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WO2007063129A2 - Isolement de peptides, polypeptides et protéines - Google Patents

Isolement de peptides, polypeptides et protéines Download PDF

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Publication number
WO2007063129A2
WO2007063129A2 PCT/EP2006/069208 EP2006069208W WO2007063129A2 WO 2007063129 A2 WO2007063129 A2 WO 2007063129A2 EP 2006069208 W EP2006069208 W EP 2006069208W WO 2007063129 A2 WO2007063129 A2 WO 2007063129A2
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WO
WIPO (PCT)
Prior art keywords
adsorbent
protein
protein solution
process according
factor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2006/069208
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English (en)
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WO2007063129A3 (fr
Inventor
Bjarke Christensen
Allan NØRGAARD
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Upfront Chromatography AS
Novozymes AS
Original Assignee
Upfront Chromatography AS
Novozymes AS
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Filing date
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Application filed by Upfront Chromatography AS, Novozymes AS filed Critical Upfront Chromatography AS
Publication of WO2007063129A2 publication Critical patent/WO2007063129A2/fr
Publication of WO2007063129A3 publication Critical patent/WO2007063129A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/473—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used alpha-Glycoproteins
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin

Definitions

  • the present invention relates to the large-scale fractionation and isolation of peptides, polypeptides and protein(s), such as human plasma or serum protein(s), from a protein solution.
  • the present invention relates to large-scale manufacture of therapeutic plasma or serum protein(s) from sources such as blood, plasma, serum or other blood derived sources using an adsorbent coupled with a ligand for the capture of the protein(s), from the protein solution.
  • Human and animal blood comprises many proteins and enzymes, which possess therapeutic and potentially life-saving properties. Some of these proteins may be found in the red blood cells whereas others are found in solution in plasma or serum. Since the middle of the 20 th century such proteins have been the target for large-scale and specific isolation with the aim of purifying and standardising the proteins for use as human therapeutic agents. Examples of prominent blood proteins that are currently available as isolated therapeutic products are: albumin, immunoglobulin G, Factor VIII and alpha-1- proteinase inhibitor. Some of these proteins are produced in the scale of several thousand kg per year (albumin and IgG) while others are produced only in the gram to kilogram per year scale. However, on a worldwide basis many million litres of blood per year are processed for the purpose of isolating these proteins.
  • Blood, blood plasma and blood serum are extremely complicated protein containing solutions that comprises many other types of compounds other than the protein(s) or enzyme(s) of interest, all carefully balanced and regulated to work in the blood-stream in a very broad range of biochemically complicated functions such as the oxygen transport, the immuno defence and the coagulation system preventing excessive bleeding from wounds.
  • biochemically complicated functions such as the oxygen transport, the immuno defence and the coagulation system preventing excessive bleeding from wounds.
  • chemical agents such as heparin and sodium citrate, can be added to increase the stability and to a certain degree prevent coagulation of the blood plasma obtained by separating the blood cells, the plasma will still be a very fragile, highly concentrated and viscous protein solution also comprising significant amounts of lipids.
  • any handling or alteration of the plasma composition involves the risk of accidental destabilisation, which may cause activation of the coagulation cascade, precipitation of e.g. lipid components as well as denaturation of the target protein(s) and thereby makes the blood very difficult to work with.
  • any method employed to isolate proteins from blood or blood derived solutions must take the inherent instability of the solution and the proteins themselves into consideration. This has proven to be a very significant challenge for the large-scale production of therapeutic products from blood.
  • the yield alpha-1 -proteinase inhibitor is as low as 10-20 % and the yield of IgG is as low as 40-50 %.
  • these products are much needed and as there is an undersupply of the product to satisfy 0 the needs of patients, new methods for isolating such products are highly needed where the loss of product is reduced.
  • WO 2005121 163 A2 unpublished at the priority date of the present application, also o describes a process for the isolation and/or fractionation of peptide, polypeptide or protein solutions in general.
  • the present invention relates to a process for the isolation and/or fractionation of peptide, 5 polypeptide or protein solutions (hereinafter commonly denoted proteins and protein solutions).
  • the process of the present invention is fast, robust, specific and safe, and provides an improved yield and purity of the product of interest during processing and thereby facilitates an improved and acceptable balance between yield of product and economy involved, compared to the conventionally used methods.
  • the process according 0 to the invention is particularly suitable for large-scale production in particular fermentations of microbial cells or production in plants.
  • the process of the invention provides means for reduction, removal and/or inactivation and/or inhibition of compounds interfering with the isolation and/or fractionation of the protein solution.
  • Such compound may be exemplified by, but not limited to, hydrolytic enzymes. It 5 has been found that such compound may seriously interfere with the isolation and/or fractionation of the protein solution and/or limit the freedom to choose condition for the isolation and/or fractionation process (for example if the optimal pH for the isolation and/or fractionation coincides with the optimal pH for hydrolytic activity). Further the process of the invention provides in some aspects means for reduction of undesired protein from the 0 source of the protein solution. Further the process of the invention provides in some aspects means for reduction of colors, salts, carbohydrates, nucleic acids, mycotoxins, and/or endotoxins.
  • the invention provides a process, preferably a large scale process, for the isolation or separation of recombinant human serum albumin (rHSA) comprising the steps of: a) providing a protein solution comprising said recombinant human serum albumin (rHSA) and optionally having a preset pH and optionally a preset ionic strength and/or conductivity; b) contacting said protein solution with an adsorbent, wherein the adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups or the adsorbent comprises a particle with at least one high density non-porous core , surrounded by a porous material; and c) Optionally washing the adsorbent, d) obtaining said recombinant human serum albumin (rHSA) from said adsorbent.
  • the present invention provides a process for the isolation of one or more protein(s) from a protein solution wherein the protein is obtained from a fungal, a prokaryot, a plant or an insect cell, said process comprising the steps of:
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups or the adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m;
  • the invention provides a process for the isolation of one or more protein(s) from a protein solution, said process comprising the steps of:
  • said adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups or adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m;
  • the present invention provides a process for the isolation of one or more protein(s) from a protein solution wherein the protein selected from the group consisting of recombinant human and/or bovine serum albumin (rHSA and/or rBSA), Betaferon (interferon- -1 b), human erythropoietin (EPO), glucocerebrosidase, insulin, in particular recombinant insulin produced in E.
  • the protein selected from the group consisting of recombinant human and/or bovine serum albumin (rHSA and/or rBSA), Betaferon (interferon- -1 b), human erythropoietin (EPO), glucocerebrosidase, insulin, in particular recombinant insulin produced in E.
  • coli Hepatitis B surface antigen, Urate oxidase, Glucagon, Granulocyte-macrophage colony stimulating factor, Hirudin, lepirudin, Platelet-derived growth factor, Antiangiogenic factor, Antibodies (mAb and/or PAb), antibody fragments (Fab), Anti microbial peptides, anti viral peptides, anti-cancer peptides, IGF-1 , EGF (epidermal growth factor), TGF (TGF-alfa) Transforming growth factor, Heparin, Interleukins, Cytokines, Hormones (sex hormones, such as Follicle Stimulating hormone and Human chorionic gonadotrophin, human growth hormone, peptide hormones, cortison hormones), Antigens (HIV, HBV, HCV, HPV, Dengue Virus, Malaria), Elastin, Gellatine, Collagen, Protein A, Lactic acid, Hyaloronic acid, human intrinsic factor, oxidoreductases
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or 0 heteroaromatic ring-system and one or more acidic groups or the adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m;
  • a process for the large-scale isolation or 0 separation of recombinant transferrin comprises the steps of:
  • Proteins can be purified from a crude protein solution without the need for separate clarification, concentration and other types of initial purification to remove particulate matter.
  • the process according to the present invention may be performed at a large-scale.
  • the term "large-scale” relates to the processing of a raw material volume of at least 1 liters per adsorption cycle, such as at least 5 liters per adsorption cycle, such as at least 10 liters per adsorption cycle, such as at least 25 liters per adsorption cycle, such as at least 100 liters per adsorption cycle, such as at least 1000 liters per adsorption cycle and thus distinguish the invention from any analytical and small scale experiments that do not relate to the severe requirements for robustness and reproducibility as in an industrial large-scale production environment.
  • the protein solution and its source such as at least 5 liters per adsorption cycle, such as at least 10 liters per adsorption cycle, such as at least 25 liters per adsorption cycle, such as at least 100 liters per adsorption cycle, such as at least 1000 liters per adsorption cycle and thus distinguish the invention from any analytical and small scale
  • the protein(s) of interest may be separated and isolated from a protein solution.
  • protein solution relates to any kind of solution in liquid form comprising the protein(s) of interest and from which the protein(s) may be separated and isolated.
  • the protein solution may be obtained from a source selected from blood, serum, plasma or other blood derived sources.
  • the blood, serum, plasma or other blood derived sources may be obtained from humans or animals such as cows, camel, pig, sheep, goat, rabbit, mouse, rat, horse, zebra, chicken, fish, or ostrich.
  • the protein may also be derived from other portions of the animal.
  • the animal selected may be capable of producing or may have been modified to produce the protein(s) of interest.
  • the protein solution may be obtained from a mammalian cell culture or a microbial fermentation broth, where the mammalian cell or the microorganism is capable of or has been modified to produce the protein(s) of interest, or a plant extract, where the plant is capable of producing or has been modified to produce the protein(s) of interest. Insect cells may also be used.
  • the protein is derived/obtained from a fungal, prokaryot, plant or insect cell.
  • the protein solution derives from a fermentation of such cells or in case of a plant cell, from farming/growth of the plant. It may be a crude broth or may be more or less purified.
  • the process of the invention preferably comprise a step of fermenting a fungal, prokaryot or insect cell or growing a transgenic plant comprising a gene encoding rHSA and capable of producing rHSA prior to contacting the protein solution with an adsorbent.
  • the protein to be isolated may in particular be derived from a plant, a transgenic plant, plant part, or plant cell thereof preferably which has been transformed with a nucleotide sequence encoding a protein of interest so as to express and produce the protein in recoverable quantities.
  • the plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot).
  • monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
  • dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • cruciferous plants family Brassicaceae
  • moss moss
  • flax seed flax seed
  • sprouts sugar cane, turnip and sunflower may be used.
  • plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, 5 parenchyme, vascular tissues, meristems.
  • Specific plant cell compartments such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part.
  • any plant cell whatever the tissue origin, is considered to be a plant part.
  • plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts, e.g.,
  • transgenic plant or plant cell expressing a protein to be isolated according to the invention may be constructed in accordance with methods known in the art.
  • the plant or plant cell is constructed by incorporating one or more expression constructs encoding a protein of interest into the plant host genome and propagating the resulting
  • the expression construct is conveniently a nucleic acid construct which comprises a polynucleotide encoding a protein of interest operably linked with appropriate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable
  • regulatory sequences such as promoter and terminator sequences and optionally signal or transit sequences is determined, for example, on the basis of
  • the expression of the gene encoding a protein of interest may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.
  • the 35S-CaMV, the maize ubiquitin 1 , and the rice actin 1 promoter may be used (Franck et al., 1980, Ce// 21 : 285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang et al., 1991 , Plant Cell 3: 1155-1165).
  • Organ- specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-
  • a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152: 708-711 ), a promoter from a seed 5 oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941 ), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772.
  • a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889)
  • the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene o promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588).
  • the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by 5 exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.
  • abiotic treatments such as temperature, drought, or alterations in salinity or induced by 5 exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.
  • a promoter enhancer element may also be used to achieve higher expression of a protein of interest in the plant.
  • the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a 0 protein of interest.
  • the first intron of the rice actin 1 gene discloses the use of the first intron of the rice actin 1 gene to enhance expression.
  • the selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
  • the nucleic acid construct is incorporated into the plant genome according to 5 conventional techniques known in the art, including Agrobacterium-medlated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et ai, 1989, Nature 338: 274).
  • Agrobacterium tumefaciens-me ⁇ ated gene transfer is the method of 0 choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants.
  • the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic 5 calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281 ; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667- 674).
  • An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21 : 415- 428.
  • the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well- known in the art.
  • the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific o excision of the selection gene by a specific recombinase.
  • the protein to be isolated may be obtained from a prokaryotic or fungal microorganism.
  • the term "obtained from” as used herein in connection with a given source shall mean that the protein encoded by a nucleotide 5 sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted.
  • the protein obtained from a given source is secreted extracellularly.
  • the protein to be isolated a unicellular and/or bacterial protein.
  • the protein may be a gram positive bacterial protein such as a 0 Bacillus protein, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,
  • Bacillus circulans Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus,
  • Bacillus licheniformis Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis protein; or a Streptomyces protein, e.g., a Streptomyces lividans or Streptomyces murinus protein; or a gram negative bacterial protein, e.g., an E. coli or a 5 Pseudomonas sp. protein.
  • the bacterial protein is obtained from Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell.
  • the protein to be isolated is a fungal protein.
  • "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and 0 Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
  • the invention encompasses 5 both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • the fungal protein is be obtained from yeast.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), o basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980). 5 Fungal proteins from yeast includes in particular protein obtained from Candida,
  • the protein is obtained from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Pichia pastoris, Saccharomyces 0 oviformis, Kluyveromyces lactis, Hansenula polymorpha or Yarrowia lipolytica.
  • the fungal protein is be obtained from a filamentous fungal cell.
  • filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are generally characterized by a mycelial wall composed of chitin, 5 cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
  • Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the protein of the filamentous fungal cell is 0 obtained from Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • the protein is obtained from Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecio
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81 : 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.
  • the protein solution used may be supplemented with an alcohol.
  • the term "supplemented with an alcohol” relates to the addition of an alcohol to the protein solution in order to achieve separation of at least two components present in the protein solution whereby one component will become present in a supernatant and the other component will become present in a fraction.
  • separation of the protein solution involves gradually increasing the amount of alcohol added to the protein solution and thereby separating the at least one serum or plasma protein from the protein solution.
  • the protein(s) of interest may be present in either the supernatant or in the fraction.
  • the protein solution is supplemented with an alcohol to comprise at least 0.1 vol.% of an alcohol, e.g.
  • At least 0.5 vol.% such as at least 0.75 vol.%, e.g. at least 1.0 vol.%, such as at least 1.5 vol.%, e.g. at least 2.0 vol.%, such as at least 3.0 vol.%, e.g. at least 5.0 vol.%, such as at least 7.5 vol.%, e.g. at least 10.0 vol.%, such as at least 20 vol.%, e.g. at least 25.0 vol.%, such as at least 40 vol.%, e.g. at least 50.0 o vol.%, such as at least 60 vol.%, e.g. at least 75.0 vol.%.
  • the protein solution has a total alcohol content of at least 0.1 vol.% of an alcohol, e.g. at least 0.5 vol.%, such as at least 0.75 vol.%, e.g. at least 1.0 vol.%, such as at least 1.5 vol.%, e.g. at least 2.0 vol.%, such as at 5 least 3.0 vol.%, e.g. at least 5.0 vol.%, such as at least 7.5 vol.%, e.g. at least 10.0 vol.%, such as at least 20 vol.%, e.g. at least 25.0 vol.%, such as at least 40 vol.%, e.g. at least 50.0 vol.%, such as at least 60 vol.%, e.g. at least 75.0 vol.%, such as at least 77 vol.%.
  • an alcohol e.g. at least 0.5 vol.%, such as at least 0.75 vol.%, e.g. at least 1.0 vol.%, such as at least 1.5 vol.%, e.g
  • the term "supernatant" relates to a liquid phase, which is lying 0 above a liquid fraction, a sediment fraction or a precipitated fraction obtained by the addition of an alcohol to the protein solution, in accordance with the present invention.
  • fraction relates to a portion of the protein solution, which may be separated from the supernatant by a fractionation process, such as filtration, 5 microfiltration, centrifugation, distillation or chromatography and the fraction may be either a combination of compounds or a pure compound.
  • the fraction may be in the form of a liquid (a liquid fraction), a sediment (a sediment fraction) or a precipitate (a precipitated fraction).
  • the protein solution may be supplemented with an alcohol selected from the group consisting of methanol, ethanol, n-propanol, i- propanol, n-butanol, i-butanol, s-butanol, t-butanol, methylene glycol, ethylene glycol, propylene glycol, diethylene glycol, methylene-ethylene glycol, and dimethylene glycol.
  • the protein solution comprises of plasma or serum, in particular from a human.
  • the protein solution comprises serum or plasma fraction(s) and/or plasma or serum supernatant(s), in particular from a human.
  • fractions obtained from a resolubilised precipitate may be obtained by gradual addition of alcohol to the protein solution.
  • a plasma or serum fraction may be provided from a resolubilised precipitate obtained by the addition of alcohol to plasma or serum.
  • a protein solution may be obtained from the protein solution and may comprise the combination of one or more supernatant(s) and/or one or more resolubilised fractions.
  • a human or animal plasma or serum protein solution may by obtained by recombination of one or more supernatants and/or one or more resolubilised precipitates obtained by the addition of alcohol to human or animal plasma or serum.
