DK2729492T3 - ALBUM INFORMATION AND USE - Google Patents

Description

DESCRIPTION

Reference to a Sequence Listing [0001] This application contains a Sequence Listing in computer readable form.

Background ofthe Invention [0002] The invention relates to a new formulation of albumin and to uses of the albumin formulation.

[0003] Albumin is the most abundant protein in plasma. Albumin has been described and characterized from a large number of mammals and birds. Albumin is believed to have a role in maintaining correct osmotic pressure and it also has a role in transport of various compounds in the blood stream. Albumin is a protein which is used to treat patients with severe burns, shock or blood loss. It is also used as an excipient for pharmacologically active compounds, many of which need to be stabilized for example to reduce the formation of soluble aggregates and/or insoluble aggregates of albumin. Furthermore, albumin is used to supplement media used for growing higher eukaryotic cells, including stem cells. Albumin fusion proteins are a fusion of a protein to albumin, or to a variant or fragment thereof, and may increase or decrease the half-life of the protein, for example increased in vivo half-life. Conjugation partners, e.g. proteins or chemicals, can be conjugated to albumin to increase or decrease the half-life ofthe conjugation partner, for example increased in vivo half-life. At present albumin is obtained from blood products, such as serum, or produced recombinantly in microorganisms such as yeast (e.g. WO1996/037515, W02000/044772) or from transgenic plants or animals. Typically, albumin is purified from the production source in order to provide a product which is sufficiently pure to meet the user's needs and/or to achieve a high yield of product. In some technical areas, such as cell culture or pharmaceuticals, there is a desire for products to be substantially free or completely free of animal derived components.

[0004] Purified albumin in a final liquid form is relatively unstable (compared to albumin in solid form) and so in order to maximize its shelf life it is either lyophilized and/or stabilizers added to the final liquid formulation. However, lyophilization can add significantly to the overall cost of the preparation and can be inconvenient to the end user who would need to resuspend the lyophilized product if they need a liquid product. For the preferred liquid product, stabilizers that are commonly added to albumin are n-acetyl-tryptophan, octanoic acid (octanoate, caprylate) and/or polysorbate 80 (e.g. Tween®). The albumin of W02000/044772 is stabilized by octanoic acid. Arakawa & Kita (2000) discloses stabilizing effects of caprylate and acetyltryptophanate on heat-induced aggregation of bovine serum albumin (Biochimica et Biophysica Acta 1479: 32-36). Hosseini et al (2002) discloses a study of the heat-treated human albumin stabilization by caprylate and acetyltryptophanate (Iranian Biomedical Journal 6(4): 135-140).

[0005] Albumin solutions are described in 'Albumin solution from bovine serum In: Products for Life Science Reseach 2008-2009', Sigma-Aldrich.

[0006] The present inventors have identified that octanoic acid, can be deleterious to mammalian cell culture particularly to stem cell culture. Furthermore, polysorbate 80 (Tween®) can be deleterious to mammalian cell culture. What is required is a stable liquid formulation of albumin which is not deleterious to mammalian cell culture.

Summary of the Invention [0007] The invention provides a liquid formulation of albumin with improved stability, where stability is shown, for example, as a reduced level of soluble aggregates of albumin or insoluble aggregates of albumin in the formulation. The invention also provides methods using the formulation and uses of the formulation, such as mammalian culture and particularly stem cell culture.

Brief Description of the Figures [0008]

Figure 1 shows the effect of pH and n-acetyl tryptophan concentration on the stability of albumin compositions (10mg/mL), as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 Absorbance Units (AU), a measure of visible (insoluble) aggregates.

Figure 2 shows the effect of pH and phosphate concentration on the stability of albumin compositions (10mg/mL), as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 3 shows the effect of pH and sodium concentration on the stability of albumin compositions (10mg/mL), as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 4 shows the effect of pH and sodium concentration (for a wide range of sodium concentrations) on the stability of albumin compositions (10mg/mL), as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 5 shows the effect of pH and sodium concentration on the stability of albumin compositions (50mg/mL), as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 6 shows the effect of sodium concentration and albumin concentration on the stability of albumin compositions at pH 6.5, as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 7 shows the relationship between sodium concentration and relative monomer content (%) for albumin compositions incubated at 40°C for 14 days.

Figure 8 shows the relationship between sodium concentration and relative polymer content (%) for albumin compositions incubated at 40°C for 14 days.

Figure 9 shows a fatty acid profile of an albumin formulation according to the invention.

Figure 10 shows a metal ion profile, by ICP-OES, of an albumin formulation according to the invention.

Figure 11 shows the effect of sodium concentration and albumin concentration on albumin stability as determined by the remaining monomer content following incubation at 40°C for 4 weeks.

Figure 12 shows the effect of cation species and cation concentration on albumin stability as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.2 AU.

Figure 13 shows the effect of sodium ion concentration and anion species on albumin stability as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 14 shows the effect of sodium ion concentration and anion species on albumin stability as determined by the remaining monomer content following incubation at 65°C for 2 hours.

Figure 15 shows the effect of sodium ion concentration in the presence of different buffer anions on albumin stability wherein the contribution of sodium from both NaCI and the buffer is included, as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 16 shows the effect of sodium ion concentration in the presence of no buffering ion or 50 mM citrate as a buffering ion on albumin stability where the contribution of sodium ion from the sodium citrate buffer is ignored, as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 17 shows the effect of sodium ion concentration on the stability of different albumins and albumin variants as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 18 shows the effect of sodium ion concentration on the stability of mouse serum albumin as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 19 shows the effect of pH and sodium ion concentration on albumin stability as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 20 shows the effect of sodium ion concentration on albumin stability at pH 5.0 as determined by the time taken (seconds) for the absorbance (A350) to increase by 0.1 AU.

Figure 21 shows the effect of sodium ion concentration on albumin stability at pH 7.0, 7.5 and 8.0 as determined by the remaining monomer content following incubation at 65°C for 2 hours.

Detailed Description ofthe Invention

Definitions [0009] The terms "cell culture medium", "culture medium" and "medium formulation" refer to a nutritive solution for culturing or growing cells.

[0010] A "serum-free" medium is a medium that contains no serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to one skilled in the art).

[0011] The term "basal medium" refers to any medium which is capable of supporting growth of cells. The basal medium supplies standard inorganic salts, such as zinc, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, an energy source, a buffer system, and essential amino acids. Suitable basal media include, but are not limited to Alpha Minimal Essential Medium (.alpha.MEM); Basal Medium Eagle (BME); Basal Medium Eagle with Earle's BSS; DME/F12; DMEM high Glucose with L-Glutamine; DMEM high glucose without L-Glutamine; DMEM:F12 1:1 with L-Glutamine; Dulbecco's Modified Eagle's Medium (DMEM); F-10; F-12; Glasgow's Minimal Essential Medium (G-MEM); G-MEM with L-glutamine; Grace's Complete Insect Medium; Grace's Insect Medium without FBS; Ham's F-10 with L-Glutamine; Ham's F-12 with L-Glutamine; IMDM with HEPES and L-Glutamine; IMDM with HEPES and without L-Glutamine; IPL-41 Insect Medium; Iscove's Modified Dulbecco's Medium.; L-15 (Leibovitz) without L-Glutamine; L-15 (Leibovitz)(2X) without L-Glutamine or Phenol Red; McCoy's 5A Modified Medium; Medium 199; MEM Eagle without L-Glutamine or Phenol Red (2X); MEM Eagle-Earle's BSS with L-glutamine; MEM Eagle-Earle's BSS without L-Glutamine; MEM Eagle-Hanks BSS without L-Glutamine; Minimal Essential Medium (MEM); Minimal Essential Medium-alpha. (MEM-alpha); NCTC-109 with L-Glutamine; Richter's CM Medium with L-Glutamine; RPMI 1640; RPMI 1640 with L-Glutamine; RPMI 1640 without L-Glutamine; RPMI 1640 with HEPES, L-Glutamine and/or Penicillin-Streptomycin; Schneider's Insect Medium; or any other media known to one skilled in the art. Preferred basal media for stem cell culture include MEF, DMEM, CTS, and DMEM/F-12.

