-PEGA resin (3 g, 0.4 mmol/g, 150-300 ⁇ m) washed with NMP (10 x).
  • the HMBA linker (548 mg, 3.6 mmol, 3 equiv) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and NEM (0.61 ml, 4.8 mmol, 4 equiv) in NMP was added to the resin and kept in a shaker for 3 h at room temperature.
  • the resin washed with NMP (10 x), DCM (10 x) and lyophilized.
  • Boc- (L)Thr(tBu)-OH (1.0 g, 3 equiv, 3.6 mmol) activated with TBTU (1.11 g, 3.5 mmol, 2.88 equiv) and NEM (0.61 ml, 4.8 mmol, 4 equiv) and added to the resin.
  • the reaction mixture stirred for 6 h at room temperature and washed with NMP (10 x) and DCM (10 x).
  • the Ligand was cleaved off from the resin by treating with 0.1 M NaOH for 2 h at room temperature.
  • the resin was filtered off and the filtrate was neutralized with 0.1 M HCI.
  • the solvent was evaporated under pressure and the residue was lyophilized.
  • Fractogel EMD-amino resin (3 ml, 0.18 mmol) washed with NMP(IO x) and suspended in NMP for 1 h.
  • the side-chain protected ligand 20 (0.39 g, 3 equiv, 0.54 mmol), HATU (0.21 g, 3 equiv, 0.54 mmol), HOAt (0.073 g, 3 equiv, 0.54 mmol) and DIPEA (125 ⁇ l, 4 equiv, 0.72 mmol) in NMP (5 ml) was added to the resin.
  • the reaction mixture stirred for 5 h at 6O 0 C.

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Abstract

L'invention porte sur un procédé de purification d'une substance médicamenteuse d'un polypeptide de facteur VII. Ce procédé comporte les étapes: (a) de mise en contact de la substance médicamenteuse avec une résine d'affinité dans des conditions qui facilitent la liaison d'une partie de ladite substance médicamenteuse à ladite résine d'affinité ; (b) facultativement de lavage de ladite résine d'affinité contenant la substance médicamenteuse liée avec un tampon de lavage, et (c) d’élution de ladite résine d'affinité contenant la substance médicamenteuse liée avec un tampon d'élution, et de récupération de la substance médicamenteuse purifiée du polypeptide de facteur VII en tant qu'éluat, ladite résine d'affinité étant un matériau en phase solide sur lequel ont été immobilisés de manière covalente un ou plusieurs ligands de la formule générale (I), chacun de R1, R2 et R3 représentant une fraction organique d'une masse moléculaire de 15 à 1 000 g/mole, la masse moléculaire totale dudit ligand étant de 200 à 2 000 g/mole, ledit ligand étant immobilisé sur ledit matériau en phase solide par l'intermédiaire de l'une desdites fractions organiques R1, R2 et R3.
PCT/EP2009/056149 2008-05-21 2009-05-20 Procédé de purification de polypeptides de facteur vii à l'aide de résines d'affinité comportant des ligands spécifiques WO2009141384A2 (fr)

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WO2012017021A3 (fr) * 2010-08-03 2012-05-10 Graffinity Pharmaceuticals Gmbh Ligands pour la purification d'anticorps et de protéine de fusion à fc par chromatographie d'affinité
WO2013117707A1 (fr) * 2012-02-08 2013-08-15 Graffinity Pharmaceuticals Gmbh Ligands pour anticorps et purification de protéine de fusion à fragment constant par chromatographie d'affinité iv
EP2690106A1 (fr) * 2011-03-24 2014-01-29 Kaneka Corporation Composé de faible poids moléculaire liant les substances protéiques
WO2014020152A1 (fr) * 2012-08-02 2014-02-06 Graffinity Pharmaceuticals Gmbh Ligands pour aphérèse et immunoabsorption
WO2014174316A1 (fr) 2013-04-26 2014-10-30 Adc Biotechnology Ltd Procédé de synthèse de conjugués anticorps-médicament au moyen de résines d'affinité
US9156879B1 (en) * 2010-10-29 2015-10-13 Merck Sharp & Dohme Corp. Chromatography media
WO2016067013A1 (fr) 2014-10-28 2016-05-06 Adc Biotechnology Ltd Procédé de synthèse de cam au moyen de résines d'affinité
WO2016067016A1 (fr) 2014-10-28 2016-05-06 Adc Biotechnology Ltd Procédé de synthèse de conjugués d'anticorps à l'aide de résines d'affinité
US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient
TWI748078B (zh) * 2017-03-30 2021-12-01 大陸商江蘇恒瑞醫藥股份有限公司 抗體藥物偶聯物的製備方法
CN113952944A (zh) * 2021-12-23 2022-01-21 浙江湃肽生物有限公司深圳分公司 一种纯化九肽-1的方法