  • the temperature of the protein solution may be in the range of -5 to 50 0 C, more preferably in the range of -5 to 40 0 C, still more preferably in the range of -5 to 30 0 C, still more preferably in the range of -5 to 20 0 C, still more preferably in the range from 0 to 10 0 C or in the range of 0 to 50 0 C, more preferably in the range of 10 to 50 0 C, still more preferably in the range of 20 to 50 0 C, still more preferably in the range of 30 to 50 0 C, still more preferably in the range from 40 to 50 0 C.
  • a stepwise fractionation of protein solutions by the addition of alcohol to obtain protein(s) may be illustrated by one or more or the following operations:
  • the protein solution such as plasma may initially be frozen and then subjected to a slow, controlled de-freezing procedure, whereby a cryoprecipitate is formed comprising Factor VIII (complexed with the von Willebrand factor, vWF) and fibrinogen and a supernatant comprising the bulk of the proteins remains in plasma (called cryo-poor plasma).
  • a cryoprecipitate comprising Factor VIII (complexed with the von Willebrand factor, vWF) and fibrinogen and a supernatant comprising the bulk of the proteins remains in plasma (called cryo-poor plasma).
  • the cryoprecipitate fraction obtained in (i) may be resolubilised to form a protein solution and the protein(s) may be isolated by contacting it with an adsorbent.
  • protein(s) such as Factor VIII, vWF and fibrinogen may be isolated from the protein solution obtained from the resolubilised fraction.
  • the supernatant from (i), cryo-poor plasma, may be supplemented with alcohol and fraction I (a precipitate) and supernatant I are formed.
  • Supernatant I may be supplemented with more alcohol and precipitated fraction Il + III and supernatant Il + III are formed.
  • Fraction Il + III may be separated and resolubilised to form a protein solution and protein(s) of interest may be isolated by contacting is with an adsorbent.
  • protein(s) such as immunoglobulins (such as IgG) may be isolated from the protein solution obtained from fractions Il + III.
  • Supernatant ll+lll will mainly comprise albumin and alpha-1 -proteinase inhibitor.
  • the supernatant Il + III may be further supplemented with alcohol and forming supernatant IV-1 and precipitated fraction IV-1.
  • Fraction IV-1 may be separated and resolubilised and form a protein solution and protein(s) may be isolated by contacting is with an adsorbent.
  • plasma protein(s) such as alpha-1 -proteinase inhibitor, anti-thrombin III and Factor IX complex may be isolated from the protein solution obtained from fraction IV-1.
  • the Supernatant IV-1 may be supplemented with more alcohol to form precipitated fraction IV-4 and supernatant IV-4.
  • Fraction IV-4 may be separated and resolubilised to form a protein solution and the protein(s) of interest may be isolated by contacting is with an adsorbent.
  • Butyrylcholinesterase may be isolated from the protein solution obtained from fraction IV-4.
  • Supernatant VI-4 may be further supplemented with alcohol to provide precipitated fraction V which may be separated and resolubilised to form a protein solution and protein(s) of interest may be isolated by contacting is with an adsorbent.
  • protein(s) of interest may be isolated by contacting is with an adsorbent.
  • albumin may be isolated from the protein solution obtained from fraction V.
  • the protein solution may be selected from the group consisting of cryo-poor plasma, supernatant I, supernatant ll+lll, supernatant IV-1 , supernatant IV-4, resolubilised cryo-precipitate, resolubilised fraction I, resolubilised fraction II+ III, resolubilised fraction IV-1 , resolubilised fraction IV-4, resolubilised fraction V and any combination thereof.
  • the protein solution may be selected from the group consisting of supernatant I, supernatant ll+lll, resolubilised fraction IV-1 , and any combination thereof.
  • the protein solution may be selected from the group consisting of supernatant I, supernatant ll+lll, supernatant l+ll+lll, and resolubilised fraction IV-1 and any combination thereof.
  • the protein solution may be selected from the group of supernatant I and resolubilised fraction ll+lll.
  • the precipitated fractions may be resolubilised in a broad range of aqueous solutions including pure water.
  • the resolubilisation medium will be an aqueous buffer having a pH and ionic strength suitable for the following downstream processing step e.g. an adsorption step according to the invention.
  • precipitated fractions may be obtained from a supernatant by filtration, centrifugation, decantation, ultrafiltration and/or sedimentation.
  • the protein solution may be obtained by the o Cohn fractionation method as described by Cohn et al, "Separation into Fractions of
  • the protein solution comprises at least one plasma or serum protein obtained from the fractionation of plasma or serum by addition of ethanol, e.g. the original Cohn fractionation process or variants hereof such as the Cohn- 0 Oncley process.
  • the protein solution is subjected to inactivation conditions, preferably prior to the step of applying the protein solution to the adsorption column.
  • inactivation conditions as used herein is to be understood as conditions inactivating or reducing activity of one or more compounds, in particular proteins, in the protein solution.
  • compounds such as proteins, in particular 0 enzymes, that degrade the protein(s) to be isolated or change its properties.
  • hydrolytic enzymes such as proteases.
  • the protein solution is subjected to inactivation conditions, by applying a heat treatment to the protein solution, such as pasteurization.
  • a heat treatment to the protein solution, such as pasteurization.
  • the protein solution is heated for a limited time to a temperature where compound to be 5 inactivated is inactivated by the heat.
  • a preferred temperature is above 50 °C, such as above 55 °C, such as above 60 °C, such as above 65 °C, such as above 70 °C, such as above 75 °C, such as above 80 °C.
  • the temperature is kept below 100 °C, such as below 95 °C, such as below 90 5 °C, such as below 85 °C.
  • a preferred range is 40-80 °C, in particular 51-80 °C, in particular 55-80 °C, in particular 60-70 °C.
  • the temperature is kept about 65 °C.
  • the time the protein solution should be heated depends on the temperature and the compound one wants to inactivate. It is preferred to keep the time from a few minutes up to hours, such as from 1 to 90 minutes, particularly 5 to 60 minutes, particularly 10-45, o particularly 20 to 30 minutes.
  • the heating step also confer the advantage that is may denature and precipitate other undesired heat labile proteins and cellular residues.
  • the protein solution is subjected to inactivation conditions, 5 by addition of an organic solvent to the protein solution.
  • the solvent should be added in concentration between about 10 to about 80 % v/v, preferably between about 20 to about 70 % v/v, preferably between about 30 to about 60 % v/v, preferably between about 35 to about 50 % v/v, most preferably about 40 % v/v.
  • Preferred solvent are ethanol and isopropanol 0
  • the protein solution is subjected to enzyme inactivation conditions, by addition of competitive or non-competitive enzyme inhibitors, in particular protease inhibitors to the protein solution.
  • Useful inhibitors include but is not limited to EDTA, Pefabloc, Aprotinin, Bestatin, Pepstatin, Phosphormidon, Leupeptin, Chymostatin, Antipain 5
  • the protein solution may be subjected stabilisation of the protein(s) to be isolated before subjecting it to inactivating conditions, so as to reduce the negative effect s on the protein to be isolated by the subsequent inactivating conditions.
  • a stabilising compound that confer heat resistance to the protein to be isolated or which confer 0 resistance to enzymatic degradation.
  • Such compounds include but is not limited to acids, in particular fatty acids, and their salts, particularly octanoic acid and salts thereof such as sodium caprylate and N-acetyl-L-tryptophan.
  • the stabilising compound is added in amounts from 0.01 to 100 mM, such as from 0.1 to 10 mM, , such as from 1 to 8 mM, , such as from 2 to 6 mM, e.g 4 mM. Addition of such stabiliser is particularly useful when 5 isolating albumins in particular human or bovine serum albumin (HSA or BSA).
  • the protein solution may also be subjected to removal of cells and/or cell debris prior to the step of applying the protein solution to the adsorption column and/or contacting the protein solution with an adsorbent and preferably also prior to subjecting the protein solution to the inactivating conditions.
  • the protein solution may be subjected to removal of material that precipitated at the inactivating conditions.
  • fractionation/isolation steps it may be advantageous to include one or more additional steps, preferably after contacting the protein solution o with an adsorbent.
  • the process of the invention comprises a colour removing and/or reduction step comprising a) applying to the protein solution, either before or after the step of applying the 5 protein solution to the adsorption column or contacting the protein solution with an adsorbent, a material capable of binding coloured compounds b) removing the colour binding material or material binding coloured compounds from the protein solution
  • Materials binding coloured compounds include but is not limited to activated carbon and chromatographic resins.
  • the material binding coloured compounds is added in amount of about 0.01 to 10 % by weigh, typically from 0.5 to 1.5 % by weight.
  • albumin such as rHSA and/or rBSA the inventors found that albumin is 5 capable of binding certain compounds that cause (mis)coloration of solutions albumin.
  • compounds that cause (mis)coloration is to be understood as compounds
  • (mis)coloration is heme (comprising Fe(II)) and/or hemin (comprising Fe (III)) and/or 0 derivatives thereof.
  • albumins such as rHSA/rBSA may be present in solution in several different reversible forms depending on the solution pH.
  • they may adapt an A-form or "aged form” near pH 10; a B-form or basic form near pH 8.5; up to 4 5 different N-forms at neutral pH; an F-form at pH 4 and an E-form below pH 3.
  • the albumin may adapt the F form at a pH between 3.5 and 5 in particular between 3.5 and 4.5 such as around pH 4.
  • the changes between the different form are reversible (Theodore Peters Jr., All about Albumin; pages 54-67, Academic Press, 1996.
  • albumin in particular rHSA and/or rBSA
  • the protein is albumin, in particular rHSA
  • the method the invention comprises a step of bringing the albumin on predominantly F-form and/or E- o form or a combination thereof allowing compounds causing (mis)coloration to be released and/or less bound and/or more weakly adsorbed to the albumin and optionally be removed from the purified protein solution.
  • Bringing albumin on predominantly F-form and/or E-form or a combination thereof means that at least 50 mole % of the albumin is on F-form and/or E-form. Particularly, at least 75 mole %, such as at least 90 mole %, such 5 as at least 95 mole %, such as 99 mole % such as 100 mole % of the albumin is on F- form and/or E-form.
  • the albumin can be brought on the F- and/or E-form before and/or during and/or after the step of the fractionation/separation method as described, supra. 0
  • Bringing the albumin on F-form may be achieved by choosing a pH of the solution of the albumin between 3 to 5, particularly between 3.5 and 4.5, more particularly around pH 4 or 4.5.
  • Bringing the albumin on E-form may be achieved by choosing a pH of the solution of the albumin below 3, but above the pH limit for allowing reversible change back to the 5 N-form.
  • the bound compounds causing (mis)coloration is removed or reduced before and/or after applying the fractionation/separation method as described, supra.
  • the protein solution is adjusted to a pH bringing the 0 albumin on F- and/or E-form to release the bound compounds causing (mis)coloration and/or less bound and/or more weakly adsorbed to the albumin e.g. followed by adding a material binding the coloured compounds such as activated carbon and chromatographic resins as described, supra.
  • the protein is rHSA or rBSA produced from a microorganism, in particular a fungus, such as a filamentous fungus, eg. Aspergilli, eg. A.
  • adding activated carbon provide the additional advantage that the activated carbon removes the 43 KDa fragment propeptide cleaved from the n-terminal of the rHSA/rBSA. 5 BChr. Describe what the 43 KDa fragment is
  • the protein(s) to be isolated is a blood protein, such as a plasma protein or a serum protein.
  • a blood protein such as a plasma protein or a serum protein.
  • plasma or serum protein(s) relates to protein(s) which is/are produced or required by the human or animal body. Normally these plasma or serum proteins are contained in the blood of humans or animals and particularly some of these proteins are originally found in the red blood cells whereas others are found in solution in the plasma or serum.
  • Plasma is a component of blood. It is the liquid in which blood cells are 5 suspended. Blood plasma contains proteins, lipids, nutrients, metabolic end products, hormones, and inorganic electrolytes. Blood plasma is typically stabilised by the addition of anti-coagulants such as sodium citrate, heparin and EDTA.
  • anti-coagulants such as sodium citrate, heparin and EDTA.
  • Serum is the same as blood plasma except that clotting factors (such as fibrinogen and Factor VIII) have been removed.
  • clotting factors such as fibrinogen and Factor VIII
  • the plasma or serum protein is a human or animal plasma or serum protein.
  • the one or more plasma or serum protein(s) to be isolated is/are selected from the group consisting of albumin, IgG, IgA, IgM, IgD, IgE, alpha-1 -proteinase inhibitor (same as ⁇ -1 -antitrypsin), blood pro- 5 coagulation protein, blood anti-coagulation protein, thrombolytic agent, anti-angiogenic protein, alpha-2-antiplasmin, C-1 esterase inhibitor, apolipoprotein, HDL, LDL, Fibronectin, beta-2-glycoprotein I, fibrinogen, plasminogen, plasmin, plasminogen activator, plasminogen inhibitor, plasma protease inhibitor, anti-thrombin III, streptokinase, inter-alpha-trypsin inhibitor, alpha-2-macroglobulin, amyloid protein, ferritin, pre-albumin, 0 GC-globulin, haemopexin, C3-comp
  • the protein is selected from the group consisting of Recombinant Human and/or bovine serum albumin, Betaferon (interferon- -1 b), human erythropoietin (EPO), Fibrin, resilin, glucocerebrosidase, the enzyme used for replacement therapy in patients with Gaucher disease, insulin, in particular recombinant insulin produced in E.
  • coli Hepatitis B surface antigen, Urate oxidase, Glucagon, Granulocyte-macrophage colony stimulating factor, Hirudin/lepirudin, Platelet-derived growth factor, Antiangiogenic factor, Antibodies (mAb and/or PAb), antibody fragments (Fab), Antimicrobial peptides such as plectasin, anti viral peptides, anti-cancer peptides, IGF-1 , EGF (epidermal growth factor), TGF (TGF-alfa) Transforming growth factor, Heparin, Interleukins, Cytokines, Hormones (sex hormones, such as Follicle Stimulating hormone and Human chorionic gonadotrophin, human growth hormone, peptide hormones, cortison hormones), statins Antigens (HIV, HBV, HCV, HPV, Dengue Virus, Malaria), elastin, gelatine, collagen, Protein A, human intrinsic factor enzymes and enzyme inhibitors such as
  • rHSA human serum albumin
  • rBSA bovine serum albumin
  • HSA human serum albumin
  • BSA bovine serum albumin
  • proteins may be recombinant proteins obtained from heterologeous expression in a microbial, particularly prokaryote or fungal, host.
  • the protein is a therapeutic glycoprotein intended for use in humans in particular fungal in particular yeast-derived glycoproteins with a humanized glycosylation.
  • the protein is an enzyme or enzyme variant. It is to be understood that enzyme variants (produced, for example, by recombinant techniques) are included within the meaning of the term "enzyme”. Examples of such enzyme variants are disclosed, e.g., in EP 251 ,446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525,610 (Solvay) and WO 94/02618 (Gist-Brocades NV). The enzyme classification here employed is in accordance with Recommendations
  • oxidoreductases EC 1.-.-.-
  • transferases EC 2.-.-.-
  • hydrolases EC 3.-.-.-
  • lyases EC 4.-.-.-
  • isomerases EC 5.-.-.-
  • ligases EC 6.-.-.-
  • oxidoreductases in the context of the invention are peroxidases (EC 1.1 1.1 ), laccases (EC 1.10.3.2) and glucose oxidase (EC 1.1.3.4)].
  • transferases are transferases in any of the following sub-classes: a) Transferases transferring one-carbon groups (EC 2.1 );
  • transferases transferring aldehyde or ketone residues (EC 2.2); acyltransferases (EC 2.3); c ) glycosyltransferases (EC 2.4); d) transferases transferring alkyl or aryl groups, other that methyl groups (EC 2.5); and e ) transferases transferring nitrogeneous groups (EC 2.6).
  • a particular type of transferase in the context of the invention is a transglutaminase (protein-glutamine ⁇ -glutamyltransferase; EC 2.3.2.13). Further examples of suitable transglutaminases are described in WO 96/06931 (Novo Nordisk A/S).
  • hydrolases in the context of the invention are: Carboxylic ester hydrolases (EC 3.1.1.-) such as lipases (EC 3.1.1.3) , such as Pancreaslipase; phytases 5 (EC 3.1.3.-), e.g. 3-phytases (EC 3.1.3.8) and 6-phytases (EC 3.1.3.26); glycosidases (EC 3.2, which fall within a group denoted herein as "carbohydrases”), such as ⁇ -amylases (EC 3.2.1.1 ); peptidases (EC 3.4, also known as proteases) such as Trypsin; and other carbonyl hydrolases.
  • Carboxylic ester hydrolases EC 3.1.1.-
  • lipases EC 3.1.1.3
  • pancreaslipase e.g. 3-phytases (EC 3.1.3.8)
  • 6-phytases EC 3.1.3.26
  • glycosidases EC
  • carbohydrase is used to denote not only 0 enzymes capable of breaking down carbohydrate chains (e.g. starches or cellulose) of especially five- and six-membered ring structures (i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizing carbohydrates, e.g. six-membered ring structures such as D-glucose to five-membered ring structures such as D-fructose.