[0012] The term "albumin" means a protein having the same and/or very similar tertiary structure as human serum albumin (HSA) or HSA domains and has similar properties of HSA or the relevant domains. Similar tertiary structures are for example the structures of the albumins from the species mentioned under parent albumin. Some ofthe major properties of albumin are i) its ability to regulate of plasma volume, ii) a long plasma half-life of around 19 days ± 5 days, iii) ligand-binding, e.g. binding of endogenous molecules such as acidic, lipophilic compounds including bilirubin fatty acids, hemin and thyroxine (see also Table 1 of Kragh-Hansen et al, 2002, Biol. Pharm. Bull. 25, 695), iv) binding of small organic compounds with acidic or electronegative features e.g. drugs such as warfarin, diazepam, ibuprofen and paclitaxel (see also Table 1 of Kragh-Hansen et al, 2002, Biol. Pharm. Bull. 25, 695). Not all of these properties need to be fulfilled in order to characterize a protein or fragment as an albumin. If a fragment, for example, does not comprise a domain responsible for binding of certain ligands or organic compounds the variant of such a fragment will not be expected to have these properties either. The term albumin includes variants, and/or derivatives such as fusions and/or conjugations of an albumin or of an albumin variant.

[0013] The term "variant" means a polypeptide derived from a parent albumin comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position. The altered polypeptide (variant) can be obtained through human intervention by modification of the polynucleotide sequence encoding the parental albumin. The variant albumin is preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2 and maintains at least one of the major properties of the parent albumin or a similar tertiary structure as HSA. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofthe EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

[0014] The variant may possess altered binding affinity to FcRn and/or an altered rate of transcytosis across endothelia, epithelia and/or mesothelia mono cell-layer when compared to the parent albumin. The variant polypeptide sequence is preferably one which is not found in nature. A variant includes a fragment, e.g. comprising or consisting of at least 100, 150, 200, 250, 300, 350, 450, 500, 550 contiguous amino acids of an albumin.

[0015] The term "wild-type" (WT) albumin means an albumin having the same amino acid sequence as the albumins naturally found in an animal or in a human being. SEQ ID NO: 2 is an example of a wild-type albumin from Homo sapiens.

[0016] The term "parent" or "parent albumin" means an albumin to which an alteration is made to produce albumin variants. The parent may be a naturally occurring (wild-type) polypeptide or an allele thereof or a variant thereof such as a variant described in PCT/EP2010/066572 or a variant or derivative described in PCT/EP2011/055577.

[0017] The term "fusion" means a genetic fusion of albumin (or a variant or fragment thereof) and a non-albumin protein. The non-albumin protein may be a therapeutic, prophylactic, or diagnostic protein. Examples of albumin fusions are provided in EP624195, WO 2001/079271, WO 2003/059934, WO 2003/060071, W02011051489, PCT/EP11/055577 and EP11164955.

[0018] The term "conjugation" means an albumin (or a variant or fragment or fusion thereof) to which a non-albumin moiety is chemically conjugated. The non-albumin moiety may be a therapeutic, prophylactic, or diagnostic protein. Examples of albumin conjugations are provided in PCT/EP11/055577 and EP11164955.

[0019] The term "suspension culture" refers to cells in culture in which the majority or all of cells in culture are present in suspension, and the minority or none of the cells in the culture vessel are attached to the vessel surface or to another surface within the vessel (adherent cells). The "suspension culture" can have greater than about 50%, 60%, 65%, 75%, 85%, or 95% ofthe cells in suspension, not attached to a surface on or in the culture vessel.

[0020] The term "adherent culture" refers to cells in culture in which the majority or all of cells in culture are present attached to the vessel surface or to another surface within the vessel, and the minority or none of the cells in the culture vessel are in suspension. The "adherent culture" can have greater than 50%, 60%, 65%, 75%, 85%, or 95% of the cells adherent.

[0021] As used herein, the term "mammal" includes any human or non-human mammal, including but not limited to porcine, ovine, bovine, rodents, ungulates, pigs, sheep, lambs, goats, cattle, deer, mules, horses, primates (such as monkeys), dogs, cats, rats, and mice.

[0022] The term "cell' includes any cell such as, but not limited to, any human or non-human mammalian cell as described herein. A cell may be a normal cell or an abnormal cell (e.g. transformed cells, established cells, or cells derived from diseased tissue samples). The cell may be a somatic cells such as a fibroblast or keratinocyte. Preferred cells are stem cells such as, but not limited to, embryonic stem cells, fetal stem cells, adult stem cells and pluripotent stem cells such as induced pluripotent stem cells. Particularly preferred cells are human embryonic stem cells, human fetal stem cells, human adult stem cells and human pluripotent stem cells such as induced human pluripotent stem cells.

[0023] A first aspect of the invention provides a composition comprising 25 to 400 g.L'1 albumin, a solvent, from 200 mM to 1000 mM cations, having a pH from about 5.0 to about 8.0 and wherein the composition comprises equal to or less than 5 mM octanoate, and wherein the albumin is human serum albumin (HSA) having the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 80% sequence identity to SEQ ID NO: 2. An advantage of such a composition is that this formulation provides an albumin which is sufficiently stable to have a useful shelf-life and is not deleterious to the health of mammalian cells (e.g. it is not toxic) when the composition is used in mammalian cell culture. To the extent that other compositions are disclosed herein, they are included merely for reference purposes.

[0024] It is preferred that the composition contains anions to balance the cations.

[0025] The solvent may be an inorganic solvent such as water or an inorganic buffer such as a phosphate buffer such as sodium phosphate, potassium phosphate, or an organic buffer such as sodium acetate or sodium citrate. The buffer may stabilize pH. Sodium phosphate (e.g. NaH2PO4) is a preferred pH buffer, such as pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0.

[0026] The inventors have observed that octanoate is deleterious to mammalian cells in cell culture. Therefore, the composition of the invention comprises low levels of octanoate equal to or less than 5 mM octanoate. A disclosed composition comprises less than 30 mM octanoate, more preferably less than about 28, 26, 24, 22, 20, 18, 16, 15, 14, 12, 10, 8 mM octanoate, even more preferably less than about 6 mM octanoate. The composition of the invention comprises less than 5, 4, 3 mM octanoate, most preferably less than about 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or 0.001 mM octanoate. It is preferred that the composition is substantially free of octanoate. That is, it is preferred that the level of octanoate in the composition is not sufficient to cause a deleterious effect on cells during culture, for example mammalian cells (particularly stem cells such as human stem cells) in cell culture such as in vitro cell culture. Most preferably the composition is free of octanoate (0 mM octanaote).