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CN103154018A (zh) * 2010-08-03 2013-06-12 葛雷菲尼迪制药有限公司 用于通过亲和色谱法的抗体和Fc-融合蛋白纯化的配体
KR102008946B1 (ko) 2010-08-03 2019-08-08 노브알리스 도이칠란트 게엠베하 친화성 크로마토그래피에 의한 항체 및 Fc-융합 단백질 정제용 리간드
KR20130103499A (ko) * 2010-08-03 2013-09-23 그라피니티 파머수티컬스 게엠베하 친화성 크로마토그래피에 의한 항체 및 Fc-융합 단백질 정제용 리간드
JP2013538798A (ja) * 2010-08-03 2013-10-17 グラフィニティ ファーマシューティカルズ ゲーエムベーハー アフィニティクロマトグラフィーによる抗体およびＦｃ融合タンパク質精製のためのリガンド
WO2012017021A3 (fr) * 2010-08-03 2012-05-10 Graffinity Pharmaceuticals Gmbh Ligands pour la purification d'anticorps et de protéine de fusion à fc par chromatographie d'affinité
CN103154018B (zh) * 2010-08-03 2016-04-27 诺瓦里克斯德国有限公司 用于通过亲和色谱法的抗体和Fc-融合蛋白纯化的配体
US9156879B1 (en) * 2010-10-29 2015-10-13 Merck Sharp & Dohme Corp. Chromatography media
US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient
EP2690106A1 (fr) * 2011-03-24 2014-01-29 Kaneka Corporation Composé de faible poids moléculaire liant les substances protéiques
US9273151B2 (en) 2011-03-24 2016-03-01 Kaneka Corporation Proteinaceous-substance-binding low-molecular-weight compound
EP2690106A4 (fr) * 2011-03-24 2015-04-22 Kaneka Corp Composé de faible poids moléculaire liant les substances protéiques
CN104395335A (zh) * 2012-02-08 2015-03-04 努艾利克斯德国股份有限公司 用于抗体和Fc-融合蛋白亲和色谱IV纯化的配体
WO2013117707A1 (fr) * 2012-02-08 2013-08-15 Graffinity Pharmaceuticals Gmbh Ligands pour anticorps et purification de protéine de fusion à fragment constant par chromatographie d'affinité iv
US9745339B2 (en) 2012-02-08 2017-08-29 Novalix Deutschland Gmbh Ligands for antibody and Fc-fusion protein purification by affinity chromotography IV
WO2014020152A1 (fr) * 2012-08-02 2014-02-06 Graffinity Pharmaceuticals Gmbh Ligands pour aphérèse et immunoabsorption
WO2014174316A1 (fr) 2013-04-26 2014-10-30 Adc Biotechnology Ltd Procédé de synthèse de conjugués anticorps-médicament au moyen de résines d'affinité
JP2016519116A (ja) * 2013-04-26 2016-06-30 エーディーシー バイオテクノロジー エル ティー ディー アフィニティー樹脂を使用するａｄｃの合成方法
WO2016067016A1 (fr) 2014-10-28 2016-05-06 Adc Biotechnology Ltd Procédé de synthèse de conjugués d'anticorps à l'aide de résines d'affinité
WO2016067013A1 (fr) 2014-10-28 2016-05-06 Adc Biotechnology Ltd Procédé de synthèse de cam au moyen de résines d'affinité
TWI748078B (zh) * 2017-03-30 2021-12-01 大陸商江蘇恒瑞醫藥股份有限公司 抗體藥物偶聯物的製備方法
CN113952944A (zh) * 2021-12-23 2022-01-21 浙江湃肽生物有限公司深圳分公司 一种纯化九肽-1的方法
CN113952944B (zh) * 2021-12-23 2022-03-29 浙江湃肽生物有限公司深圳分公司 一种纯化九肽-1的方法

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