  • Carbohydrases of relevance include the following (EC numbers in parentheses): ⁇ -amylases (EC 3.2.1.1 ), ⁇ - 5 amylases (EC 3.2.1.2), glucan 1 ,4- ⁇ -glucosidases (EC 3.2.1.3), endo-1 ,4-beta-glucanase (cellulases, EC 3.2.1.4), endo-1 ,3(4)- ⁇ -glucanases (EC 3.2.1.6), endo-1 ,4- ⁇ -xylanases (EC 3.2.1.8), dextranases (EC 3.2.1.11 ), chitinases (EC 3.2.1.14), polygalacturonases (EC 3.2.1.15), lysozymes (EC 3.2.1.17), ⁇ -glucosidases (EC 3.2.1.21 ), ⁇ -galactosidases (EC 3.2.1.22), ⁇ -galactosidases (EC 3.2.1.23), amy
  • the protein is an antimicrobial enzyme.
  • the antimicrobial enzyme may be, e.g., a muramidase, a lysozyme, a protease, a lipase, a phospholipase, a chitinase, a glucanase, a cellulase, a peroxidase, or a laccase.
  • the biological compound may be an enzyme synthesizing conventional antibiotics, e.g. polyketides or penicillins.
  • the protein is an antimicrobial peptide (AMP).
  • the peptide may be a peptide compound interacting/binding/sequestering essential cellular targets.
  • the antimicrobial peptide (AMP) may be, e.g., a membrane-active antimicrobial peptide, or an antimicrobial peptide affecting/interacting with intracellular targets, e.g. binding to cell DNA.
  • the AMP is generally a relatively short peptide, consisting of less than 100 amino acid residues, typically 20-80 residues.
  • the antimicrobial peptide has bactericidal and/or fungicidal effect, and it may also have antiviral or antitumour effects. It generally has low cytotoxicity against normal mammalian cells.
  • the antimicrobial peptide generally has a highly cationic portion (depending on pH) and a hydrophobic portion. It typically contains several arginine and lysine residues, and it may not contain a single glutamate or aspartate. It usually contains a large proportion of hydrophobic residues.
  • the peptide generally has an amphiphilic structure, with one surface being highly positive and the other hydrophobic.
  • bioactive peptide and the encoding nucleotide sequence may be derived from plants, invertebrates, insects, amphibians and mammals, or from microorganisms such as bacteria and fungi.
  • the antimicrobial peptide may act on cell membranes of target microorganisms, e.g. through nonspecific binding to the membrane, usually in a membrane-parallel orientation, interacting only with one face of the bilayer.
  • the antimicrobial peptide typically has a structure belonging to one of five major classes: ⁇ helical, cystine-rich (defensin-like), ⁇ -sheet, peptides with an unusual composition of regular amino acids, and peptides containing uncommon modified amino acids.
  • alpha-helical peptides are Magainin 1 and 2; Cecropin A, B and P1 ; CAP18; Andropin; Clavanin A or AK; Styelin D and C; and Buforin II.
  • cystine- rich peptides are ⁇ -Defensin HNP-1 (human neutrophil peptide) HNP-2 and HNP-3; ⁇ - Defensin-12, Drosomycin, ⁇ 1-purothionin, and Insect defensin A.
  • ⁇ -sheet peptides are Lactoferricin B, Tachyplesin I, and Protegrin PG1-5.
  • peptides with an unusual composition are Indolicidin; PR-39; Bactenicin Bac5 and Bac7; and Histatin 5.
  • peptides with unusual amino acids are Nisin, Gramicidin A, and Alamethicin.
  • AFP antifungal peptide
  • the process of the invention may even be used for isolation of 5 non-proteins such as Lactic acid, hyaluronic acid, chitin, chitosan, glucan, cellulose and polygalactosamine.
  • the adsorbent capable of capturing the one or more protein(s) may be held within a o column or it may not be held within a column.
  • the term "column” relates to any kind of container which can be supplied with at least one inlet and at least one outlet for the application of the protein solution to the column and subsequent to elute the protein.
  • the inlet and the outlet may for certain columns be the same (e.g. for batch adsorption tanks).
  • the column may be in the form of an Expanded bed adsorption (EBA) 5 column, packed bed column, a fluidized bed adsorption column, a suspended bed adsorption column, , membrane reactor, or a batch adsorption tank.
  • the adsorbent column may be used in either a batch system or in a continuous system.
  • packed bed columns and expanded bed adsorption columns operate under plug flow conditions
  • suspended bed 0 columns and batch adsorption tanks operate with a high degree of mixing at least in the major part of the column volume.
  • EBA electrowetting-on-adsorption
  • EBA may offer a robust process comprising fewer steps and thus result in increased yields and an improved process economy. Due to the expansion of the adsorbent bed during execution of an EBA process, EBA columns may further be scaled up to industrial scale without any significant considerations regarding increased back pressures or breakdown of the process due to clogging of the system 0 which often is a problem when using packed bed columns.
  • the protein solution is applied to a packed bed column or an expanded bed column comprising an adsorbent.
  • packed bed relates to embodiments wherein the adsorbent particles are employed in columns operating with the particles in a sedimented or packed state wherein all particles are fixed on top of each other. Often packed bed columns are equipped with top and bottom adaptors defining and fixing the whole adsorbent bed to avoid any movement of the particle during operation.
  • the term "expanded bed” relates to embodiments wherein the adsorbent particles are employed in columns allowing the adsorbent to expand with an upward liquid flow through the column.
  • the column will be designed to avoid excessive liquid mixing and turbulence in the column while the individual adsorbent particles are kept in a non-fixed, dynamic state moving only in a narrow local zone in the column.
  • preferred expanded beds have a small mixing zone in the bottom part of the column where incoming liquid is distributed throughout the cross-section of the column, expanded beds generally operate under plug flow conditions in similarity with packed beds.
  • the adsorbent is held in an Expanded bed adsorption column and preferably used for the large-scale isolation of one or more protein(s) from a protein solution.
  • the adsorbent in the case where the adsorbent is not held within a column it may be a solid phase, such as for membrane based adsorption, e.g. a membrane filter, fibers or sheets, whereto the ligand is coupled.
  • membrane based adsorption e.g. a membrane filter, fibers or sheets, whereto the ligand is coupled.
  • the contacting between the adsorbent and the protein solution may generally be performed by pumping/forcing the protein solution across the surface and/or through a porous structure of the membrane or sheet to ensure that the one or more plasma or serum protein may be coming in close contact with the covalently attached functional groups on the surface and/or in the adsorbents.
  • the adsorption may be characterised by the use of selective adsorbent characteristics and/or ligand chemistry enabling the specific binding and subsequent elution of substantially only one biomolecular substance, or alternatively enabling a group specific binding of a few biomolecular substances followed by selective and consecutive elution of one or more substances from the adsorbent.
  • the adsorbent comprises a ligand suitable for binding to the one or more protein(s) of interest.
  • the adsorbent may optionally be washed and/or equilibrated with one or more washing buffer and/or equilibration buffers.
  • the adsorbent is a particle having combined characteristics in terms of size and density. It has thus been found that for highly concentrated protein solutions such as plasma or serum it is highly desirable to employ particles having a volume mean particle diameter of less than 150 ⁇ m in order to obtain a fast and efficient protein-binding 5 (which is important for the productivity and thus the economy of a production plant). However it has further been found that it is the combination of the small diameter of the adsorbent particles (below 150 ⁇ m) with a certain minimum density (more than 1.5 g/ml)) of the adsorbent particles that enables significant improvements in production plant productivity.
  • the high liquid flow rates obtainable with the adsorbents according to the invention may be significant.
  • the small adsorbent particles have a high density providing fast 5 sedimentation during the packing and re-packing procedure, which otherwise is a slow and demanding process step.
  • a smaller mean volume diameter of the particles may desire a higher density of the particles.
  • Examples of commercial adsorbent particles that may be employed for some of the 0 embodiments of the present invention are:
  • STREAMLINE SP Amersham Biosciences, Sweden, having a volume mean particle diameter of 200 ⁇ m and a density of 1.2 g/ml.
  • STREAMLINE Direct CST-1 Amersham Biosciences, Sweden having a volume mean particle diameter of 135 ⁇ m and a density of 1.8 g/ml.
  • the degree of expansion may be determined as H/HO, where HO is the height of the bed in packed bed mode (without 5 flow through the column) and H is the height of the bed in expanded mode (with a given flow through the column).
  • H/HO is in the range of 1.1-6, such as 1.1-5, e.g.
  • the degree of expansion H/HO is at most 1.2, e.g. at the most 1.3, such as at 0 most 1.5, e.g. at most 1.8 such as at most 2, such as at most 2.5, e.g. at lmost 3, such as at most 3.5, e.g. at 4, such as at most 4.5.
  • the flow-rate is 5 cm/min and the degree of expansion H/HO is at most 1.2, e.g. at the most 1.3, such as at most 1.5, e.g.
  • the flow-rate is 7 cm/min and the degree of expansion H/HO is at most 1.2, e.g. at the most 1.3, such as at most 1.5, e.g. at most 1.8 such as at most 2, such as at most 2.5, e.g. at most 3, such as at most 3.5, e.g. at 4, such as at most 4.5.
  • the flow-rate is 10 cm/min and the degree of expansion H/HO is at most 1.2, e.g.
  • the flow-rate is 15 cm/min and the degree of expansion H/HO is at most 1.2, e.g. at the most 1.3, such as at most 1.5, e.g. at most 1.8 such as at most 2, such as at most 2.5, e.g. at lmost 3, such as at most 3.5, e.g. at 4, such as at most 4.5.
  • the flow-rate is 15 cm/min and the degree of expansion H/HO is at most 1.2, e.g. at the most 1.3, such as at most 1.5, e.g. at most 1.8 such as at most 2, such as at most 2.5, e.g. at lmost 3, such as at most 3.5, e.g. at 4, such as at most 4.5.
  • the flow-rate is 20 cm/min and the degree of expansion H/HO is at most 1.2, e.g. at the most 1.3, such as at most 1.5, e.g. at most 1.8 such as at most 2, such as at most 2.5, e.g. at lmost 3, such as at most 3.5, e.g. at 4, such as at most 4.5.
  • the linear flow rate of the packed bed column 5 or the expanded bed column may be at least 2 cm/min, more preferably at least 3 cm/min, still more preferably at least 4 cm/min, still more preferably at least 5 cm/min, still more preferably at least 6 cm/min, still more preferably at least 7 cm/min, still more preferably at least 8 cm/min, still more preferably at least 10 cm/min, still more preferably at least 12 cm/min, still more preferably at least 15 cm/min, still more preferably at least 20 cm/min,
  • the linear flow rate is in the range of 1-75 cm/min, such as 2-75 cm/min, e.g. 5-75 cm/min, such as 7-75 cm/min, e.g. 10-75 cm/min, such as 15-75 cm/min, e.g. 20-75 cm/min, such as 30-75 cm/min, e.g. 40-75 cm/min, such as 50-
  • the application of protein solution to the adsorbent column may be performed with a linear flow rate of at least 200 cm/hour, 25 such as at least 300 cm/hour, more preferably at least 400 cm/hour, such as at least 500 or 600 cm/hour, such as at least 900 cm/hour.
  • the column may comprise a high-density adsorbent.
  • high-density adsorbent relates to part of the
  • adsorbent particle are used interchangeably with the term “particle” and relates to the individual single particles which makes up the adsorbent in the column.
  • the preferred shape of a single adsorbent particle is substantially spherical.
  • the overall shape of the particles is, however, normally not extremely critical, thus, the
  • 35 particles can have other types of rounded shapes, e.g. ellipsoid, droplet and bean forms. However, for certain applications (e.g. when the particles are used in a fluidised bed setup), it may be preferred that at least 95% of the particles are substantially spherical.
  • particle diameter and “particle size” are used interchangeable and relates to the diameter of a circle which may be made around the particle and 5 therefore, may be regarded as the diameter of the particle on the widest part of the particle.
  • the density of an adsorbent particle is meant to describe the density of the adsorbent particle in its fully solvated (e.g. hydrated) state as opposed to the density of a dried o adsorbent.
  • the density of the particle may be measured by performing the following procedure: 1 ) Draining a sample of the adsorbent particles by gentle suction on a vacuum glass filter to remove the interstitial water occupying the space between the individual beads. 2) Weighing the drained particle sample to determine the total mass of the particles. 3) Adding the entire amount of drained particle sample to a 5 known amount of water in a measuring cylinder and reading out the increase in total volume obtained by the addition of the drained particles. 4) Calculating the density by dividing the total mass of the drained particles with the volume increase determined under Item 3.
  • the density of the adsorbent particle may be in the range of 1.5 g/ml to 20 g/ml, more preferably in the range from 1.9-20, more preferably in the range from 2.0 g/ml to 20 g/ml, more preferably in the range from 2.1 g/ml to 20 g/ml, more preferably in the range from 2.3 g/ml to 20 g/ml, even more preferably in the range of 2.5 g/ml to 20 g/ml, even more preferably in the range of 2.8 5 g/ml to 20 g/ml, e.g.
  • the density of the EBA adsorbent particle may be significant for the applicable flow rates in relation to the maximal degree of expansion of the adsorbent bed possible inside a 5 typical EBA column (e.g. H/H0 max 3-5) and may be at least 1.3 g/mL, more preferably at least 1.5 g/mL, still more preferably at least 1.8 g/mL, still more preferably at least 1.9 g/mL, even more preferably at least 2.0 g/mL, still more preferably at least 2.1 g/mL, most preferably at least 2.3 g/mL, even more preferably at least 2.5 g/ml, even more preferably at least 2.8 g/ml, even more preferably at least 2.9 g/ml, still more preferably at least 3.0 5 g/ml, still more preferably at least 3.5 g/ml in order to enable a high productivity of the process.
  • 85 % by volume of the individual particles of the adsorbent have a diameter within the range of 5 to 200 micron ( ⁇ m), more o preferably within the range of 10 to 150 micron, still more preferably within the range of 15 to 120 micron, still more preferably within the range of 20 to 100 micron, still more preferably within the range of 20 to 80 micron, still more preferably within the range of 80 to 150 micron, and even still more preferably within the range of 40 to 120 micron.
  • the mean particle diameter of the adsorbent may 5 be 150 micron or less, preferably 120 micron or less, even more preferably 100 micron or less, still more preferably 80 micron or less, still more preferably 70 micron or less, still more preferably 60 micron or less, still more preferably 50 micron or less, still more preferably 40 micron or less.
  • EBA column containing a certain EBA adsorbent at very high flow rates in terms of the physical fluidisation and expansion properties, while the applied high flow rate results in a poor and 0 inefficient adsorption (i.e. a low dynamic capacity) due to the fact that the target molecules to be bound cannot diffuse in and out of the adsorbent particles to match this flow rate (i.e. the mass transfer kinetics is the limiting factor).
  • the mean volume particle diameter is 150 ⁇ m or less.
  • the mean volume particle diameter is below 120 ⁇ m, preferably below 90 ⁇ m.
  • the mean volume particle diameter is preferably below 85 ⁇ m, more preferably below 75 ⁇ m.
  • the mean volume diameter (or volume mean diameter) referred to in the present context relates to the volume mean diameter labelled "D(4,3)" by
  • the particles have a particle diameter in the range of X-Y ⁇ m" it is meant to be understood as at least 90 % of the total volume of particles have a diameter in the range of X-Y ⁇ m, such as at least 95%, e.g. at least 98%, such as at least 99%.
  • the adsorbent density, particle diameter and the mean volume particle diameter as described above may be combined in any way possible to provide the most suitable adsorbent for the isolation of the one or more protein(s) of interest.
  • the density of the adsorbent may be in the range of 1.5 to 10.0, 85% by volume of the individual particles of the adsorbent may have a diameter within the range of 10 to 150 micron, and the mean volume particle diameter may be in the range of 15 to 100 micron.
  • the density of the adsorbent may be in the range of 2.0 to 5.0,
  • 85% by volume of the individual particles of the adsorbent may have a diameter within the range of 20 to 140 micron, and the mean volume particle diameter may be in the range of
  • the density of the adsorbent may be in the range of 2.5 to 3.5, 85% by volume of the individual particles of the adsorbent may have a diameter within the range of 40 to 120 micron, and the mean volume particle diameter may be in the range of 60 to 80 micron.
  • the particle density is at least 1.6 g/mL, more preferably at least 1.9 g/mL.
  • the mean volume particle diameter is less than 90 ⁇ m the density must be at least 1.8 g/mL or more preferable at least 2.0 g/mL.
  • the mean volume particle diameter is less than 75 ⁇ m the density must be at least 2.0 g/mL, more preferable at least 2.3 g/mL and most preferable at least 2.5 g/mL.
  • the adsorbent particle comprises a particle having a mean volume particle diameter of at the most 150 ⁇ m and a particle density of at least 1.5 g/ml; such as a particle density of at least 1.6 g/ml; e.g. a particle density of at least 1.9 g/ml; such as a particle density of at least 2.0 g/ml; e.g. a particle density of at least 2.3 g/ml; such as a particle density of at least 2.5 g/ml; e.g. a particle density of at least 2.8; e.g.