[0027] Preferred parameters for fatty acids are provided below. The fatty acid content is preferably an average of multiple samples, for example 2, 3, 4 or 5 samples:

[0028] It is also preferred that the overall fatty acid content of the composition is less than or equal to 20 mM, more preferably less than or equal to 15, 10, 5, 4, 3, 2 or 1 mM. It is more preferred that the composition is substantially free of fatty acids, more preferably free of fatty acids.

[0029] Afatty acid profile and a metal ion profile of an albumin formulation comprising 100g.L"1 albumin, < 1 mM octanoate, 250 mM Na+ and having a pH of about 6.5 are provided in Figures 9 and 10, respectively. These are particularly preferred profiles. The albumin composition may comply with one or both of the profiles of Fig 9 and Fig 10.

[0030] Cations may be present from at least about 175 mM, for example from at least about 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mM. Maximum cation concentrations may include 1000, 950, 900, 850, 800, 750, 700, 650, 600, 575, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275 and 250 mM. In the context ofthe present invention, the composition comprises from 200 mM to 1000 mM cations. Preferred cation concentrations include 200 to 500 mM. More preferred is a cation concentration of about 200 to 350 mM. Most preferred is a cation concentration of about 250 mM.

[0031] The pH of a composition may be between about 5.0 and about 9.0, for example from about 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, or 8.5 to about 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75 or 9.0. In the context of the present invention, the pH of the composition is from about 5.0 to 8.0, such as from about 6.0 to about 8.0, more preferably from about 6.0 to about 7.0 or 6.0 to 6.5. Most preferred the pH is about 6.5.

[0032] The cations ofthe composition may be provided by any cation and may be provided by one or more (several) classes or species as described below. For example, the cations may be either mono or bivalent, monoatomic or polyatomic and may be provided by one or more (several) of an alkali metal (such as sodium, potassium), an alkaline earth metal (such as calcium, magnesium) or ammonium. It is preferred that the cations are provided by sodium and/or potassium and/or magnesium, most preferably sodium or magnesium.

[0033] Cations may be provided by a salt of an inorganic acid (e.g. a group 1 or 2 metal or ammonium salt such as sodium chloride), a salt of a divalent acid (e.g. a group 1 or group 2 metal or ammonium sulphate or phosphate such as sodium sulphate) or a salt of an organic acid (e.g. a group 1 or group 2 metal or ammonium salt of acetate or citrate such as sodium acetate).

[0034] Cations and anions used to stabilize the albumin may be provided by (i) salts and/or (ii) pH buffers such as described herein. Therefore, there may be more than one (several) species of cation or anion, such as 2, or 3 species. There may be more than one (several) source of a single cation, for example Na which may be provided by both a pH buffer (such as sodium phosphate) and a salt (such as NaCI).

[0035] Anions useful to the invention include inorganic anions such as phosphate, and halides such as chloride, and organic anions such as acetate and citrate. Anions may be either mono or bivalent, monoatomic or polyatomic. Preferred anions include sulphate, acetate phosphate and chloride, particularly chloride, sulphate and acetate.

[0036] Therefore, the composition may comprise one or more (several) of an alkali metal phosphate or chloride (such as sodium phosphate, potassium phosphate, sodium chloride or potassium chloride), an alkaline earth metal phosphate (such as calcium phosphate, magnesium phosphate, calcium chloride, magnesium chloride) or ammonium phosphate or ammonium chloride.

[0037] The composition may have an overall ionic strength of at least 175 mmol.L"1. For example, from about 175 to 1000 mmol.L"1 such as from about 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mmol.L"1 to about 1000, 950, 900, 850, 800, 750, 700, 650, 600, 575, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250 mmol.L"1. More preferred is an overall ionic strength of about 200 to 350 mmol.L"1. Most preferred is an ionic strength of about 250 mmol.L"1.

[0038] The inventors have realized that the presence of stabilizers such as detergents (e.g. polysorbate 80 ('Tween®')) can be deleterious to mammalian cells in cell culture. Therefore, it is preferred that the composition comprises less than 20 mg.L"1 detergent (e.g. polysorbate 80), preferably less than 15, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.01, 0.001 mg.L"1 detergent (e.g. polysorbate 80). Even more preferably, the composition is substantially free of detergent (e.g. polysorbate 80). That is, it is preferred that the level of detergent (e.g. polysorbate 80) in the composition is not sufficient to cause a deleterious effect on cells during culture, for example mammalian cells (particularly stem cells such as human stem cells) in cell culture such as in vitro cell culture. Most preferably the composition is free of detergent (e.g. polysorbate 80). Detergent (e.g. polysorbate 80) levels can be assayed by techniques known to the skilled person for example, but not limited to, the assay disclosed in WO 2004/099234.

[0039] For some cell media, it is preferred that the media is substantially free or free of tryptophan (e.g. tryptophan-free RPMI 1640 as disclosed by Lee et al., (2002), Immunology Vol 107(4): 452-460). An albumin composition may be added to a medium. Therefore, in order to maintain the tryptophan free character of a medium, an albumin composition which has low levels of amino acids (e.g. N-acetyl tryptophan), is substantially free of amino acids (e.g. N-acetyl tryptophan) or is free of amino acids (e.g. N-acetyl tryptophan) is useful. Therefore, it is preferred that the albumin composition comprises less than 5 mM amino acids (e.g. N-acetyl tryptophan), preferably less than 4, 3, 2, 1, 0.5, 0.1, 0.01, 0.005, 0.001 mM amino acids (e.g. N-acetyl tryptophan). Even more preferably, the composition is substantially free of amino acids (e.g. N-acetyl tryptophan). That is, it is preferred that the level of amino acids (e.g. N-acetyl tryptophan) in the composition is not sufficient to cause a deleterious effect on cells during culture, for example mammalian cells (particularly stem cells such as human stem cells) in cell culture such as in vitro cell culture. Most preferably the composition is free of amino acids (e.g. N-acetyl tryptophan).

[0040] It is even more preferred that the composition is substantially free of, or completely free of, octanoate, amino acids (e.g. N-acetyl tryptophan) and detergent (e.g. polysorbate 80).

[0041] In order to identify whether or not there is a deleterious or toxic effect of the albumin formulation on cell culture, a test may be carried out by preparing a first cell culture medium containing the albumin formulation ofthe invention and preparing one or more (several) control cell culture media and monitoring their effect on cell lines. A control cell culture medium is identical to the first cell culture medium except that the albumin formulation of the invention is replaced with another albumin formulation, e.g. an albumin formulation stabilized with octanoate, a detergent (e.g. polysorbate 80) and/or an amino acid (e.g. n-acetyl tryptophan). The test media and controls may be used to cultivate one or more (several) cell lines (e.g. a cell line as described herein) and the effect of the albumin on the cells monitored e.g. by monitoring cell growth, cell morphology and/or cell differentiation. It is preferred that the test is carried out over multiple passages of the cell line, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 passages. Suitable methods are known in the art. It is preferred that the albumin formulation of the present invention is less toxic or deleterious to cells than an albumin stabilized with higher levels of octanoate, detergent or amino acids. For example, a medium comprising the albumin composition ofthe invention may show at least a 2-, 5-, 10-, 100-, 1000-, 10000-, or 100000-fold improvement over a control medium comprising another albumin formulation, e.g. an albumin formulation stabilized with octanoate, a detergent (e.g. polysorbate 80) and/or an amino acid (e.g. n-acetyl tryptophan). The 2, 5, 10, 100, 1000, 10000, or 100000-fold improvement may relate to viable cell numbers, correct or healthy cell morphology and/or to the number or relative number of differentiated cells, particularly cells showing differentiation to a desired cell class or type.