  • the adsorbent particle comprises a particle having a mean volume particle diameter of at the most 120 ⁇ m and a particle density of at least 1.5 g/ml; such as a particle density of at least 1.6 g/ml; e.g. a particle density of at least 1.9 g/ml; such as a particle density of at least 2.0 g/ml; e.g.
  • the adsorbent particle comprises a particle having a mean volume particle diameter of at the most 100 ⁇ m and a particle density of at least 1.5 g/ml; such as a particle density of at least 1.6 g/ml; e.g.
  • a particle density of at least 1.9 g/ml such as a particle density of at least 2.0 g/ml; e.g. a particle density of at least 2.3 g/ml; such as a particle density of at least 2.5 g/ml; e.g. a particle density of at least 2.8; e.g. a particle density of at least 3.0 g/ml; such as a particle density of at least 3.5 g/ml; e.g. a particle density of at least 4.0 g/ml; such as a particle density of at least 4.5 g/ml.
  • the adsorbent particle comprises a particle having a mean volume particle diameter of at the most 90 ⁇ m and a particle density of at least 1.5 g/ml; such as a particle density of at least 1.6 g/ml; e.g. a particle density of at least 1.9 g/ml; such as a particle density of at least 2.0 g/ml; e.g. a particle density of at least 2.3 g/ml; such as a particle density of at least 2.5 g/ml; e.g. a particle density of at least 2.8; e.g. a particle density of at least 3.0 g/ml; such as a particle density of at least 3.5 g/ml; e.g.
  • the adsorbent particle comprises a particle having a mean volume particle diameter of at the most 75 ⁇ m and a 5 particle density of at least 1.5 g/ml; such as a particle density of at least 1.6 g/ml; e.g. a particle density of at least 1.9 g/ml; such as a particle density of at least 2.0 g/ml; e.g. a particle density of at least 2.3 g/ml; such as a particle density of at least 2.5 g/ml; e.g. a particle density of at least 2.8; e.g.
  • the adsorbent particle comprises a particle having a mean volume particle diameter of at the most 50 ⁇ m and a particle density of at least 1.5 g/ml; such as a particle density of at least 1.6 g/ml; e.g. a particle density of at least 1.9 g/ml; such as a particle density of at least 2.0 g/ml; e.g.
  • the adsorbent particle comprises a particle having a mean volume particle diameter of at the most 40 ⁇ m and a particle density of at least 1.5 g/ml; such as a particle density of at least 1.6 g/ml; e.g.
  • a particle density of at least 0 1.9 g/ml such as a particle density of at least 2.0 g/ml; e.g. a particle density of at least 2.3 g/ml; such as a particle density of at least 2.5 g/ml; e.g. a particle density of at least 2.8; e.g. a particle density of at least 3.0 g/ml; such as a particle density of at least 3.5 g/ml; e.g. a particle density of at least 4.0 g/ml; such as a particle density of at least 4.5 g/ml. 5
  • the adsorbent particle used according to the invention must be at least partly permeable to the biomolecular substance to be isolated in order to ensure a significant binding capacity in contrast to impermeable particles that can only bind the target molecule on its surface resulting in relatively low binding capacity.
  • the adsorbent particle may be of an 0 array of different structures, compositions and shapes.
  • the high density of the adsorbent particle is, to a great extent, achieved by inclusion in a porous polymer phase, of a certain proportion of a dense non-porous core material.
  • the non-porous core preferably has a density of at least 4.0 g/mL, such as at least 5.0 g/mL, 5 e.g. at least 8.0 g/mL, such as at least 10 g/mL, e.g. at least 15 g/mL.
  • the non- porous core material has a density in the range of about 4.0-25 g/ml, such as about 4.0- 20 g/ml, e.g. about 4.0-15 g/mL, such as 12-19 g/ml, e.g. 14-18 g/ml, such as about 6.0- 15.0 g/mL, e.g. about 6.0-10 g/ml.
  • high density adsorbent particles are based on particles made out of a porous high density material, such as zirconium oxide, in which pores ligands for adsorption may be immobilised either directly to the high density material or to porous polymer networks filled into the pores of the high density material, see e.g. US 6,036,861 and WO 99/51316.
  • a porous high density material such as zirconium oxide
  • the adsorbent particle employed according to the invention has a high accessible protein binding volume.
  • particle accessible protein binding volume relates to the relative pore volume of any specific particle type and is expressed as volume percent relative to the volume of the entire bead (i.e. the volume occupied by pores/the total volume of the bead x 100 %). Thus if too much of the particle volume is 0 occupied by the high density material only low column productivities can be achieved.
  • the particle accessible protein binding volume of the adsorbent may be at least 20 %, more preferably at least 30 %, still more preferably at least 40 %, still more preferably at least 50 %, still more preferably at least 55 %, still 5 more preferably at least 60 %, still more preferably at least 65 %, still more preferably at least 70 %, still more preferably at least 75 %, still more preferably at least 80 %, still more preferably at least 85 % and still more preferably at least 90 %.
  • the adsorbent may have a dynamic binding 0 capacity at 10 % break-through for said at least one specific protein of at least 5 g per litre sedimented adsorbent, more preferably at least 10 g per litre, even more preferably at least 15 g per litre, still more preferably at least 20 g per litre, still more preferably at least
  • the ratio between the adsorbent particle present in the column and the protein solution may be optimized in order to retain a high capacity of the adsorbent and to obtain a high purity of the protein or proteins to be isolated.
  • the adsorbent present in the column relative to the protein solution to be loaded on to the column are provided at a ratio of at least 1 :100, such as at least 1 :50, e.g. at least 1 :30, such as at least 1 :15, e.g. 1 :10, such as 1 :5, such as 1 :1 , such as 1 :0,5 measured on a volume/volume basis.
  • the adsorbent particles may be constituted by a number of chemically derivatised porous materials having the necessary density, diameter and/or binding capacity to operate at the given flow rates per se.
  • the particles are either of the conglomerate type, as described in WO 92/00799, having at least two non-porous cores surrounded by a porous material, or of the pellicular type having a single non-porous core surrounded by a porous material.
  • the term "conglomerate type” relates to a particle of a particulate material, which comprises beads of core material of different types and sizes, held together by the polymeric base matrix, e.g. an core particle consisting of two or more high density particles held together by a surrounding polymeric base matrix (e.g. agarose).
  • pellicular type relates to a composite of particles, wherein each particle consists of only one high density core material coated with a layer of the porous polymeric base matrix, e.g. a high density stainless steel bead coated with agarose.
  • At least one high density non-porous core relates to either a pellicular core, comprising a single high density non-porous particle or it relates to a conglomerate core comprising more that one high density non-porous particle.
  • the adsorbent particle may comprise a high density non-porous core with a porous material surrounding the core, and said porous material optionally comprising a ligand at its outer surface.
  • core relates to the non-porous core particle or core particles which are present inside the adsorbent particle.
  • the core particle or core particles may be incidental distributed within the porous material and is not limited to be located in the centre of the adsorbent particle.
  • the non-porous core constitutes typically of at most 70% of the total volume of the adsorbent particle, such as at most 60%, preferably at most 50%, preferably at most 40%, preferably at most 30%, preferably at most 20%, preferably at most 15%, preferably at most 10% preferably at most 5%.
  • suitable non-porous core materials are inorganic compounds, metals, heavy metals, elementary non-metals, metal oxides, non metal oxides, metal salts and metal alloys, etc. as long as the density criteria above are fulfilled.
  • core materials are metal silicates metal borosilicates; ceramics including titanium diboride, titanium carbide, zirconium diboride, zirconium carbide, tungsten carbide, silicon carbide, aluminum nitride, silicon nitride, titanium nitride, yttrium oxide, silicon metal powder, and molybdenum disilide; metal oxides and sulfides, including magnesium, aluminum, titanium, vanadium, chromium, zirconium, hafnium, manganese, iron, cobalt, nickel, copper and silver oxide; non-metal oxides; metal salts, including barium sulfate; metallic elements, including tungsten, zirconium, titanium, hafnium, vanadium, chrom
  • the porous material is a polymeric base matrix used as a means for covering and keeping multiple (or a single) core materials together and within the adsorbent particle and as a means for binding the adsorbing ligand.
  • the polymeric base matrix may be sought among certain types of natural or synthetic organic polymers, typically selected from i) natural and synthetic polysaccharides and other carbohydrate based polymers, including agar, alginate, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, agaroses, celluloses, pectins, mucins, dextrans, starches, heparins, chitosans, hydroxy starches, hydroxypropyl starches, carboxymethyl starches, hydroxyethyl celluloses, hydroxypropyl celluloses, and carboxymethyl celluloses; ii) synthetic organic polymers and monomers resulting in polymers, including acrylic polymers, polyamides, polyimides, polyesters, polyethers, polymeric vinyl compounds, polyalkenes, and substituted derivatives thereof, as well as copolymers comprising more than one such polymer functional
  • a preferred group of polymeric base matrices are polysaccharides such as agarose.
  • the adsorbent particle has a mean volume particle diameter of at the most 150 ⁇ m, typically a mean volume particle diameter in the range of about 40 ⁇ m to 150 ⁇ m.
  • the adsorbent particle typically has a mean volume particle diameter of at most 120 ⁇ m, particularly at most 100 ⁇ m, more preferably at most 90 ⁇ m, 80 ⁇ m or 75 ⁇ m, more preferably at 70 ⁇ m and most preferably at most 60 ⁇ m.
  • adsorbent is able to bind a high amount of the biomolecular substance per volume unit of the adsorbent.
  • adsorbents having a polymeric phase i.e. the permeable polymeric network where a ligand is positioned and whereto the actual adsorption is taking place
  • adsorbents having a polymeric phase which constitutes at least 50% of the adsorbent particle volume, preferably at least 70%, more preferably at least 80% and most preferably at least 90% of the volume of the adsorbent particles.
  • the isolation process of the one or more protein(s) may be provided and facilitated by attaching a suitable ligand to the adsorbent.
  • the adsorbent comprises a functionalised matrix polymer carrying a plurality of ligands comprising covalently attached functional groups.
  • the ligand comprises an aromatic or heteroaromatic ring-system and one or more acidic groups.
  • functionalised matrix polymer relates to the anchoring site for the ligand promoting the desired protein adsorption characteristics.
  • the matrix polymer may form the backbone or skeleton 5 defining the physical shape of the adsorbent particle or it may be a polymer that is occupying the pores of another material that serve as the particle backbone or skeleton.
  • the functionalised matrix polymer is a synthetic or natural organic polymer, such as a polysaccharide (e.g. poly-acrylic polymers, agarose or cellulose), or it may be an inorganic polymer, such as silica.
  • the matrix o polymer itself may constitute the protein adsorption site in which case it in not necessary to immobilise further ligands onto the polymer.
  • the adsorbent comprises a functionalised matrix polymer carrying a plurality of covalently attached functional groups, said groups 5 having the general formula:
  • M designates the adsorbent polymer
  • SP1 designates an optional spacer 0 optionally substituted with -A-SP2-ACID, -A, or -ACID
  • X designates — O— , -S-, —
  • AIk may be absent, -A-SP2-ACID, -A, -ACID or Ci -4 alkyl, where Ci -4 alkyl may be optionally substituted with -A-SP2-ACID, -A, or -ACID;
  • A designates an optionally substituted aromatic or heteroaromatic moiety;
  • SP2 designates an optional spacer; and
  • ACID designates one or more acidic groups; wherein at least one of SP1 or 5 AIk is substituted with -A-SP2-ACID or -A, and at least one of SP1 or AIk comprise -ACID and wherein at least one of SP1 or AIk is present. If AIk is absent, X will also be absent.
  • the adsorbent may be coupled with a ligand carrying a positive charge at pH value at pH 10 or lower, such as pH 9 or lower, e.g. pH 8 0 or lower, such as pH 7 or lower, e.g. pH 6 or lower, such as pH 5 or lower, e.g. pH 4 or lower.
  • the functional groups should not be too large in size and complexity in order to obtain a high binding capacity and a high chemical stability of the 5 adsorbent.
  • a larger size in terms of molecular weight and number of ring-systems present in the functional group in many instances only increase the cost of the adsorbent without giving the benefit of a higher binding capacity in terms of the amount of protein that can be bound per litre adsorbent.
  • the molar concentration of the covalently attached functional group achievable on the adsorbent may be lower if a 5 large molecular size of the functional group is employed (presumably due to steric hindrance).
  • the covalently attached functional groups comprise a maximum of three mono- or bicyclic aromatic or heteroaromatic ring-systems o for each functional group attached to the matrix polymer, more preferably a maximum of two mono- or bicyclic aromatic or heteroaromatic ring-systems and even more preferably a maximum of one mono- or bicyclic aromatic or heteroaromatic ring-systems for each functional group attached to the matrix polymer.
  • the covalently attached functional groups comprise a maximum of three acidic 5 groups, preferably a maximum of 2 acidic groups and most preferably a maximum of one acidic group attached to each aromatic or heteroaromatic ring-system present in the covalently attached functional groups.
  • the one or more acidic groups are chosen from 0 the group of carboxylic acids, sulfonic acids, phosphonic acids, boronic acids and combinations hereof.
  • the ligand may be derived from a diethylaminoethyl group, a polyalkylene imine, an alkyl-amine, an alkyl-diamine or a 5 polyallylamine.
  • alkyl-amine or alkyl-diamine having a chain-length of 3-14 atoms and 1-5 functional amine groups may be suitable. Atoms to form part of the chain may involve C (carbon), N (nitrogen), O (oxygen) and/or S (sulfur).
  • the adsorbent may comprise a ligand, 0 having both aromatic groups and amino groups such as an aromatic amine or an aromatic diamine.
  • the aromatic diamine is 1 ,4-xylene-diamine or isomers of 1 ,4-xylene- diamine.
  • the adsorbent may be coupled with a ligand 5 having an acid group, an aromatic or heteroaromatic moiety, a bicyclic substituted heteroaromatic group or any combination hereof, such as a ligand having an acid group and an aromatic or heteroaromatic moiety, a ligand having an acid group and a bicyclic substituted heteroaromatic group or an aromatic or heteroaromatic moiety and a bicyclic substituted heteroaromatic group.
  • the ligand comprises a bicyclic substituted heteroaromatic group which may be derived from compounds selected from the group consisting of benzimidazoles, benzothiazoles, and benzoxazoles.
  • the ligand may be an aromatic or heteroaromatic acid selected from the group consisting of carboxylic acids, sulfonic acids, phosphonic acids, and boronic acids.
  • the ligand may be selected from the group consisting of 2-mercaptobenzoic acid, 2-mercaptonicotinic acid, 2-aminobenzoic acid, 3-aminobenzoic acid, and 4-aminobenzoic acid, 4-hydroxyphenyl-mercapto-acetic acid, 4-hydroxyphenyl-mercapto-propionic acid, 4-hydroxyphenyl-mercapto-butanoic acid, 2,3 - dihydroxy-benzoic acid, 2,4 dihydroxy-benzoic acid, 2,5 di-hydroxy-benzoic acid, 2,6 dihydroxy-benzoic acid, 3,4-dihydroxy-benzoic acid, 3,5-dihydroxy-benzoic acid, mercaptobenzimidazole sulfonic acid, orthanilic acid, metanilic acid, s
  • the ligand may be an N-benzoyl amino acid or an N-benzoyl amino acid comprising thiol or mercapto groups.
  • the ligand may be coupled to the adsorbent through a thio-ether linkage, an amine linkage, or an oxygen-ether linkage.
  • the optimal concentration of the covalently attached functional groups (the ligands) on the polymeric adsorbent backbone (also frequently referred to as the density of functional groups or the ligand concentration) will depend on the detailed structure of the functional group and the type of adsorbent material used to prepare the adsorbent. In order to ensure an optimal adsorption strength and productivity of the adsorbent it has been found that the ligand concentration on the adsorbent may be significant.
  • the adsorbent carries ligands for adsorption of the biomolecular substances in a concentration of at least 20 mM, such as at least 30 mM or at least 40 mM, preferably at least 50 mM and most preferably at least 60 mM.
  • the adsorbent has a concentration of covalently attached functional groups in the range of 5 - 500 millimole per liter adsorbent in its sedimented (packed) bed state, more preferably in the range of 10-250 millimole per liter, still more preferably in the range of 10-125 millimole per liter, still more preferably in the range of 15 - 100 millimole per liter, still more preferably in the range of 20-80 millimole per liter still more preferably in the range of 25-75 millimole per liter still more preferably in the range of 30-60 millimole per liter.
  • the covalently attached functional groups may be attached to the adsorbent by any type of covalent bond known per se to be applicable for this purpose, either by a direct chemical reaction between the ligand and the adsorbent or by a preceding activation of the adsorbent or of the ligand with a suitable reagent known per se making it possible to link the polymeric matrix backbone and the functional group.