[0042] It is preferred that the stability of the albumin composition is higher than that of equivalent albumin in water or in 150 mM Na. One method to compare stability, particularly related to the formation of insoluble aggregates of albumin, is: 1. i) place an aliquot (e.g. 1 ml.) ofthe albumin composition in a cuvette (e.g. a polystyrene cuvette, such as Sarstedt 10x4x45 mm); 2. ii) place the cuvette in a temperature controlled spectrophotometer that has been preequilibrated and controlled at a desired temperature, e.g. 65°C; 3. iii) Monitor I measure the absorbance of the composition at 350 nm, referenced against an empty cuvette over a desired time period (e.g. 2 hours) by taking a reading at defined intervals (e.g. every 18 seconds) 4. iv) Process the data by taking the first several (e.g. seven) data points, average the data point readings and subtract this data point from all data points in order to provide base absorbance values of around 0. 5. v) Determine and/or record the time taken for the processed absorbance values to increase by 0.1 AU (Absorbance Units) above this baseline.

[0043] It is preferred that stability analysis is performed in duplicate.

[0044] It is preferred that the stability ofthe albumin composition ofthe invention is sufficiently high so that the time taken for the measured absorbance to increase by 0.1 AU above the baseline (according to the above described test carried out at 65°C), compared to a control solution of albumin at the same concentration in a solvent such as 150 mM Na or water and measured under the same conditions is at least 10% better. It is more preferred that the stability is at least 20, 30, 40, 50, 60, 70, 80, 90 or 100% better.

[0045] An alternative or additional stability test, particularly for the formation of soluble aggregates of albumin, is to monitor the formation of soluble albumin polymer by GP-HPLC over time at a set temperature. One suitable stability study with measurement by GP HPLC includes: 1. i) Placing 10ml_ sterilely (e.g. by filtration through a sterile 0.22pm filter) of each sample to be investigated into sterile vials (e.g. baked 10ml_ glass vials) which are then stoppered (e.g. with a sterile butyl rubber seal and optionally over-sealed). 2. ii) A TO sample of ~200pL is then taken and the vial is incubated at a specified temperature (e.g. placed in a water bath that is set at a specified temperature (e.g. at 40°C)). 3. iii) Samples (~200pL) are then taken from each of the vials after certain time points (e.g. 14 days). 4. iv) injecting an aliquot (e.g. 25pL) of the albumin sample taken out of the vial (at <50mg/ml_) onto a GP-HPLC column (e.g. 7.8mm id x 300mm length TSK G3000SWXL column, (Tosoh Bioscience), with a 6.0mm id x 40mm length TSK SW guard column (Tosoh Bioscience)); 5. v) chromatographing the aliquot in a suitable buffer (e.g. 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% (w/v) sodium azide, pH 7.0) at a suitable speed (e.g. 1 mL/min) 6. vi) monitoring the chromatograph procedure e.g. by UV detection at 280nm; 7. vii) quantifying one or more (several), or all, of monomer, dimer, trimer and polymer content of the aliquot as % (w/w) by identifying their respective peak area relative to the total peak area.

[0046] It is preferred that the test is carried out in triplicate.

[0047] Therefore, the invention also provides an albumin composition having a stability as defined in one or both of the above mentioned tests. A method for producing an albumin composition may include one or both ofthe above mentioned tests.

[0048] Albumin has been described and characterized from a large number of mammals and birds (e.g. albumins listed in WO2010/092135 (particularly Table 1) and PCT/EP11/055577 (particularly page 9 and SEQ ID No: 2, 4-19 and 31).

[0049] The composition of the invention may comprise one or more (several) albumins. In the context of the composition of the present invention, the albumin is HSA having the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 80% sequence identity to SEQ ID NO: 2. To the extent that other albumins, variants or derivatives are mentioned herein they are included merely for reference purposes. Albumin may be selected from human albumin (e.g. AAA98797 or P02768-1, SEQ ID NO: 2 (mature), SEQ ID NO: 3 (immature)), non-human primate albumin, (such as chimpanzee albumin (e.g. predicted sequence XP_517233.2 SEQ ID NO: 4), gorilla albumin or macaque albumin (e.g. NP_001182578, SEQ ID NO: 5), rodent albumin (such as hamster albumin (e.g. A6YF56, SEQ ID NO: 6), guinea pig albumin (e.g. Q6WDN9-1, SEQ ID NO: 7), mouse albumin (e.g. AAH49971 or P07724-1 Version 3, SEQ ID NO: 8, or the mature sequence SEQ ID NO: 19) and rat albumin (e.g. AAH85359 or P02770-1 Version 2, SEQ ID NO: 9)), bovine albumin (e.g. cow albumin P02769-1, SEQ ID NO: 10), equine albumin such as horse albumin (e.g. P35747-1, SEQ ID NO: 11) or donkey albumin (e.g. Q5XLE4-1, SEQ ID NO: 12), rabbit albumin (e.g. P49065-1 Version 2, SEQ ID NO: 13), goat albumin (e.g. ACF10391, SEQ ID NO: 14), sheep albumin (e.g. P14639-1, SEQ ID NO: 15), dog albumin (e.g. P49822-1, SEQ ID NO: 16), chicken albumin (e.g. P19121-1 Version 2, SEQ ID NO: 17) and pig albumin (e.g. P08835-1 Version 2, SEQ ID NO: 18). Mature forms of albumin are particularly preferred and the skilled person is able to identify mature forms using publicly available information such as protein databanks and/or by using signal peptide recognition software such as SignalP (e.g., SignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6)). SignalP Version 4.0 is preferred (Petersen et al (2011) Nature methods (8): 785-786).

[0050] Human albumin as disclosed in SEQ ID NO: 2 or any naturally occurring allele thereof, is the preferred albumin of the albumin composition according to the invention. SEQ ID No: 2 may be encoded by the nucleotide sequence of SEQ ID No: 1.

[0051] The albumin, particularly the human albumin, may be a variant. A derivative of albumin includes a fusion of conjugation of an albumin or of an albumin variant. Albumin variants may have at least 70% identity to HSA (SEQ ID No: 2), such as at least 72, 73, 75% identity to HSA. In the context ofthe present invention, the albumin has at least 80, 85, 90, 95, 96, 97, 98, 99 or 99.5 % identity to HSA. The albumin variant may have one or more point (several) mutations, e.g. K573P, K573Y, K573W, K500A compared to a parent albumin such as those provided in the sequence listing, particularly SEQ ID No: 2 (mutations are described in relation to SEQ ID No: 2 and the skilled person can identify equivalent mutations in other albumins by aligning an albumin sequence against SEQ ID No: 2 using the EMBOSS software described herein). For an albumin having about 70 to 80 % identity to SEQ ID No: 2 (such as mouse albumin e.g. SEQ ID NO: 19), it is more preferred that the cation is present from at least 250 mM.

[0052] Albumin may be present in a composition at a concentration of from about 1 g.L"1 to about 400 g.L"1. For example, the concentration may be from about 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 g.L·1 to about 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375 or 400 g.L·1. In the context of the present invention albumin is present at a concentration from 25 to 400 g.L·1. It is preferred that the concentration of albumin is from about 50 g.L·1 to about 200 g.L·1 [0053] Advantageously, the composition may comprise a recombinant albumin. That is, the albumin may be sourced from a recombinant organism such as a recombinant microorganism, recombinant plant or recombinant animal. Since some users prefer animal-free ingredients, it is more preferred that the albumin is sourced from a non-animal recombinant source, such as a recombinant microorganism or recombinant plant. Preferred microorganisms include prokaryotes and, more preferably, eukaryotes such as animals, plants, fungi or yeasts, for example, but not limited to, the following species in which albumins have been successfully expressed as recombinant proteins: • fungi (including but not limited to Aspergillus (WO 2006/066595), Kluyveromyces (Fleer 1991, Bio/technology 9, 968-975), Pichia (Kobayashi 1998 Therapeutic Apheresis 2, 257-262) and Saccharomyces (Sleep 1990, Bio/technology 8, 42-46)), bacteria (Pandjaitab 2000, J. Allergy Clin. Immunol. 105, 279-285)), • animals (Barash 1993, Transgenic Research 2, 266-276) • plants (including but not limited to potato and tobacco (Sijmons 1990, Bio/technology 8, 217 and Farran 2002, Transgenic Research 11,337-346) and rice e.g. Oryza sativa) • mammalian cells such as CHO and HEK.

[0054] The albumin of the invention is preferably produced recombinantly in a suitable host cell. Non-animal host cells are preferred. A preferred host is yeast, preferably selected among Pichia or Saccharomycacae, more preferred Saccharomyces cerevisiae.

[0055] A preferred composition comprises 50 to 250 g.L-1 albumin, 200 to 300 mM Na+, 20 to 30 mM phosphate, comprises less than 2 mM octanoate and has a pH between about 6.0 and 7.0. A particularly preferred composition comprises 50 to 150 g.L'1 albumin, 225 to 275 mM Na+, 20 to 30 mM phosphate, comprises less than 1 mM octanoate and has a pH of about 6.5.

[0056] Also disclosed is a composition comprising albumin, a solvent, at least 175 mM cations, having a pH from about 5.0 to about 8.0 or 9.0. An advantage of such a composition is that this formulation provides an albumin which is sufficiently stable to have a useful shelf-life and is not deleterious to the health of mammalian cells when the composition is used in mammalian cell culture. Preferred parameters for the solvent, cations, ionic strength, and pH are the same as those disclosed in relation to the first aspect ofthe invention.

[0057] The albumin composition according to the invention may be provided in a flexible polymeric container, such as a bag. Suitable container volumes include from about 50 ml. to about 10 000 mL, e.g. 50 mL, 1000 ml_, 5000 mL and 10 000 mL. It is preferred that the container comprises one or more (several) inlets or outlets to allow filling of the container and/or dispensing from the bag. The albumin composition may be sterilized, e.g. prior to or after being filled in the container.

[0058] The production of recombinant albumin is known in the art and numerous hosts such as

Escherichia coli (EP 73, 646), yeast has been reported in WO 00/44772, EP 0683233 A2, and US 5,612,196, and Bacillus subtillis (Saunders et al., 1987, J. Bacteriol, 169, 2917-2925), Aspergillus. Production of albumin has been demonstrated in transgenic plants such as but not limited to tobacco, rice, and maize and in transgenic animals such as but not limited to chicken and bovine [0059] Also disclosed is a cell culture medium comprising a composition of the invention as described herein and a basal medium.

[0060] A second aspect of the invention provides a method for preparing a cell culture medium comprising supplementing a basal medium with the composition of the first aspect of the invention. The cell culture medium may, for example, be for the culture of mammalian cells such as human cells. The cell culture medium may, for example, be for the culture of stem cells or of gametes or of embryos for example cell culture for assisted reproductive technology (ART) purposes.

[0061] It is preferred that the cell culture medium is substantially free of animal-derived components. It is more preferred that the cell culture medium is free of animal-derived components. In this context, 'animal-derived' component means a component which has been obtained from an animal. It does not include a component which is identical or substantially identical to an animal-derived component but which, instead of being obtained from an animal, is obtained as a recombinant component from a non-animal. A non-animal includes a plant, such as rice, a microorganism such as a yeast or bacterium.

[0062] Examples of cell culture media in which the albumin formulation may be used include those described in WO 2008/009641.

[0063] A cell culture medium comprising the albumin formulation of the first aspect of the invention may or may not comprise one or more (several) fatty acids, such as provided by a fatty acid supplement. Fatty acid supplements are commercially available, e.g. F7050 Fatty Acid Supplement (Animal-component free, liquid, sterile-filtered, suitable for cell culture) available from Sigma-Aldrich.

[0064] A third aspect of the invention relates to use of an albumin formulation, composition or cell culture medium as described herein to culture cells, such as cells described with reference to the second aspect of the invention and/or described below.

[0065] A fourth aspect of the invention relates to a method of culturing cells comprising incubating cells in a culture medium as described herein. The cells may be the cells described with reference to the second aspect of the invention and/or described below.

[0066] The albumin formulation of the first aspect of the invention may be used in pharmaceutical products. Therefore, a pharmaceutical composition may comprise the albumin formulation and an active pharmaceutical ingredient (API).

[0067] Also disclosed is the use of a high cation concentration to stabilize albumin, e.g. from at least 175 mM cations as described for the first aspect ofthe invention.

[0068] The compositions and media of the present invention may be used to culture a variety of cells. In one embodiment, the medium is used to culture eukaryotic cells such as plant and/or animal cells. The cells can be mammalian cells, fish cells, insect cells, amphibian cells or avian cells. The medium can be used to culture cells selected from the group consisting of MK2.7 cells, PER-C6 cells, NSO, GS-NSO, CHO cells, HEK 293 cells, COS cells and Sp2/0 cells.MK2.7 (ATCC Catalogue Number CRL 1909) is an anti-murine VCAM IgGI expressing Hybridoma cell line derived from the fusion of a rat splenocyte and a mouse Sp2/0 myeloma. MK2.7 is a non-adherent cell line that can be grown in serum-free media. Other types of cells can be selected from the group consisting of 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2/0 cells; and/or the hybridoma cells listed in Table 2, WO 2005/070120 or any other cell type disclosed herein or known to one skilled in the art.

[0069] Preferred cells includes stem cells such as but not limited to, embryonic stem cells, fetal stem cells, adult stem cells and pleuripotent stem cells such as induced pleuripotent stem cells. Particularly preferred cells are human embryonic stem cells, human fetal stem cells, human adult stem cells and human pleuripotent stem cells such as induced human pleuripotent stem cells. The cell line may be derived from a blastocyst. The cell line may test positive for one or more (several) of the following cell markers: POU5F1 (OCT-4), SSEA-3, SSEA-4, TRA1-60, TRA1-81, ALPL, telomerase activity, and/or hES-Cellect™ (Cellartis AB, Gothenburg Sweden). The cell line may test negative for cell marker ALPL and/or SSEA-1. Particularly preferred cell lines include SA121 and SA181 (Cellartis AB, Gothenburg, Sweden).

[0070] Additional mammalian cell types can include, but are not limited to, including primary epithelial cells (e.g. keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells) and established cell lines and their strains (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 91 1 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-PK.sub.2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.1 cells, GH3 cells, L2 cells, LLC-RC 256 cells, MH.sub.IC1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-2 1 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, Wl- 26 cells, MiCl.sub.1 cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H/IOTI/2 cells, HSDM.sub.IC3 cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, Cll cells, and Jensen cells, or derivatives thereof).