  • activating reagents examples include epichlorohydrin, epibromohydrin, allyl glycidylether; bis-epoxides such as butanedioldiglycidylether; halogen-substituted aliphatic compounds such as di-chloro-propanol, carbonyldiimidazole; aldehydes such as glutaric dialdehyde; quinones; periodates such as sodium-meta- periodate; carbodiimides; sulfonyl chlorides such as tosyl chlorides and tresyl chlorides; N-hydroxy succinimides; 2-fluoro-1-methylpyridinium toluene-4-sulfonates; oxazolones; maleimides; pyridyl disulfides; and hydrazides.
  • the activating reagents leaving a spacer group SP1 different from a single bond e.g. epichlorohydrin, epibromohydrin, allyl-glycidylether; bis-epoxides; halogen-substituted aliphatic compounds; aldehydes; quinones; cyanogen bromide; chloro-triazines; oxazolones; maleimides; pyridyl disulfides; and hydrazides, are preferred.
  • activating reagents are epoxy-compounds such as epichlorohydrin, allyl-glycidylether and butanedioldiglycidylether and polyglycidylethers such as glycerol polyglycidylether.
  • the activating reagent may be based on triazine derived reagents e.g chloro-triazines such as cyanuric chloride.
  • the spacer SP1 may be considered as being part of the activating reagent, which forms the link between the matrix polymer and the functional group.
  • the spacer SP1 corresponds to the activating reagents and the coupling reactions involved.
  • the activating reagent forms an activated form of the matrix polymer or of the functional group reagent. After coupling no parts of the activating reagent is left between the functional group and the matrix polymer, and, thus, SP1 is simply a single bond.
  • the spacer SP1 may be an integral part of the functional group effecting the binding characteristics, i.e. the functional group, and this will be especially significant if the spacer SP1 comprises functionally active sites or substituents such as thiols, amines, acidic groups, sulfone groups, nitro groups, hydroxy groups, nitrile groups or other groups able to interact through hydrogen bonding, electrostatic bonding or repulsion, charge 0 transfer or the like.
  • functionally active sites or substituents such as thiols, amines, acidic groups, sulfone groups, nitro groups, hydroxy groups, nitrile groups or other groups able to interact through hydrogen bonding, electrostatic bonding or repulsion, charge 0 transfer or the like.
  • the spacer SP1 may comprise an aromatic or heteroaromatic ring, which plays a significant role for the binding characteristics of the adsorbent. This would for example be the case if quinones or chlorotriazines where used as activation agents for 5 the adsorbent or the functional group.
  • the spacer SP1 may be a single bond or a biradical derived from an activating reagent selected from epichlorohydrin, allyl-glycidylether, allylbromide, bis- epoxides such as butanedioldiglycidylether, halogen-substituted aliphatic compounds 0 such as 1 ,3-dichloropropan-2-ol, aldehydes such as glutaric dialdehyde, quinones, cyanogen bromide, chloro-triazines such as cyanuric chloride, 2-fluoro-1-methylpyridinium toluene-4-sulfonates, maleimides, oxazolones, and hydrazides.
  • an activating reagent selected from epichlorohydrin, allyl-glycidylether, allylbromide, bis- epoxides such as butanedioldiglycidyl
  • the spacer SP1 may be a short chain aliphatic 5 biradical, e.g. having the formula: -CH 2 -CH(OH)-CH 2 - (derived from epichlorohydrin), - (CH 2 )S-O-CH 2 -CH(OH)-CH 2 - (derived from allyl-glycidylether) or -CH 2 -CH(OH)-CH 2 -O- (CH 2 ) 4 -O-CH 2 -CH(OH)-CH 2 - (derived from butane-dioldiglycidylether; or a single bond.
  • the adsorbents typically comprises a ligand comprising aromatic or heteroaromatic groups (radicals) selected from the groups comprising i) ligands comprising the following types as functional groups: benzoic acids such as 2-aminobenzoic acids, 3-aminobenzoic acids, 4-aminobenzoic acids, 2- mercaptobenzoic acids, 4-amino-2-chlorobenzoic acid, 2-amino-5-chlorobenzoic acid, 2- amino-4-chlorobenzoic acid, 4-aminosalicylic acids, 5-aminosalicylic acids, 3,4- diaminobenzoic acids, 3,5-diaminobenzoic acid, 5-aminoisophthalic acid, 4-aminophthalic acid; cinnamic acids such as hydroxy-cinnamic acids; nicotinic acids such as 2- mercaptonicotinic acids; naphthoic acids such as 2-hydroxy-1 -naphtho
  • M is agarose
  • SP1 is derived from vinyl sulfone
  • L is 4-aminobenzoic acid
  • amino- benzoic acids like 2-amino-benzoic acid, 2-mercapto-benzoic acid, 3-aminobenzoic acid
  • 4-aminobenzoic acid 4-amino-2-chlorobenzoic acid, 2-amino-5-chlorobenzoic acid, 2- amino-4-chlorobenzoic acid
  • 4-aminosalicylic acids 5-aminosalicylic acids, 3,4- diaminobenzoic acids, 3,5-diaminobenzoic acid, 5-5-aminoisophthalic acid, 4- aminophthalic acid.
  • the coupling using divinyl sulphone may not be suitable because the divinyl sulphone coupling is unstabil when contacted with an alkaline and as alkalines are presently the most suitable and used cleaning agents, adsorbents coupled with divinyl sulphone are not being considered industrial relevant; ii) ligands comprising 2- hydroxy-cinnamic acids, 3-hydroxy-cinnamic acid and 4-hydroxy-cinnamic acid iii) ligands comprising a carboxylic acid and an amino group as substituents such as 2-amino- nicotinic acid, 2-mercapto-nicotinic acid, 6-amino-nicotinic acid and 2-amino-4- hydroxypyrimidine-carboxylic acid iv) ligand comprising radicals derived from a benzene ring fused with a heteroaromatic ring system, e.g.
  • a ligand selected from benzimidazoles such as 2-mercapto-benzimidazol and 2-mercapto-5-nitro-benzimidazol; benzothiazols such as 2-amino-6-nitrobenzothiazol, 2-mercaptobenzothiazol and 2-mercapto-6- ethoxybenzothiazol; benzoxazols such as 2-mercaptobenzoxazol;and v) ligands chosen 5 from the group of thiophenols such as thiophenol and 2-aminothiophenol.
  • the adsorbents typically have a dynamic binding capacity of at least 10 g of biomolecular substance per litre, more preferably at least 20 g per litre, still more preferable at least 30 1 o g per litre when tested according to the process conditions used in the relevant application.
  • the binding capacity of the adsorbent may be determined in terms of its binding capacity to bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the binding capacity is typically such that at least 10g/L of BSA binds according to test Method A.
  • Method A is a method used for determination of the bovine albumin binding capacity of selected adsorbents consisting of the following process:
  • Bovine serum albumin solution pH 4.0 (BSA pH 4.0): Purified bovine serum albumin (A 20 7906, Sigma, USA) is dissolved to a final concentration of 2 mg/ml in 20 mM sodium citrate pH 4.0. Adsorbents are washed with 50 volumes of 20 mM sodium citrate pH 4.0 and drained on a suction filter.
  • a sample of 1.0 ml suction drained adsorbent is placed in a 50 ml test tube followed by 25 the addition of 30 ml of BSA, pH 4.0.
  • test tube is then closed with a stopper and the suspension incubated on a roller mixer for 2 hours at room temperature (20-25 0 C).
  • the test tube is then centrifuged for 5 min. at 2000 RPM in order to sediment the adsorbent completely.
  • the supernatant is then 30 isolated from the adsorbent by pipetting into a separate test tube, avoiding the carry-over of any adsorbent particles and filtered through a small non-adsorbing 0.2 ⁇ m filtre (Millipore, USA). Following this a determination of the concentration of non-bound BSA in the supernatant is performed by measuring the optical density (OD) at 280 nm on a spectrophotometer.
  • OD optical density
  • mg BSA bound per ml suction drained adsorbent (1-(OD of test supernatant/OD of BSA starting solution)) x 60 mg BSA/ml adsorbent.
  • the protein solution comprising one or more protein(s) of interest may be adjusted to having a preset pH and a preset ionic strength or conductivity.
  • preset relates to the adjustment of the pH, ionic strength or conductivity, respectively, to a specific and predetermined value for the purpose of selecting the ability of the adsorbent for binding the one or more protein(s) of interest and thereby increasing the efficiency of the adsorbent for protein(s) isolation.
  • the preset pH is in the range of the lowest pH for allowing reversible change back to the N-form to pH 10, preferably pH 3.0 to pH 10.0, preferably in the range of pH 4 to pH 9, more preferably in the range of pH 4 to pH 8, even more preferably in the range of pH 4 to pH 7, still more preferably in the range of pH 4 to pH 6, still more preferably in the range of pH 4.5 to pH 5.5 or in the range of pH 4 to pH 10, preferably in the range of pH 5 to pH 9, more preferably in the range of pH 6 to pH 9, even more preferably in the range of pH 6 to pH 8.
  • the preset ionic strength is in the range of 0.0001 to 12.0, preferably in the range of 0.0001 to 5, more preferably in the range of
  • 0.0001 to 1 even more preferably in the range of 0.0001 to 0.1 , still more preferably in the range of 0.0001 to 0.075, still more preferably in the range of 0.01 to 0.05 or in the range of 0.1 to 12.0, preferably in the range of 0.5 to 12, more preferably in the range of 1 to 12, even more preferably in the range of 1.5 to 12, still more preferably in the range of 2 to 12, still more preferably in the range of 4 to 12.
  • the preset conductivity is in the range of 0.01 to 1000 mS/cm, preferably in the range of 0.01 to 200 mS/cm, more preferably in the range of 0.05 to 100 mS/cm, more preferably in the range of 0.1 to 50 mS/cm, more preferably in the range of 0.5 to 20 mS/cm, more preferably in the range of 1.0 to 10 mS/cm, still more preferably in the range of 1.0 to 5 mS/cm or in the range of 10 to 1000 mS/cm, preferably in the range of 100 to 1000 mS/cm, more preferably in the range of 200 to 1000 mS/cm, more preferably in the range of 300 to 1000 mS/cm, more preferably in the range of 400 to 1000 mS/cm, more preferably in the range of 500 to 1000 mS/cm, 5 still more preferably in the range of 600 to 1000 mS/cm, more
  • the ionic strength and conductivity of the protein solutions according to the present o invention are related entities in that both entities are functions of the concentration of ions in the solution. There is, however, no direct theoretical correspondence between them.
  • the one or more protein(s) may be eluted with one or more buffer(s).
  • adsorbent may be washed with a washing buffer before being subjected to the elution buffer.
  • the adsorbent is washed with a washing buffer to wash out non-bound material before eluting one or more protein(s) from the adsorbent.
  • albumin in particular rHSA and/or rBSA, is less capable of binding compounds causing (mis)coloration of solution such as heme/hemin when adapting the F- and/or E-form. It has been found that albumin in the F- and/or E- form is fully capable of binding to the adsorbent/ligand in the fractionation/separation 5 method.
  • the method of fractionation/separation the albumin preferably comprises a step of bringing the albumin predominantly on F-form and/or E-form and allowing compounds causing (mis)coloration to be released and/or less bound and/or more weakly adsorbed and optionally separated from the albumin.
  • the albumin is 5 brought predominantly on the F- and/or E-form while attached to the ligand/adsorbent and the released and/or less bound and/or more weakly adsorbed (mis)coloring compounds is separated from the albumin by one or more or several washing steps.
  • At least 75 mole % such as at least 90 mole %, such as at least 95 mole %, such as 99 mole % such as 100 mole % of the albumin is brough on F-form o and/or E-form.
  • albumin with less bound (mis)coloring compounds giving color to the solution can be liberated from the ligand/adsorbent.
  • the albumin is brought on the F- and/or E-form releasing the (mis)coloring compounds 5 before loading the protein solution to column so that only the albumin will bind to the ligand/adsorbent separating it from the (mis)coloring compounds which will continue to flow through the column. Further separation may be achieved by employing one or more or several washing steps.
  • Bringing the albumin on F-form may be achieved by choosing a pH of the solution surrounding the albumin attached to the ligand/adsorbent between 3 to 5, particularly between 3.5 and 4.5, more particularly around pH 4.5.
  • Bringing the albumin on E-form may be achieved by choosing a pH of the solution 5 surrounding the albumin attached to the ligand/adsorbent below 3, but above the pH limit for allowing reversible change back to the N-form.
  • the albumin is brought on the F or E-form, e.g. by choosing a pH from above the lowest allowing reversible change back to the N-form to 5, 0 preferably from pH 3 to 5, more preferably from pH 3.5 to 4.5, most preferably around pH 4 or 4.5, during the step of contact with an adsorbent, in particular while being bound to the ligand/adsorbent, and during the an optional washing step, while choosing another or the same form and/or pH for the step of eluating the albumin from the ligand/adsorbent.
  • the pH for the step of eluating the albumin from the ligand/adsorbent is kept on or above pH 5, particularly from pH 6-8, more particularly around pH 7.
  • the invention provides a process comprising a) providing a protein solution comprising recombinant human serum albumin (rHSA) obtained from a fungal cell, b) optionally subjecting the protein solution to stabilisation of proteins by addition of a compound selected from acids, in particular fatty acids, and their salts, particularly octanoic acid and salts thereof such as sodium caprylate and N-acetyl-L- tryptophan, c) optionally inactivating hydrolytic enzymes in the protein solution by heating the protein solution, by adding organic solvent or by adding a competitive or noncompetitive enzyme inhibitor to the protein solution, d) optionally removing cells and/or debris from the fungal cells, e) adjusting pH of the protein solution bringing at least 95 mole % of the rHSA on F- or E-form or a combination thereof and optionally selecting a preset ionic strength and/or conductivity; f) contacting said protein solution with an adsorbent, wherein the adsorbent
  • the protein solution may be subjected to at least one virus elimination treatment.
  • at least one virus elimination treatment may be performed prior to contacting the protein solution with the adsorbent.
  • the virus elimination treatment may involve the addition of an organic solvent such as tri- n-butylphosphate and/or a detergent such as non-ionic or anionic surfactants such as tween and triton, to the protein solution.
  • virus elimination steps may be performed and preferably one virus elimination treatment may be performed prior to contacting the protein solution with the adsorbent.
  • virus elimination steps may be performed and preferably one virus elimination treatment is performed prior to contacting the protein solution with the adsorbent and, during the adsorption step, any substances, such as detergents and/or organic solvents added to the protein solution remain unbound to the adsorbent and is washed out of the column prior to elution of the protein to be isolated.
  • one virus elimination treatment is performed prior to contacting the protein solution with the adsorbent and, during the adsorption step, any substances, such as detergents and/or organic solvents added to the protein solution remain unbound to the adsorbent and is washed out of the column prior to elution of the protein to be isolated.
  • a process for eliminating viruses in biological fluids may be preformed by a treatment with organic solvents and/or detergents; especially treatment with tri(n-butyl)phosphate (TNBP) and non-ionic detergents such as Tween 80 or Triton X-100.
  • TNBP tri(n-butyl)phosphate
  • This method may result in excellent recovery of labile proteins, e.g., coagulation factor VIII and IX, while achieving a high level of virus kill, e.g., the killing of ⁇ 10 6 to ⁇ 10 8 ID, of enveloped viruses; however, little elimination of non-enveloped viruses.
  • TNBP tri(n-butyl)phosphate
  • Tween 80 or Triton X-100 Triton X-100.
  • viruses elimination commonly applied, and applicable in the present invention, to biological fluids to be used in a transfusion treatment may also involve a treatment with heat at temperatures at 60°C or more, or treatment with UVC together alone or together with B-propiolactone (B-PL).
  • B-PL B-propiolactone
  • the method further involves the step of subjecting the adsorbent to an elution buffer (a first elution buffer) to elute at least one of said one or more protein(s).
  • the ligand coupled to the adsorbent may comprise a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an 5 aromatic or heteroaromatic ring-system and one or more acidic groups being negatively charged above pH 4.0.
  • the adsorbent may be subjected to a washing buffer.
  • the one or more protein(s) to be isolated from the protein solution may be washed out of the adsorbent with the washing buffer. This washing may be performed before subjecting the adsorbent to an elution buffer as described above.
  • alpha-1 -proteinase inhibitor may be washed out as a non-bound protein with the washing buffer.
  • the process conditions, the adsorbent and the 5 ligand could easily be changed by the person skilled in the art in such a way that one or more other human serum or plasma protein(s) (other than alpha-1 -proteinase inhibitor) may be washed out with the washing buffer and alpha-1 -proteinase inhibitor may be bound.
  • washing and/or elution may be performed with a washing buffer and/or an elution buffer having a higher pH and/or a higher ionic strength than the preset pH and preset ionic strength of the protein solution.
  • the washing buffer and/or the elution buffer may comprise one 5 or more compounds having a hydrophobic as well as a negatively charged group within the same molecule e.g. negatively charged detergent such as octyl sulfate, bromphenol blue, octane sulfonate, sodium laurylsarcosinate, hexane sulfonate, sodium dodecyl sulfate, sodium caprylate.
  • negatively charged detergent such as octyl sulfate, bromphenol blue, octane sulfonate, sodium laurylsarcosinate, hexane sulfonate, sodium dodecyl sulfate, sodium caprylate.
  • the process according to the present invention further comprises the step of eluting with one or more additional elution buffer(s) to elute remaining protein(s).
  • additional elution buffer(s) relates to the buffer(s) o subsequently used for the elution of one or more protein(s), which remains bound to the adsorbent after the elution with the first elution buffer.
  • the term "remaining protein(s)” relates to the one or more protein(s) which remains bound to the adsorbent after being subjected to a first elution buffer and 5 which protein(s) may subsequently be eluted by the addition of an additional elution buffer.
  • the adsorbent may be washed with an additional washing buffer between each elution step.
  • protein fraction relates to the collections obtained from the adsorbent wherein the one or more protein(s) to be isolated may be located. This protein fraction may be subjected to further downstream processing for further isolation of the one or more protein(s) present.
  • the further downstream processing may involve 5 operations like filtration, centrifugation, sedimentation, microfiltration, precipitation and chromatography.
  • chromatography involves ion exchange chromatography, gel filtration, affinity chromatography, hydrophobic interaction chromatography and reversed phase chromatography, where the protein(s) may be bound to a second adsorbent in subsequent down stream processing.
  • the further downstream processing may comprise the adsorption of the protein in the protein fraction(s), such as alpha-1- proteinase inhibitor, to a positively charged ion exchanger
  • the further downstream processing may comprise the adsorption of the protein in the protein fraction(s), such as alpha-1 - proteinase inhibitor, to an alkyl-amine such as an alkyl-diamine such as diamino-hexane, diamino-heptane, diamino-octane, diamino-nonane, diamino-decane and isomers or derivatives hereof.
  • alpha-1 -proteinase inhibitor is unbound in the first adsorption step and the further downstream processing comprise the adsorption of the alpha-1 -proteinase to an alkyl-amine such as an alkyl-diamine such as diamino- hexane, diamino-heptane, diamino-octane, diamino-nonane, diamino-decane or isomers or derivatives hereof
  • the further downstream processing may comprise the adsorption of the protein in the protein fraction, such as alpha-1 -proteinase inhibitor, to an adsorbent in the presence of a lyotropic salt such as, but not limited to ammonium sulfate, potassium sulfate, sodium sulfate, ammonium phosphate, potassium phosphate, and sodium citrate.
  • a lyotropic salt such as, but not limited to ammonium sulfate, potassium sulfate, sodium sulfate, ammonium phosphate, potassium phosphate, and sodium citrate.
  • concentration of lyotropic salt in the solution comprising alpha-1 -proteinase inhibitor is at least 0.1 M, at least 0.25 M, such as at least 0.5 M, at least 0.75 M, at least 1 M, at least 1.25 M, at least 1.5 M, at least 1.75 M or at least 2 M.
  • the further downstream processing comprise the adsorption of the protein in the protein fraction, such as alpha-1 -proteinase inhibitor, to an adsorbent in the presence or absence of a lyotropic salt wherein the adsorbent comprise a hydrophobic ligand such as an uncharged ligand comprising long, optionally substituted, alkyl chains (e.g. butyl-, hexyl-, octyl-, decyl-, dodecyl- derived groups) and/or aromatic and heteroaromatic structures (e.g. phenyl-, naphthyl-, benzimidazole derived groups).
  • a hydrophobic ligand such as an uncharged ligand comprising long, optionally substituted, alkyl chains (e.g. butyl-, hexyl-, octyl-, decyl-, dodecyl- derived groups) and/or aromatic and heteroaromatic structures (e
  • ligands such as those described in US 6,610,630 wherein there is disclosed chromatography adsorbents utilizing mercapto- heterocyclic ligands, hereby incorporated by reference.
  • Further preferred ligands are mixed mode ligands comprising a positive charge at, and below, about pH 4 such as positive charged ligands comprising long, optionally substituted, alkyl chains (e.g. butyl-, hexyl-, octyl-, decyl-, dodecyl- derived groups) and/or aromatic and heteroaromatic structures (e.g. phenyl-, naphthyl-, benzimidazole derived groups).
  • Non-limiting examples of such ligands are butylamine, hexylamine-, octylamine-, benzylamine and phenyl- butylamine-.
  • a process for the large-scale isolation of one or more protein(s) according to the present invention comprises the steps of:
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups;
  • a process for the large-scale isolation of one or more protein(s) according to the present invention comprises the steps of:
  • adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material and/or said particle having a particle density of at least 1.5 g/ml and a mean volume particle size of at the most
  • 2 or more proteins from the protein solution are isolated by the means of a cascade of 2 or more adsorbents, such as 3 adsorbents, e.g. 4 adsorbents, such as 5 adsorbents, e.g. 6 adsorbents, such as 7 adsorbents, e.g. 8 adsorbents, such as 9 adsorbents, e.g. 10 adsorbents.
  • 3 adsorbents e.g. 4 adsorbents, such as 5 adsorbents, e.g. 6 adsorbents, such as 7 adsorbents, e.g. 8 adsorbents, such as 9 adsorbents, e.g. 10 adsorbents.
  • adsorbents such as 3 adsorbents, e.g. 4 adsorbents, such as 5 adsorbents,
  • the first adsorbent is capable of capturing one or more blood protein(s), serum protein(s) or plasma protein(s);
  • the second adsorbent is capable of capturing one or more blood protein(s), serum protein(s) or plasma protein(s) different for the one or more blood protein(s), serum protein(s) or plasma protein(s) capable of being captured to the first adsorbent;
  • the third adsorbent is capable of capturing one or more blood protein(s), serum protein(s) or plasma protein(s) different for the one or more blood protein(s), serum protein(s) or plasma protein(s) capable of being captured to the first adsorbent or second adsorbent;
  • one or more coagulation factor(s) or anticoagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and Protein S, is/are bound to a first adsorbent.
  • at least 2 of the coagulation or anti-coagulation factors binds to the adsorbent, such as at least 3 of the coagulation or anti-coagulation factors, e.g. at least 4 of the coagulation or anti-coagulation factors, such as at least 5 of the coagulation or anticoagulation factors, e.g. at least 6 of the coagulation or anti-coagulation factors, such as at least 7 of the coagulation or anti-coagulation factors, e.g. 8 of the coagulation or anticoagulation factors.
  • At least one of the proteins selected from albumin, IgG, tranferrin, fibrinogen is/are bound to a second adsorbent.
  • at least 2 of the proteins binds to the adsorbent, such as at least 3 of proteins, e.g. 4 of the proteins.
  • At least one of the proteins ⁇ -1 -proteinase inhibitor or ⁇ -1-acid-glycoprotein binds to a third adsorbent.
  • 2 of the proteins binds to the adsorbent.
  • coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S
  • the degree of cross-contamination of the individual protein in the protein fraction is at the most 20%, such as at the most 15%, e.g. at the most 10%, such as at the most 5%, e.g. at the most 3%, such as at the most 1 %, e.g. at the most 0.5%, such as at the most 0.1 %, e.g. at the most 0.01 %.
  • the individual protein fraction are obtained within a single adsorption circle.
  • the one or more protein(s) to be isolated may be isolated by one of the following:
  • One or more protein(s) to be isolated may be washed through the adsorbent without specifically binding to the adsorbent collected in the washing buffer.
  • One or more protein(s) to be isolated may bind specifically to the adsorbent and subsequently be eluted using one or more elution buffer(s) and collected in the one or more elution buffer(s).
  • One or more of the protein(s) to be isolated may be washed through adsorbent and another one or more protein to be isolated may be specifically bound to the adsorbent and collected in the washing buffer, and one or more of the protein(s) of interest may subsequently be eluted with one or more elution buffer(s) and collected in the one or more elution buffer(s).
  • the fibrinogen may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as o at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • albumin and IgG may be bound to the adsorbent and simultaneously ⁇ -1 -proteinase inhibitor may be obtained as non-bound material from the adsorbent. Subsequently, albumin and IgG may be obtained from the 5 adsorbent by stepwise elution.
  • at least 50% of the albumin may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • at least 50% of the IgG may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • stepwise elution relates to a gradual but discontinuous change of the properties of an elution buffer added to the adsorbent in terms of, but not limited to, changes of ionic strength or conductivity, pH, polarity, temperature, concentration of competing substances and so on. 5
  • fibrinogen and IgG may be bound to the adsorbent and simultaneously albumin may be obtained as non-bound material from the adsorbent. Subsequently, fibrinogen and IgG may be obtained from the adsorbent by stepwise elution.
  • at least 50% of the IgG may bind to the adsorbent, such as 0 at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • at least 50% of the fibrinogen may bind to the adsorbent, such as at least 60%, e.g.
  • fibrinogen, albumin and IgG may be bound to the adsorbent and simultaneously ⁇ -1 -proteinase inhibitor may be obtained as non-bound material from the adsorbent. Subsequently, fibrinogen, albumin and IgG may be obtained from the adsorbent by stepwise elution. Preferably, at least 50% of the IgG 5 may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g.
  • At least 90% such as at least 95%, e.g. at least 98%.
  • at least 50% of the fibrinogen may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • at least 50% of the albumin may bind to the adsorbent, such as at least 60%, e.g. at least 70%, 1 o such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • At least 1 such as at least 2 e.g. 3 of fibrinogen, albumin and IgG may be bound to the adsorbent and simultaneously ⁇ -1-acid glycoprotein may be obtained as non-bound material from the adsorbent. Subsequently,
  • fibrinogen, albumin and/or IgG may be obtained from the adsorbent by stepwise elution.
  • at least 50% of the IgG may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • at least 50% of the fibrinogen may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least
  • At least 50% of the albumin may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • At least 1 such as at least 2 e.g. 3 of 25 fibrinogen, albumin and IgG may be bound to the adsorbent and simultaneously ⁇ -1-acid glycoprotein and/or ⁇ -1 -proteinase inhibitor may be obtained as non-bound material from the adsorbent.
  • fibrinogen, albumin and/or IgG may be obtained from the adsorbent by stepwise elution.
  • at least 50% of the IgG may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 30 90%, such as at least 95%, e.g. at least 98%.
  • At least 50% of the fibrinogen may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • at least 50% of the albumin may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • At least 50% ⁇ -1 -proteinase inhibitor, albumin, IgG, fibrinogen or one or more coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S may be 5 obtained from the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S
  • the adsorbent such as at least 60%,
  • albumin may be bound to the adsorbent and simultaneously ⁇ -1 -proteinase inhibitor may be obtained as non-bound material from the o adsorbent.
  • at least 50% of the albumin may bind to the adsorbent, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
  • ⁇ -1 -proteinase inhibitor may be isolated from 5 a protein solution, such as plasma or serum, by a process comprising the steps of (i) contacting an aqueous solution of plasma proteins containing IgG, albumin and ⁇ -1- proteinase inhibitor with an anion exchange adsorbent under conditions such that the albumin and alpha-1 -proteinase inhibitor bind to the adsorbent and the IgG remains unbound, wherein the protein solution is selected from the group consisting of pretreated 0 cryopoor plasma, pretreated cryopoor serum, pretreated supernatant I or pretreated supernatant ll+lll, pretreated supernatant l+ll+lll; (ii) optionally recovering the unbound IgG to obtain an IgG rich protein fraction; (iii) eluting albumin from the anion exchange medium to obtain an albumin rich protein fraction; and (iv) eluting from the anion exchange medium to obtain an ⁇ -1
  • the non-bound protein fraction may comprise the protein(s) washed through the adsorbent, such as ⁇ -1 -protease inhibitor, ⁇ - 1-acid-glycoprotein, albumin, IgG or fibrinogen in high yield.
  • the yield will be more than 70 %, more preferably more than 80 %, more preferably more than 90 % of the 0 alpha-1 -protease inhibitor.
  • the adsorbent such as ⁇ -1-protease inhibitor, ⁇ -1-acid-glycoprotein, albumin, IgG or fibrinogen present in high yield in the non-bound protein fraction may subsequently be isolated from the non-bound protein fraction by further downstream processing, e.g. by employing a second chromatographic adsorption step.
  • each of these protein fractions comprises a high yield of individual proteins without significant cross-contamination of the protein fraction(s) between the at least 2 proteins, such as at least 3 proteins e.g. at least 4 proteins, such as at least 5 proteins e.g. at least 6 proteins within the same protein fraction.
  • the amount of cross contamination in a protein fraction is less than 20%, such as less than 15%, e.g. less than 10%, such as less than 5%, e.g. less than 3%, such as less than 1 %, e.g. less than 0.5%, such as less than 0.1 %, e.g. less than 0.01 %.
  • cross-contamination relates to the amount or content of protein not of interest which is present in the protein fraction. In some cases it is of interest to elute two or more proteins simultaneously in one elution circle and in this case the proteins intentionally eluted together are not considered contaminating.
  • the degree of cross-contamination of the individual protein in the protein fraction is at the most 20%, such as at the most 15%, e.g. at the most 10%, such as at the most 5%, e.g. at the most 3%, such as at the most 1 %, e.g. at the most 0.5%, such as at the most 0.1 %, e.g. at the most 0.01 %.
  • Fig 1 illustrates the overall stepwise fractionation of human plasma proteins by gradual addition of ethanol to obtain a series of supernatants and precipitates comprising various human plasma proteins.
  • Fig 2 illustrates the difference between a packed bed adsorption column comprising tightly packed adsorbent particles and an expanded bed adsorption column comprising adsorbent particles, which are fluidised by an upward flow of liquid.
  • the expanded bed adsorption column still having plug flow with minimal back-mixing.
  • Rg 3 illustrates a SDS-PAGE of el ⁇ ate from DEAE ion exchanger performed in Example 7 and it shows that alpha-1 -proteinase inhibitor-eluate from the DEAE ion exchanger has a high degree of purity as estimated by SDS-PAGE (estimated at > 80 %).
  • Fig 4 illustrates elastase binding activity where lane 1 comprises protein solution without elastase incubation and lane 2 comprises protein solution + elastase incubation.
  • the experiment showed that substantially all the alpha-1 -proteinase inhibitor in the protein solution (non-bound fraction from example 5) is active and binds to elastase.
  • Fig 5 illustrates the result in example 8 showing a very high degree of purity of the alpha- 1-pr ⁇ teinase-inhlbitor-eluate from example 5 using b ⁇ nzylamine as the ligand, and estimated by SDS-PAGE (estimated purity at > 95 %).
  • Fig 6 illustrates the comparison of sample starting material (without activated carbon treatments (black) and a standard (grey) to material that has been, treated with activated carbon at oH 4.5 Mark ⁇ rev ⁇ and at DH 7.
  • Fi ⁇ 7 illustrates chromato ⁇ rams of samples of rHSA (FPX25932 ⁇ before and after carbon treatment at pH 4.5.
  • said adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m;
  • a process according to item 2 wherein at least one virus elimination treatment is performed prior to contacting the protein solution with the adsorbent.
  • virus elimination treatment involves addition of nonionic detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • nonionic detergent and/or an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • the adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and/or one or more acidic groups.
  • the blood, serum, plasma or other blood derived sources is obtained from humans or animals such as cows, fish, camel, pig, sheep, goat, rabbit, mouse, rat, horse, chicken, zebra or ostrich.
  • the one or more protein(s) is one or more human blood protein, such as one or more human plasma protein or one or more human serum protein.
  • said one or more human blood protein(s) is/are selected from the group consisting of albumin, IgG, IgA, IgM, IgD, IgE, alpha-1 -proteinase inhibitor, blood pro-coagulation protein, blood anti-coagulation protein, thrombolytic agent, anti-angiogenic protein, alpha-2-antiplasmin, C-1 esterase inhibitor, apolipoprotein, HDL, LDL, Fibronectin, beta-2-glycoprotein I, fibrinogen, plasminogen, plasmin, plasminogen activator, plasminogen inhibitor, plasma protease inhibitor, anti- thrombin III, streptokinase, inter-alpha-trypsin inhibitor, alpha-2-macroglobulin, amyloid protein, ferritin, pre-albumin, GC-globulin, haemopexin, C3-complement, transferrin, urokinase,
  • a process for the large-scale isolation of one or more blood protein(s), such as one or more serum protein(s) or one or more plasma protein(s), from a protein solution comprising the steps of: b) optionally adjusting the pH of the protein solution to a preset pH;
  • said adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at
  • a process according to item 13, wherein the blood, serum, plasma or other blood derived sources is obtained from humans or animals such as cows, fish, camel, pig, sheep, goat, rabbit, mouse, rat, horse, chicken, zebra or ostrich.
  • the adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and/or one or more acidic groups.
  • IgE alpha-1 -proteinase inhibitor, blood pro-coagulation protein, blood anti-coagulation protein, thrombolytic agent, anti-angiogenic protein, alpha-2-antiplasmin, C-1 esterase inhibitor, apolipoprotein, HDL, LDL, Fibronectin, beta-2-glycoprotein I, fibrinogen, plasminogen, plasmin, plasminogen activator, plasminogen inhibitor, plasma protease
  • inhibitors such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1
  • a process according to item 21 wherein one non-bound protein is alpha-1 -proteinase inhibitor.