[0071] Cells include cancer cells such, but not limited to, the following cancer cell lines: human myeloma (e.g., KMM-1, KMS-11, KMS-12-PE, KMS-12-BM, KMS-18, KMS-20, KMS-21-PE, U266, RPMI8226); human breast cancer (e.g., KPL-1, KPL-4, MDA-MB-231, MCF-7, KPL-3C, T47D, SkBr3, HS578T, MDA4355, Hs 606 (CRL-7368), Hs 605.T (CRL-7365) HS 742.T (CRL-7482), BT-474, HBL-100, HCC202, HCC1419, HCC1954, MCF7, MDA-361, MDA-436, MDA-453, SK-BR-3, ZR-75-30, UACC-732, UACC-812, UACC-893, UACC-3133, MX-1 and EFM-192A); ductal (breast) carcinoma (e.g., HS 57HT (HTB-126), HCC1008 (CRL-2320), HCC1954 (CRL-2338; HCC38 (CRL-2314), HCC1143 (CRL-2321), HCC1187 (CRL-2322), HCC1295 (CRL-2324), HCC1599 (CRL-2331), HCC1937 (CRL-2336), HCC2157 (CRL-2340), HCC2218 (CRL-2343), Hs574.T (CRL-7345), Hs 742.T (CRL-7482); skin cancer (e.g., COLO 829 (CRL-1974), TE 354.T (CRL-7762), Hs 925.T (CIU-7677)); human prostate cancer (e.g., MDA PCa 2a and MDA PCa 2b); bone cancer (e.g., Hs 919.T (CRL-7672), Hs 821.T (CRL-7554), Hs 820.T (CRL-7552)y HS 704.T (CRL-7444), HS 707(A).T (CRL-7448), HS 735.T (CRL-7471), HS 860.T (CRL-7595)y HS 888.T.(CRL-7622); HS 889.T (CRL-7626); HS 890.T (CRL-7628), Hs 709.T (CRL-7453)); human lymphoma (e.g., K562); human cervical carcinoma (e.g., HeLA); lung carcinoma cell lines (e.g., H125, H522, H1299, NCI-H2126 (ATCC CCL-256), NCI-H1672 (ATCC CRL-5886), NCI-2171 (CRL-5929); NCI-H2195 (CRL05931); lung adenocarcinoma (e.g., NCI-H1395 (CRL-5856), NCI-H1437 (CRL-5872), NCI-H2009 (CRL-5911), NCI-H2122 (CRL-5985), NCI-H2087 (CRL-5922); metastatic lung cancer (e.g., bone) (e.g., NCI-H209 (HTB-172); colon carcinoma cell lines (e.g., LN235, DLD2, Colon A, LIM2537, LIM1215, LIM1863, LIM1899, LIM2405, LIM2412, SK-CO1 (ATCC HTB-77), HT29 (ATCC HTB38), LoVo (ATCC CCL-229), SW1222 (ATCC HB-11028), and SW480 (ATCC CCL-228); ovarian cancer (e.g., OVCAR-3 (ATCC HTB-161) and SKOV-3 (ATCC HTB-77); mesothelioma (e.g., NCI-h2052 (CRL-5915); neuroendocrine carcinoma (e.g., HCI-H1770 (e.g., CRL-5893); gastric cancer (e.g., LIM1839); glioma (e.g., T98, U251, LN235); head and neck squamous cell carcinoma cell lines (e.g., SCC4, SCC9 and SCC25); medulloblastoma (e.g., Daoy, D283 Med and D341 Med); testicular non-seminoma (e.g., TERA1); prostate cancer (e.g., 178-2BMA, Du 145, LNCaP, and PC-3). Other cancer cell lines are well known in the art.

[0072] The media disclosed herein can be used to culture cells in suspension or adherent cells. The media of the present invention are suitable for adherent, monolayer or suspension culture, transfection, and/or cultivation of cells, and for expression of proteins or antibodies in cells in monolayer or suspension culture.

[0073] Cell culture can be performed using various culture devices, for example, a fermenter type tank culture device, an air lift type culture device, a culture flask type culture device, a spinner flask type culture device, a microcarrier type culture device, a fluidized bed type culture device, a hollow fiber type culture device, a roller bottle type culture device, a packed bed type culture device or any other suitable device known to one skilled in the art.

[0074] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention. To the extent that the examples refer to subject matter that falls outside of the scope of the present invention, it is included merely for reference purposes.

Examples

Example 1: Effect of n-acetyl-tryptophan, phosphate concentration and sodium concentration on the stability of albumin.

[0075] Aim: Previous work indicated that monitoring the formation of insoluble aggregates at 65°C, through an increase in absorption at 350nm, is a valid method for screening ofthe effect of different formulation (composition) parameters on the stability of albumin. Since octanoate and polysorbate 80 appear to be detrimental to stem cell growth, it is preferred that an albumin formulation is substantially free of these components. This Example analyzes the effect of pH, sodium ion and buffer concentration on the stability of albumin. A common stabilizer for albumin is n-acetyl-tryptophan, therefore it is included in this Example as a test constituent.

[0076] Method: Albumin at 100 mg/mL in 145 mM NaCI (albumin batch 1401) was diluted to 10 mg/mL according to Table 3. The buffers used for dilution are shown in Tables 1 and 2.

Table 2

[0077] Once diluted the samples were adjusted to their target pH with 1 M HCI, the volume of which was insignificant and does not alter the final albumin or constituent concentrations. An aliquot (1 ml.) of the resulting solution was then placed in a polystyrene cuvette (Sarstedt 10x4x45 mm). The cuvette was then placed into a temperature controlled spectrophotometer that had been pre-equilibrated and controlled at 65°C. The absorbance at 350 nm, referenced against an empty cuvette, was then monitored over a 2 hour period with a reading being taken every 18 seconds. The data were processed by taking the first 7 data points, averaging them (calculating the mean) and then subtracting this from all data points in order to give base absorbance values of around 0. The time taken for this absorbance to then increase by 0.1 AU (Absorbance Units) above this baseline was then recorded for that particular formulation sample. Each formulation sample was performed in duplicate and the time for the absorbance to increase by 0.1 AU for each replicate averaged.

[0078] Results: The processed data with the time for each sample to increase by 0.1 AU, were plotted for time for absorbance increase against pH for each of the formulation constituents tested; n-acetyl-tryptophan, phosphate concentration and sodium concentration. Values above 7200 sec were extrapolated. The data are presented in Figs 1 (pH and n-acetyl tryptophan), 2 (pH and phosphate) and 3 (pH and sodium).

Conclusions:

[0079] • For all the data, except for 50 mM sodium, the optimum pH was between pH 6 and 7. For 50 mM sodium, although insoluble aggregates were not forming, it is possible that high levels of soluble oligomers were being generated and these were not coalescing to form insoluble aggregates. Soluble aggregates can be identified by GP-HPLC. • For the phosphate buffer concentration, there was no significant difference between 50 and 100 mM. However, 0 mM phosphate did appear to be slightly more stable between pH 6 to 8. Although using no phosphate would be best in terms of stability, the use of a buffer aids pH control, for example because it reduces or eliminates the requirement to pH-adjust an albumin prior to formulation. • Increasing sodium levels had a significant effect on stability with a large increase in stability between 145 and 300 mM sodium.

Example 2: Effect of increasing sodium concentration on albumin stability.