  • a process for the large-scale isolation of one or more protein(s) from a protein solution comprising the steps of:
  • said adsorbent comprises a 5 particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m;
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • 35 comprising an aromatic or heteroaromatic ring-system and/or one or more acidic groups.
  • 31 A process according to any one of items 24-30, wherein the one or more protein(s) is one or more human blood protein, such as a human plasma protein or a human serum protein.
  • said one or more human blood protein(s) is/are selected from the group consisting of albumin, IgG, IgA, IgM, IgD, IgE, alpha-1 -proteinase inhibitor, blood pro-coagulation protein, blood anti-coagulation protein, thrombolytic agent, anti-angiogenic protein, alpha-2-antiplasmin, C-1 esterase
  • apolipoprotein apolipoprotein, HDL, LDL, Fibronectin, beta-2-glycoprotein I, fibrinogen, plasminogen, plasmin, plasminogen activator, plasminogen inhibitor, plasma protease inhibitor, anti-thrombin III, streptokinase, inter-alpha-trypsin inhibitor, alpha-2- macroglobulin, amyloid protein, ferritin, pre-albumin, GC-globulin, haemopexin, C3- complement, transferrin, urokinase, ⁇ -1-acid-glycoprotein, and the coagulation or anti-
  • coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S.
  • a process for the large-scale isolation of one or more protein(s) from a protein solution comprising the steps of:
  • the adsorbent comprises a functionalised matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring- system and one or more acidic groups,
  • a process according to item 36, wherein the blood, serum, plasma or other blood 5 derived sources is obtained from humans or animals such as cows, fish, camel, pig, sheep, goat, rabbit, mouse, rat, horse, chicken, zebra or ostrich.
  • virus elimination 5 treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups 0 comprising an aromatic or heteroaromatic ring-system and/or one or more acidic groups.
  • a process for the large-scale isolation of one or more protein(s) from a protein solution comprising the steps of:
  • adsorbent comprises a functionalised matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups,
  • virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • a process according to item 50, wherein the blood, serum, plasma or other blood derived sources is obtained from humans or animals such as cows, fish, camel, pig, 0 sheep, goat, rabbit, mouse, rat, horse, chicken, zebra or ostrich.
  • adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • a process for the large-scale isolation of one or more protein(s) from a protein solution comprising the steps of:
  • adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material
  • virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant,
  • a process according to item 61 wherein the blood, serum, plasma or other blood derived sources is obtained from humans or animals such as cows, fish, camel, pig, sheep, goat, rabbit, mouse, rat, horse, chicken, zebra or ostrich.
  • IgE alpha-1 -proteinase inhibitor, blood pro-coagulation protein, blood anti-coagulation protein, thrombolytic agent, anti-angiogenic protein, alpha-2-antiplasmin, C-1 esterase inhibitor, apolipoprotein, HDL, LDL, Fibronectin, beta-2-glycoprotein I, fibrinogen, plasminogen, plasmin, plasminogen activator, plasminogen inhibitor, plasma protease
  • inhibitors such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1
  • adsorbent comprises a functionalised matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups
  • the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • a process for the large-scale isolation or separation of alpha-1 proteinase inhibitor comprising the steps of:
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups;
  • a process according to item 71 wherein at least one virus elimination treatment is performed prior to contacting the protein solution with the adsorbent.
  • the virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • a process according to item 74, wherein the blood, serum, plasma or other blood derived sources is obtained from humans or animals such as cows, fish, camel, pig, sheep, goat, rabbit, mouse, rat, horse, chicken, zebra or ostrich.
  • the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • a process for the large-scale isolation or separation of alpha-1 proteinase inhibitor comprising the steps of:
  • said adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material;
  • adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • a process according to item 81 wherein at least one virus elimination treatment is performed prior to contacting the protein solution with the adsorbent.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • alpha-1 -proteinase inhibitor is 30 washed out as a non-bound protein with one or more washing buffer(s).
  • a process for the large-scale isolation or separation of human albumin comprising the steps of: a) providing a protein solution comprising said human albumin and having a preset pH and optionally a preset ionic strength or conductivity;
  • the 5 adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups;
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material.
  • the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • a process for the large-scale isolation or separation of fibrinogen comprising the steps of:
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups;
  • virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • the blood, serum, plasma or other blood derived sources is obtained from humans or animals such as cows, fish, camel, pig, sheep, goat, rabbit, mouse, rat, horse, chicken, zebra or ostrich.
  • adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous o material.
  • adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m. 5
  • a process for the large-scale isolation or separation of fibrinogen comprising the steps of:
  • said adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material; and 5 c) obtaining said fibrinogen from said adsorbent.
  • the virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • a process for the large-scale isolation or separation of transferrin comprising the steps of:
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • a process for the large-scale isolation or separation of transferrin comprising the 25 steps of:
  • said adsorbent comprises a particle with at least one high density non-porous core , surrounded by a porous material;
  • the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • a process according to item 131 wherein the blood, serum, plasma or other blood derived sources is obtained from humans or animals such as cows, fish, camel, pig,
  • a process for the large-scale isolation or separation of ⁇ -1-acid-glycoprotein comprising the steps of:
  • the 5 adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups;
  • virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • a process according to any one of items 135-141 wherein said adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent comprises a particle density of at least 1.5 g/ml and a mean
  • a process for the large-scale isolation or separation of ⁇ -1-acid-glycoprotein comprising the steps of:
  • said adsorbent comprises a particle with at least one high density non-porous core , surrounded by a porous material; and 5 c) obtaining said ⁇ -1-acid-glycoprotein from said adsorbent.
  • the adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m. 0
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • the protein solution is obtained from a source selected from the group consisting of blood, serum, plasma, and other blood derived sources.
  • a process for the large-scale isolation or separation of one or more coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S, said process comprises the steps of:
  • the 25 adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and one or more acidic groups;
  • the virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • a process for the large-scale isolation or separation of one or more coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S, said process comprises the steps of:
  • adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material;
  • adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups comprising an aromatic or heteroaromatic ring-system and/or one or more acidic groups.
  • a process according to any one of items 154-171 wherein one or more coagulation factor(s) is washed out as a non-bound protein with one or more washing buffer(s).
  • virus elimination treatment involves addition of detergent and/or an organic solvent, such as non-ionic or anionic surfactant, tri-n-butylphosphate, to the protein solution.
  • an organic solvent such as non-ionic or anionic surfactant, tri-n-butylphosphate
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional o groups comprising an aromatic or heteroaromatic ring-system and/or one or more acidic groups.
  • adsorbent comprises a particle density of at least 1.5 g/ml and a mean volume particle diameter of at most 150 ⁇ m.
  • 30 particle diameter is in the range of 15 to 100 micron.
  • adsorbent has a dynamic binding capacity at 10 % break-through for said at least one specific protein of at least 5 g per liter sedimented adsorbent.
  • adsorbent comprises a functionalized matrix polymer carrying a plurality of covalently attached functional groups, said groups having the general formula:
  • M designates the adsorbent polymer
  • SP1 designates an optional spacer optionally substituted with -A-SP2-ACID, -A, or -ACID
  • X designates — O— , -S-, — NH-, or — NAIk-
  • AIk may be absent, -A-SP2-ACID, -A, -ACID or Ci -4 alkyl, where Ci -4 alkyl may be optionally substituted with -A-SP2-ACID, -A, or -ACID
  • A designates an optionally substituted aromatic or heteroaromatic moiety
  • SP2 designates an optional spacer
  • ACID designates one or more acidic groups; wherein at least one of SP1 or AIk is substituted with -A-SP2-ACID or -A, and at least one of SP1 or AIk comprise -ACID and wherein at least one of SP1 or AIk is present. If AIk is absent, X will also be absent.
  • a process according to item 201 wherein the ligand is an aromatic or heteroaromatic acid selected from the group consisting of carboxylic acids, sulfonic acids, phosphonic acids, and boronic acids.
  • the ligand is chosen from the group consisting of 2-mercaptobenzoic acid, 2-mercaptonicotinic acid, 2- aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 4-hydroxyphenyl- mercapto-acetic acid, 4-hydroxyphenyl-mercapto-propionic acid, 4-hydroxyphenyl-
  • adsorbent is 1 o coupled to a ligand comprising a bicyclic substituted heteroaromatic group.
  • ligand is derived from a diethylaminoethyl group, a polyalkylene imine, an alkyl-amine, an alkyl-diamine or
  • alkyl-amine or alkyl-diamine has a chain-length of 3-14 atoms and 1-5 functional amine groups.
  • adsorbent comprises a ligand which is an aromatic amine or an aromatic diamine.
  • fibrinogen is bound to the adsorbent and simultaneously one or more of the coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor
  • a process according to any one of the preceding items, wherein fibrinogen and IgG are bound to the adsorbent and simultaneously albumin may be obtained as non-bound material from the adsorbent.
  • a process according to item 219, wherein fibrinogen and IgG may be obtained from the adsorbent by stepwise elution.
  • at least 1 such as at 35 least 2 e.g. 3 of fibrinogen, albumin and IgG is/are bound to the adsorbent and simultaneously ⁇ -1-acid glycoprotein and/or ⁇ -1 -proteinase inhibitor is/are obtained as non-bound material from the adsorbent.
  • a process according to item 230, wherein fibrinogen, albumin and/or IgG may be 5 obtained from the adsorbent by stepwise elution.
  • coagulation or anticoagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1
  • inhibitor, protein C and/or Protein S is/are obtained from the adsorbent.
  • coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S , is/are isolated in at least 2 individual protein fractions, such as 3 individual protein fractions, e.g. 4 individual protein fractions, such as 5 individual protein fractions, e.g. 6 individual
  • the protein solution may comprise a supernatant and a precipitated fraction after being supplemented with an alcohol.
  • the precipitated fraction is a resolubilised precipitate obtained by the addition of alcohol to blood, plasma, serum or other blood derived sources.
  • the first adsorbent is capable of capturing one or more blood protein(s), serum protein(s) or plasma protein(s);
  • the second adsorbent is capable of capturing one or more blood protein(s), serum protein(s) or plasma protein(s) different for the one or more blood protein(s), serum protein(s) or plasma protein(s) capable of being captured to the first adsorbent;
  • the third adsorbent is capable of capturing one or more blood 5 protein(s), serum protein(s) or plasma protein(s) different for the one or more blood protein(s), serum protein(s) or plasma protein(s) capable of being captured to the first adsorbent or second adsorbent;
  • one or more coagulation or anti-coagulation factor(s) such as Factor II, Factor V, Factor VII, Factor VIII, von Willebrand factor, Factor VIII - von Willebrand factor complex, Factor IX, Factor X, Factor Xl, C1 inhibitor, protein C and/or Protein S, is/are bound to a first adsorbent.
  • the adsorbent was based on agarose with tungsten carbide particles incorporated, the density of the o conglomerate particles was 2.9 g/ml and the particle diameter was in the range of 40-120 ⁇ m with a volume mean particle diameter (D (4,3) of 70 ⁇ m (as determined on the Mastersizer 2000E, Malvern Instruments, Worcestershire, UK).
  • the adsorbent comprised 2-mercaptonicotinic acid as the ligand and had a ligand concentration of 40 micromoles per ml sedimented adsorbent. 5
  • the protein solution comprised of Cohn fraction, Supernatant I, II, III, Conductivity 5,74 mS, pH 7.2, comprising approx. 10 % ethanol.
  • the protein solution was diluted with demineralised water in a ratio of one volume of Cohn supernatant I, II, III to 2 volumes of water and pH was adjusted to pH 5.0 with 1 M hydrochloric acid.
  • the conductivity after dilution was 3.8 mS.
  • the column was packed with 50 cm of adsorbent (157 ml) and equilibrated at room temperature (20-25 0 C) with 160 ml 1 M NaOH (first equilibration buffer), 400 ml 40 mM sodium citrate buffer pH 4.5 (second equilibration buffer), and 400 ml 40 mM sodium 0 citrate buffer pH 5.0 (third equilibration buffer).
  • Two experiments were performed with a linear flow rate of 450 cm/hr.:
  • SRI Single Radial Immunodiffusion
  • a standard curve was performed with the protein solution loaded onto the column in the concentration of 100 %, 80 %, 60%, 40 % and 20 %. Each of the three fractions was read relative to the standard curve.
  • alpha-1-PI human alpha-1 -protease inhibitor
  • human albumin molecules No breakdown or denaturation of the human alpha-1 -protease inhibitor (alpha-1-PI) or human albumin molecules could be detected by sodiumdodecyl-gelelctrophoresis (SDS- PAGE). The purity of the eluted human albumin was found to be higher than 95 % as determined by SDS-PAGE.
  • Example 2 Separation of human albumin and alpha-1 -protease inhibitor from Cohn fraction (SUP I, Il and III) by expanded bed adsorption at different pH values.
  • the protein solution comprised a Cohn fraction Supernatant I, II, III, conductivity 5,74 mS, pH 7.2, comprising approx 10 % ethanol.
  • the protein solution was diluted with demineralised water or sodium acetate 20 mM pH 5 in a ratio of one volume of Cohn supernatant I, II, III to 5 volumes of water or sodium acetate 20 mM pH 5 and pH was adjusted to different pH values with 1 M hydrochloric acid.
  • Experiment B 400 ml 40 mM sodium citrate buffer pH 5.3
  • Experiment C 400 ml 40 mM sodium citrate buffer pH 5.5
  • the bound proteins were eluted in two steps. 5 - Fraction 2, first elution step: Human albumin was eluted with sodium caprylate 5 mg/ml, pH 6
  • SRI Single Radial Immunodiffusion
  • the adsorbent was based on agarose with tungsten carbide particles incorporated, the density of the conglomerate particles was 2.9 g/ml and the particle diameter was in the range of 40-120 ⁇ m with a mean particle diameter of 70 ⁇ m.
  • the adsorbent comprised 0 diethylaminoethyl (DEAE) groups as the ligand and a ligand concentration of 150 micromoles per ml sedimented adsorbent.
  • Protein fraction 1 (the non-bound material) from the separation of albumin and alpha-1 - 5 proteinase inhibitor as described in example 1 , experiment A.
  • the pH in the protein fraction was adjusted to pH 8.2 with 1 M NaOH.
  • the conductivity was hereafter 6.16 mS.
  • the column was packed with 1 ml of ion exchanger and equilibrated at room temperature (20-25 0 C) for 30 minutes with 5 ml 1 M potassium phosphate pH 8.2. After incubation the ion exchanger was washed with 10 ml 10 mM potassium phosphate pH 8.2.
  • the bound alpha-1 -protease inhibitor was eluted with 10 mM potassium phosphate +1 M NaCI to pH 8.2. 5
  • Single Radial Immunodiffusion was performed in order to quantify the relative yield in percent of alpha-1 -protease inhibitor in the fractions from the column as described in Scand. J. Immunol. Vol. 17, Suppl. 10, 41-56, 1983.
  • the RDI was performed with: Rabbit anti-human alpha-1 -protease inhibitor from Dako Cytomation, Denmark, Cat. No.: A0012 (0.6 ⁇ l per cm 2 ).
  • a standard row was performed with the protein fraction loaded onto the column in the 5 concentration of 100 %, 80 %, 60%, 40 % and 20 %. Each of the fractions was read relative to the standard curve.
  • Example 3 Expanded bed adsorption of alpha-1 -proteinase inhibitor.
  • Example 3 was repeated, however, this time using an expanded bed adsorption column using the same ion exchange adsorbent at a settled bed height of 5 cm and a linear flow rate of 5 cm/min.
  • the adsorbent was based on agarose with tungsten carbide particles incorporated, the density of the conglomerate particles was 2.9 g/ml and the particle diameter was in the range of 40-120 ⁇ m with a mean particle diameter of 70 ⁇ m.
  • the adsorbent comprised 2-mercaptonicotinic acid as the ligand and a ligand concentration of 40 micromoles per ml sedimented adsorbent.
  • the protein solution was diluted with demineralised water in a ratio of one volume of human plasma to 2 volumes of water and pH was adjusted to pH 5.0 with 1 M hydrochloric acid.
  • the conductivity was hereafter 5,25 mS/cm 2
  • the column was loaded with adsorbent to reach a settled bed height (HO) of 50 cm (corresponding to 157 ml adsorbent) and washed and equilibrated at 20-25 0 C with the following buffers in successive order 1 ) 160 ml 1 M NaOH, 2) 400 ml 40 mM citric acid buffer pH 4.5 3) 400 ml 40 mM citric acid buffer pH 5.0.
  • the experiment was performed with a linear flow rate of 450 cm/hr in all steps and the outlet from the column was connected to an UV monitor and recorder.
  • Fraction 1 (unbound fraction) was collected as one fraction according to the UV monitoring of the column effluent.
  • SRI Single Radial Immunodiffusion
  • the SRI was performed with the following antibodies, all from Dako Cytomation, Denmark:
  • a standard curve was established with the protein solution (100 % reference) loaded onto the column in the concentration of 100 %, 80 %, 60%, 40 % and 20 %. Each of the four fractions was determined relative to this standard curve.