[0080] Aim: Example 1 indicated that increased levels of sodium had a beneficial effect on albumin stability. To investigate this further, increasing concentrations of sodium over a wider range than in Example 1 at the optimum pH range were investigated.

[0081] Method: Albumin at 100 mg/mL in 145 mM NaCI (albumin batch 1401) was diluted to 10 mg/mL according to the Table 6. The buffers used for dilution are shown in Tables 4 and 5.

Table 5

[0082] The dilution was performed by first mixing the albumin and buffer as a bulk and then

adjusting it to the correct pH by the addition of 1 M HCI. This was then divided and water and 5 M NaCI added as appropriate. This ensured that all samples were at exactly the same pH. An aliquot (1 mL) of the resulting solution was then placed in a polystyrene cuvette (Sarstedt 10x4x45 mm). The cuvette was then placed into a temperature controlled spectrophotometer that had been pre-equilibrated and controlled at 65°C. The absorbance at 350 nm, referenced against an empty cuvette, was then monitored over a 2 hour period with a reading being taken every 18 seconds. The data were processed by taking the first 7 data points, averaging them (calculating the mean) and then subtracting this from all data points in order to give base absorbance values of around 0. The time taken for this absorbance to then increase by 0.1 AU above this baseline was then recorded for that particular formulation sample. Each formulation sample was performed in duplicate and the time for the absorbance to increase by 0.1 AU for each replicate averaged.

[0083] Results: The processed data with the time for each sample to increase by 0.1 AU, were plotted for time for absorbance increase against Na concentration for each pH. Values above 7200 sec were extrapolated. The data are shown in Fig. 4.

Conclusion: [0084] • Consistent with Example 1, increasing levels of sodium increased albumin stability. This was particularly the case around 200 mM where there was a sudden increase in stability. This was the case for all pHs, although it was less obvious at pH 6 since even at <200 mM increasing salt was still having a beneficial effect. The fact that the increase was around 200 mM maybe the reason that it has not been observed previously, since most other albumin formulations are 150 mM or lower in order to keep them approximately physiological. For an albumin used in cell culture media, this should not be an issue as the albumin will be diluted down into the media and the overall salt concentration of the media will be suitable for cell culture. • pH 6 was slightly better than pH 6.5, both being significantly better than pH 7.

Example 3: Effect of sodium concentration on the stability of different concentrations of albumin.

[0085] Aim: Example 2 shows that sodium concentration is important to stability of albumin. Example 2 was done at an albumin concentration of 10 mg/mL. In order to confirm that this effect is also true at higher concentrations the effect of sodium at higher albumin concentrations was investigated.

[0086] Method: Albumin at 100 mg/mL in 145 mM NaCI (albumin batch 1401) was diluted to 50 or 90 mg/mL according to Table 9. The buffers used for dilution are shown in Tables 7 and 8.

[0087] The dilution was performed by first mixing the albumin and buffer as a bulk and then adjusting it to the correct pH by the addition of 1 M HCI. This was then divided and water and 5 M NaCI added as appropriate. This ensured that all samples were at exactly the same pH.

[0088] An aliquot (1 mL) of the resulting solution was then placed in a polystyrene cuvette (Sarstedt 10x4x45 mm). The cuvette was then placed into a temperature controlled spectrophotometer that had been pre-equilibrated and controlled at 65°C. The absorbance at 350 nm, referenced against an empty cuvette, was then monitored over a 2 hour period with a reading being taken every 18 seconds. The data were processed by taking the first 7 data points, averaging them (calculating the mean) and then subtracting this from all data points in order to give base absorbance values of around 0. The time taken for this absorbance to then increase by 0.1 AU above this baseline was then recorded for that particular formulation sample. Each formulation sample was performed in duplicate and the time for the absorbance to increase by 0.1 AU for each replicate averaged.

[0089] Results: The processed data with the time for each sample to increase by 0.1 AU were plotted for time for absorbance increase against Na concentration for each pH (6.0, 6.5 and 7.0) at 50 mg/mL albumin and then for 3 different albumin concentrations (10, 50 and 90 mg/mL) at pH 6.5. The data are shown in Figs 5 and 6.

Conclusions:

[0090] • At 50 mg/mL albumin the trend of increasing sodium concentration improving albumin stability was confirmed at all 3 pHs. In this instance pH 6.5 was the best. • At pH 6.5, the trend of increased sodium improving stability was again confirmed at all albumin concentrations. The trend was not as pronounced at 90 g/L, but it was still the case that sodium concentrations above 200 mM significantly improved the albumin stability.

Example 4: Effect of sodium concentration on the production of soluble aggregates in albumin.

[0091] Aim: Examples 1 to 3 show, using the formulation screening assay that measures insoluble aggregates, that increasing sodium concentrations improved albumin stability. In order to look at soluble aggregates (albumin polymer) GP-HPLC needs to be used as the measurement tool with polymer formation monitored in an accelerated stability trial at 40°C over a 2 week period. The tests were carried out at pH 6.5 because this was shown, by Examples 1 to 3, to be a preferred pH. A control of albumin in previous formulation conditions (pH 8.6, 150 mM Na) was also used to confirm that a new formulation would be significantly beneficial. The actual albumin concentration was 90 mg/mL instead of the anticipated 100 mg/mL as this was the highest that could be used allowing for dilution into the various formulations. However, it is thought that observed trends at this slightly lower concentration will be the same at higher albumin concentrations.

[0092] Method: Albumin at 100 mg/mL in 145 mM NaCI (albumin batch 1401) was diluted to 90 mg/mL according to Table 12. The buffers used for dilution are shown in Tables 10 and 11.

[0093] The dilution was performed by first mixing the albumin and buffer as a bulk and then adjusting it to the correct pH by the addition of 1 M HCI. This was then divided into appropriate sized aliquots and water and 5 M NaCI added as required. This ensured that all samples were at exactly the same pH.

[0094] 10 mL of each sample was then sterile filtered into a baked 10 mL glass vial stopped with a sterile butyl rubber seal and then over-sealed. A TO sample of -200 pL was then taken and the vial placed in a water bath that was set at 40°C. Samples (-200 pL) were then taken from each of the vials after 14 days, diluted 2 fold and injected in triplicate on the GP-HPLC system.

[0095] The GP-HPLC system was run, by injecting (25 pL) onto a 7.8 mm id x 300 mm length TSK G3000SWxl column (Tosoh Bioscience), with a 6.0 mm id x 40 mm length TSK SW guard column (Tosoh Bioscience). Samples were chromatographed in 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% (w/v) sodium azide, pH 7.0 at 1 mL/min and monitored by UV detection at 280 nm. Monomer, dimer, trimer and polymer content were quantified as % w/w by their respective peak area relative to the total peak area. Results from the triplicate injections were averaged to get a mean result for each sample.

[0096] Results: The data for the 14 day time point for monomer (Fig. 7) and polymer (Fig. 8) were plotted against sodium concentration.