  • the adsorbent was based on agarose with tungsten carbide particles incorporated, the density of the 5 conglomerate particles was 2.9 g/ml and the particle diameter was in the range of 40-120 ⁇ m with a mean particle diameter of 70 ⁇ m.
  • the adsorbent comprised 2-mercaptonicotinic acid as the ligand and a ligand concentration of 40 micromoles per ml sedimented adsorbent.
  • Human plasma was adjusted to 8 % ethanol by volume with 99 % ethanol at - 3° C and incubated at - 3° C for 1.5 hour. After incubation the plasma was centrifuged 10 minuets. The supernatant was diluted demineralised water in a ratio of 1 part of plasma to two parts of water and the pH was adjusted to pH 5.0 with 1 M hydrochloric acid. The 5 conductivity was hereafter 4.16 mS/cm 2
  • the ion exchanger was based on 4 % agarose with tungsten carbide particles incorporated.
  • the conglomerate beads had a density of approximately 2.9 g/ml and a particle size in the range of 40-120 ⁇ m with a mean particle size (diameter) of 70 ⁇ m.
  • the adsorbent comprised DEAE (diethylaminoethyl-) groups and a concentration of o approx.150 millimole per litre sedimented adsorbent
  • the starting material for this experiment was protein fraction 1 (the non-bound material) obtained from isolation of human plasma proteins using expanded bed adsorption as 5 described in example 5.
  • the pH in the protein fraction was carefully adjusted to pH 8.2 with 1 M NaOH.
  • the conductivity was hereafter 5,15 mS/cm 2
  • the experiment was performed as packed bed experiments in Poly-Prep columns product 0 number 731-1550, BioRad, USA.
  • the column was packed with 1 ml of DEAE ion exchanger and equilibrated, at 20-25 0 C, 5 with 5 ml 1 M K 2 HPO 4 adjusted to pH 8.2 with 1 M NaOH. After equilibration the ion exchanger was washed with 10 ml 10 mM potassium phosphate pH 8.2.
  • SRI Single Radial Immunodiffusion
  • the SRI was performed with Rabbit anti-human API from Dako Cytomation, Denmark, Cat. No.: A0012 (0.6 ⁇ l per cm 2 ).
  • a standard curve was performed with the protein fraction (100 % reference) loaded onto the column in the concentration of 100 %, 80 %, 60%, 40 % and 20 %.
  • the alpha-1-PI concentration in each of the fractions was determined against the standard curve and the relative yield in that fraction was calculated from the volume of the fraction relative to the volume and concentration of alpha-1-PI in the applied protein fraction.
  • Sample preparation 25 ⁇ l sample and 25 ⁇ l sample buffer tris-glycine Invitrogen (cat no. LC2676) was mixed and boiled for 5 minutes in a water bath.
  • the running buffer 0.024 M 5 tris (Sigma T1378), 0.19 M glycine (Merck 5001901000), 0.1 % SDS (sodium dodecyl sulphate, JT Baker 281 1 ) pH 8.6 was added.
  • Protein fraction 1 (the non-bound material) from isolation of human plasma proteins using expanded bed adsorption from example 5. The protein fraction was added ammonium sulphate to a final concentration of 2 M ammonium sulphate followed by careful adjustment to pH 8.2 with 1 M NaOH. Process parameters
  • Each of the columns was packed with 1 ml of adsorbent and the adsorbent was washed in the column with subsequently 1) 5 ml demineralised water and 2) 4 ml 2 M ammonium sulphate pH 8.2.
  • SRI Single Radial Immunodiffusion
  • the SRI was performed with the following antibodies:
  • the relative concentration of the specific proteins in the fractions collected was determined against the standard curve.
  • the relative yield of the specific proteins in each fraction was calculated from the volume of the fraction relative to the total amount of the o protein applied to the column.
  • Sample preparation 25 ⁇ l sample and 25 ⁇ l sample buffer tris-glycine Invitrogen (cat no. LC2676) was mixed and boiled for 5 minutes in a water bath.
  • the running buffer 0.024 M tris (Sigma T1378), 0.19 M glycine (Merck 5001901000), 0.1 % SDS (sodium dodecyl 0 sulphate, JT Baker 281 1 ) pH 8.6 was added.
  • Concentration of the eluate from experiment 5 (benzylamine as the ligand) by 5 ultrafiltration followed by two viral inactivation steps: 1 ) solvent-detergent treatment and 2) viral filtration (nano-filtration) provide a product suitable for therapeutic use.
  • Example 9 Isolation of human API and human orosomucoid (alpha-1-acid glycoprotein).
  • the particle size was in the range of 80-150 ⁇ m.
  • Adsorbents were based on 6 % agarose beads cross-linked and activated with epichlorohydrin prior to coupling of the following ligands:
  • Protein fraction 1 (the non-bound material) from isolation of human plasma proteins using expanded bed adsorption from example 5. The protein fraction was added ammonium sulphate to a final concentration of 2 M ammonium sulphate followed by careful adjustment to pH 8.2 with 1 M NaOH. 0
  • SRI Single Radial Immunodiffusion
  • the relative concentration of the specific proteins in the fractions collected was determined against the standard curve.
  • the relative yield of the specific proteins in each fraction was calculated from the volume of the fraction relative to the total amount of the protein applied to the column. 5
  • the adsorbent was based on agarose beads with tungsten carbide particles incorporated, the density of the conglomerate particles was 2.9 g/ml and the particle diameter was in the range of 40-120 ⁇ m with a mean volume particle diameter of 70 ⁇ m.
  • the adsorbent was activated and cross-linked with epichlorohydrin and coupled with the ligand p- 5 Xylylenediamine (final concentration of ligand was 25 micromoles per ml sedimented adsorbent).
  • the raw human plasma (standard citrate plasma) was adjusted to pH 6.7 with 1 M acetic acid.
  • the conductivity was hereafter 1 1.5 mS/cm 2
  • the column was loaded with adsorbent to reach a settled bed height (HO) of 25 cm o (corresponding to approx. 20 ml settled adsorbent) and washed and equilibrated at 20-25 0 C with 20 mM sodium citrate buffer pH 6.7.
  • HO settled bed height
  • the experiment was performed with a linear flow rate of 300 cm/hr in all steps and the outlet from the column was connected to an UV monitor and recorder. 5
  • the raw plasma, the combined run-through and washing fraction and the eluate were then measured for the activity of a range of specific coagulation and anti-coagulation factors 0 using the DiaMed CD-X analyzer (Cresser, Switzerland).
  • Von Willebrand factor (vWF) biological activity was assessed by the ristocetin cofactor assay (vWFRco).
  • Von Willebrand Factor antigen (vWFAg) was quantified using a turbidimetric assay. Protein S, Protein C and C1 -inhibitor were measured with a functional assay.
  • Other proteins such as albumin, IgG, alpha-1 -antitrypsin, fibrinogen, transferrin and alpha- 1-acid-glycoprotein were determined by single radial immunodiffusion.
  • the relative yield in each fraction can be determined.
  • the total recovery is defined as the sum of the yields found in the run-through, washing and elution fractions.
  • the eluate was further analysed by size exclusion chromatography on a Superdex G200 (Amersham Biosciences). Fractions from the analysis were analysed for Factor VIII o activity.
  • the four adsorbents employed in this experiment were all based on agarose beads with tungsten carbide particles incorporated, the density of the conglomerate particles was 2.9 g/ml while the volume mean particle diameter was varied between 40 to 200 ⁇ m.
  • the 0 adsorbents were activated and cross-linked with epichlorohydrin and coupled with the ligand p-Xylylenediamine (final concentration of ligand was 25 micromoles per ml sedimented adsorbent).
  • Four different adsorbent preparations were tested having the following volume mean particles diameters:
  • the raw human plasma (standard citrate plasma) was adjusted to pH 6.7 with 1 M acetic acid.
  • the conductivity was hereafter 1 1.5 mS/cm 2
  • the experiment was performed with a linear flow rate of 350 cm/hr in all steps and the 5 outlet from the column was connected to an UV monitor and recorder.
  • wash 1 5 mM sodium citrate pH 6.7
  • wash 2 5 mM sodium citrate + 0.2 M sodium chloride pH 6.7
  • wash 2 5 mM sodium citrate + 0.8 M sodium chloride pH 6.7
  • the raw plasma, the combined run-through and washing fraction and the eluate were then measured for the activity of Factor VIII using the DiaMed CD-X analyzer (Cresser, 0 Switzerland).
  • the adsorbent binding capacity for Factor VIII increases significantly with a decrease in 5 volume mean particle diameter.
  • the experiment illustrates the superior performance of adsorbents having a volume mean particle diameter below 150 ⁇ m.
  • the adsorbents were all based on agarose with tungsten carbide particles incorporated, the density of the conglomerate particles was 2.9 g/ml and the particle diameter was in 5 the range of 40-120 ⁇ m with a volume mean particle diameter of 70 ⁇ m.
  • the adsorbents were cross-linked and activated with epichlorohydrin and coupled with the following different ligands: 2-mercaptonicotinic acid, 2-mercapto-benzoic acid, 3,4-diamino-benzoic acid, 2,4-dihydroxy-benzoic acid, 3,5-dihydroxy-benzoic acid, 2-(4-aminophenylthio)acetic acid, 2-mercapto-benzimidazole sulphonic acid, N-benzoyl-cysteine. 0
  • the ligand concentration on all the individual adsorbents was determined by acid-base titration to be in the range of 25 - 40 micromoles per ml sedimented adsorbent.
  • the protein solution human plasma
  • demineralised water in a ratio of one volume of plasma to 2 volumes of water and pH was adjusted to pH 5.0 with 1 M hydrochloric acid.
  • the conductivity was hereafter 5,25 mS/cm 2
  • the column was loaded with adsorbent to reach a settled bed height (HO) of 50 cm o (corresponding to approx 40 ml settled adsorbent) and washed and equilibrated at 20-25 0 C with the following buffers in successive order 1) 1 M NaOH, 2) 40 mM citric acid buffer pH 4.5 3) 40 mM citric acid buffer pH 5.0.
  • HO settled bed height
  • the experiment was performed with a linear flow rate of 600 cm/hr in all steps and the 5 outlet from the column was connected to an UV monitor and recorder.
  • the SRI was performed with the following antibodies, all from Dako Cytomation, o Denmark:
  • a standard curve was established with the protein solution (100 % reference) loaded onto the column in the concentration of 100 %, 80 %, 60%, 40 % and 20 %. Each of the four fractions was determined relative to this standard curve and the relative yield of the 5 specific protein in each fraction was determined. If the yield of a specific protein in a specific fraction relative to the amount of protein added to the column is above 5 % the protein is defined to distribute into said fraction.

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  • Organic Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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Abstract

L'invention concerne un procédé d'isolement de rHSA d'une solution protéique. Le procédé comprend les étapes consistant: a) à utiliser une solution protéique comprenant une ou plusieurs protéines spécifiques et possédant un pH établi au préalable et une force ionique ou une conductivité établie au préalable, b) à faire passer la solution protéique dans une colonne à lit fixe ou expansé comprenant un adsorbant, et c) à obtenir une ou plusieurs protéines à partir de la colonne.
PCT/EP2006/069208 2005-12-02 2006-12-01 Isolement de peptides, polypeptides et protéines Ceased WO2007063129A2 (fr)

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WO2009025754A3 (fr) * 2007-08-17 2009-04-30 Csl Behring Gmbh Procédés de purification d'alpha-1-antitrypsine et d'apolipoprotéine a-i
CN102533553A (zh) * 2011-12-29 2012-07-04 湖南省微生物研究所 一种快速降解稻草秸秆的有机物料腐熟组合菌剂及其应用方法
EP2574618A1 (fr) * 2011-08-19 2013-04-03 EMD Millipore Corporation Procédés de réduction du niveau d'une ou plusieurs impuretés dans un échantillon pendant la purification de protéines
WO2015117093A1 (fr) * 2014-01-31 2015-08-06 Fina Biosolutions, Llc Expression et purification de crm197 et de protéines associées
WO2015114664A1 (fr) 2014-01-29 2015-08-06 Hemarus Therapeutics Ltd Procédé intégré de production d'agents thérapeutiques (albumine humaine, immunoglobulines intraveineuses, facteur de coagulation viii et facteur de coagulation ix) â partir de plasma humain
CN109182145A (zh) * 2018-09-29 2019-01-11 武汉友芹种苗技术有限公司 一种棘孢曲霉菌株及其应用
US11060123B2 (en) 2014-01-31 2021-07-13 Fina Biosolutions, Llc Production of soluble recombinant protein without n-terminal methionine
WO2023154467A1 (fr) * 2022-02-11 2023-08-17 Clara Foods Co. Compositions protéiques et produits de consommation s'y rapportant
US12104161B2 (en) 2014-01-31 2024-10-01 Fina Biosolutions Llc Production of soluble recombinant proteins without N-terminal methionine in E-coli

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GB9902000D0 (en) * 1999-01-30 1999-03-17 Delta Biotechnology Ltd Process
WO2005121163A2 (fr) * 2004-06-07 2005-12-22 Upfront Chromatography A/S Isolation de proteines du plasma ou du serum

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JP2010536758A (ja) * 2007-08-17 2010-12-02 ツェーエスエル ベーリンク ゲゼルシャフト ミット ベシュレンクテル ハフツング α−１−抗トリプシンおよびアポリポタンパク質Ａ−Ｉを精製する方法
WO2009025754A3 (fr) * 2007-08-17 2009-04-30 Csl Behring Gmbh Procédés de purification d'alpha-1-antitrypsine et d'apolipoprotéine a-i
EP2520583A1 (fr) * 2007-08-17 2012-11-07 CSL Behring GmbH Procédés de purification de l'alpha 1-antitrypsine et apolipoprotéine a-i
EP2522674A1 (fr) * 2007-08-17 2012-11-14 CSL Behring GmbH Procédés de purification de l'alpha 1-antitrypsine et apolipoprotéine a-i
US8962802B2 (en) 2007-08-17 2015-02-24 Csl Behring Gmbh Methods for purification of alpha-1-antitrypsin and apolipoprotein A-1
US8436152B2 (en) 2007-08-17 2013-05-07 Csl Behring Gmbh Methods for purification of alpha-1-antitrypsin andapolipoprotein A-1
US9096648B2 (en) 2011-08-19 2015-08-04 Emd Millipore Corporation Methods of reducing level of one or more impurities in a sample during protein purification
EP2574618A1 (fr) * 2011-08-19 2013-04-03 EMD Millipore Corporation Procédés de réduction du niveau d'une ou plusieurs impuretés dans un échantillon pendant la purification de protéines
US11634457B2 (en) 2011-08-19 2023-04-25 Emd Millipore Corporation Methods of reducing level of one or more impurities in a sample during protein purification
US10287314B2 (en) 2011-08-19 2019-05-14 Emd Millipore Corporation Methods of reducing level of one or more impurities in a sample during protein purification
EP2942353A1 (fr) * 2011-08-19 2015-11-11 EMD Millipore Corporation Procédés de réduction du niveau d'une ou plusieurs impuretés dans un échantillon pendant la purification de protéines
CN102533553B (zh) * 2011-12-29 2013-06-26 湖南省微生物研究所 一种快速降解稻草秸秆的有机物料腐熟组合菌剂及其应用方法
CN102533553A (zh) * 2011-12-29 2012-07-04 湖南省微生物研究所 一种快速降解稻草秸秆的有机物料腐熟组合菌剂及其应用方法
US9663553B2 (en) 2014-01-29 2017-05-30 Hemarus Therapeutics Limited Integrated process for the production of therapeutics (human albumin, immunoglobulins, clotting factor VIII and clotting factor IX) from human plasma
WO2015114664A1 (fr) 2014-01-29 2015-08-06 Hemarus Therapeutics Ltd Procédé intégré de production d'agents thérapeutiques (albumine humaine, immunoglobulines intraveineuses, facteur de coagulation viii et facteur de coagulation ix) â partir de plasma humain
US10093704B2 (en) 2014-01-31 2018-10-09 Fina Biosolutions, Llc Expression and purification of CRM197 and related proteins
US11060123B2 (en) 2014-01-31 2021-07-13 Fina Biosolutions, Llc Production of soluble recombinant protein without n-terminal methionine
WO2015117093A1 (fr) * 2014-01-31 2015-08-06 Fina Biosolutions, Llc Expression et purification de crm197 et de protéines associées
US12104161B2 (en) 2014-01-31 2024-10-01 Fina Biosolutions Llc Production of soluble recombinant proteins without N-terminal methionine in E-coli
CN109182145A (zh) * 2018-09-29 2019-01-11 武汉友芹种苗技术有限公司 一种棘孢曲霉菌株及其应用
CN109182145B (zh) * 2018-09-29 2021-05-11 武汉友芹种苗技术有限公司 一种棘孢曲霉菌株及其应用
WO2023154467A1 (fr) * 2022-02-11 2023-08-17 Clara Foods Co. Compositions protéiques et produits de consommation s'y rapportant

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