Conclusions: [0097]

• The formulation at pH 6.5 was significantly better than that at pH 8.6 used for albumin batch 1401. The level of polymer significantly increased at pH 8.6, rising to approximately 20% after 2 weeks at 40°C compared to -2% for the same sodium concentration at pH 6.5. • The proposed trend of increasing sodium increasing albumin stability observed with the screening assay is confirmed here for soluble aggregates with a significant trend of reduced polymer formation with increasing sodium concentration. Going from 150 mM, a standard albumin concentration due to it being close to physiological conditions, to 200 mM sodium the level of polymer decreases by >2 fold with then a further decrease of ~2 fold going from 200 to 250 mM. Although the polymer decreases even further with higher salt concentrations up to 350 mM (and potentially beyond) the rate of decrease is slower. These results are matched in an increase in monomer remaining with increased sodium. Overall there is a > 4 fold decrease in polymer formation going from 150 to 250 mM sodium. Consequently, a preferred albumin formulation is 25 mM phosphate buffer pH 6.5, 250 mM sodium. The phosphate is present to aid pH control. Notably, sodium will come from both sodium chloride and sodium phosphate (including any NaOH used to ensure the pH of the phosphate is correct) and therefore the buffer is not 250 mM NaCI. • Although this work has all been performed with sodium, similar monovalent or bivalent metal ions are expected to have a similar effect. However, sodium is a preferred metal ion because it is known to be compatible with stem cell culture.

Example 5: Effect of albumin concentration and sodium ion concentration on stability of albumin [0098] Method: A sample of purified albumin containing low octanoate (-0.2 mM octanotae, 100 g/L albumin) was diafiltered against a minimum of 10 continuous volumes of 25 mM phosphate, 50 mM sodium pH 6.5 and then concentrated to 338 g/L using a 10 KDa Pall Omega crossflow UF to generate a 50 mM sodium starting material. The sample was then diluted with water, 5 M NaCI and 0.5 M sodium phosphate pH 6.5 as shown in Table 13:

[0099] The samples were then aseptically filtered (0.22 pm filter) into sterile 5 mL glass vials and the vials placed in a 40°C incubator for 4 weeks. An aliquot from each sample was taken out at intervals, diluted to 40 g/L with water and assayed for soluble aggregates by GP-HPLC as per Example 4.

[0100] Results: Figure 11 shows monomer levels after a 4 week incubation. A higher monomer content shows better stability.

Conclusions: [0101] • All points follow a trend apart from the 150 g/L albumin, 100 mM sodium sample. It is unclear why this sample is out of trend but is likely to be an outlier and does not detract from the overall conclusions of the experiment. • For all albumin concentrations tested, there is a clear correlation of increasing monomer content i.e. increasing stability with increasing sodium content. • The majority of the improved stability comes with increasing the sodium ion concentration up to -200 mM. Above this concentration, although there is some further increase in stability it has mostly levelled off. Consequently the optimum sodium ion concentration is 200 mM or higher.

Example 6: Effect of different cations on stability of albumin [0102] Method: A sample of purified albumin containing low octanoate (-0.2 mM @ 100 g/L albumin) was diluted initially to 50 mg/mL with water such that it contained 50 mg/mL albumin, 75 mM NaCI and no pH buffer constituent. The pH was adjusted with 0.5 M HCI to pH 6.43, the amount of HCI added was insignificant and would not have altered the albumin or other constituent concentrations. The samples were then diluted further to 10 mg/mL in UV transparent microtitre plate wells using 1 M cation stocks (KCI, NH4CI, CaCl2, MgCl2, NaCI) as shown in Table 14.

[0103] Samples for each of KCI, NH4CI, CaCI2, MgCI2, NaCI were prepared according to Table 14. Therefore, in total, 40 different samples were prepared. Each sample was tested in duplicate on a microtitre plate.

[0104] The microtitre plate was gently rocked to mix the contents of each well, centrifuged to remove any air bubbles and placed in a Biotek Synergy Mx (Potton, UK) plate reader that had been pre-equilibrated and controlled at 65°C. The plate was then read at 350 nm every minute over a total incubation time of 8 hours. Gen5 software (Biotek software for the plate reader version 2.00.18) was used to calculate the time taken for the A350nm absorbance to increase by 0.2 adsorption units above a base line. The base line was calculated from the mean of the first 5 data points.

[0105] Results: Figure 12 shows the time taken for the absorbance of samples to increase to 0.2 units above the baseline. A longer time shows better stability.

Conclusions: [0106] • The control using NaCI shows the same trend as per the other examples i.e. increasing sodium levels improves the stability. This confirms that this microtitre plate method is suitable for testing stability effects. • For all the different cations, both single and dual valency (group 1 metals and group 2 metals, respectively) there was a clear increase in albumin stability with increasing cation concentration up to 500 mM and probably beyond. • These data indicate that while all cations improve stability albumin, MgCI2 is very good.

Example 7: Effect of different anions on the stability of albumin [0107] Method: A sample of purified albumin containing a low concentration of octanoate (-0.2 mM, 100 g/L albumin) was diluted initially to 50 mg/mL with water such that it contained 50 mg/mL albumin, 75 mM NaCI and no pH buffer constituent. The sample was pH adjusted with 0.5 M HCI to 6.43, the amount of HCI added was insignificant and would not have altered the albumin or constituent concentrations. 1 M sodium anion stock solutions were prepared according to Table 15.

Table 15: Anion stocks

[0108] The albumin and anion stocks were used as detailed below, being made to a final volume of 1 mL in a polystyrene cuvette (Sarstedt 10x4x45 mm). The samples were gently mixed prior to the cuvettes being placed into a temperature controlled spectrophotometer that had been pre-equilibrated and controlled at 65°C. The absorbance at 350 nm, referenced against an empty cuvette, was then monitored over a 2 hour period with a reading taken every 30 seconds. The data was processed by taking the first 9 data points (~ the first 4 minutes), calculating the mean (average) and then subtracting this from all data points in order to give a baseline absorbance. The time taken for the absorbance to increase by 0.1 AU above this baseline was recorded for that particular sample. If the absorbance did not go above 0.1 AU in 2 hours (7200 seconds), then the data was extrapolated in order to get an approximate time. Samples for which the absorbance does not go above 0.1 AU in 2 hours are significantly improved in stability compared to samples having lower cation concentrations.

[0109] A six cuvette holder in the spectrophotometer was used with the first sample always being a control and the other five samples using an increasing excipient (i.e. test material such as NaCI, Na2SO4) concentration. The control was always a pH 6.5 sample containing 250 mM NaCI, this needed to remain in solution with no insoluble aggregates over the full 2 hour 65°C incubation for the test to be considered to be valid.

Table 16:

[0110] For the citrate samples, the stabilizing effect of the sodium was inconclusive when measuring the insoluble aggregates as detected by the A350nm absorbance increase. Therefore after the 2 hour, 65°C incubation in the spectrophotometer, the samples were removed, centrifuged to remove any large particles and the samples were analyzed for soluble aggregates by GP-HPLC (as per Example 4). The data was expressed as % monomeric

albumin remaining (the higher the value the more stable the formulation). This was also done for the phosphate samples.

[0111] Results: All controls were valid. Figure 13 shows the effect of sodium ion concentration and anion species on the time taken for the A350 absorbance to increase to 0.1 AU above the base line. Figure 14 shows, the effect of citrate, phosphate and sodium on the stability of albumin following a 65°C, 2 hour incubation. A higher monomer level shows a higher stability. The results for the pH 6.5 controls (250 mM sodium) run at the same time gave a mean result of 81 % monomer content.

Conclusions: [0112] • Sodium chloride (sodium salt of an inorganic acid), sodium sulphate (sodium salt of a divalent acid) and sodium acetate (sodium salt of an organic acid) all gave a strong increase in albumin stability with increasing sodium concentration. • For sodium dihydrogen phosphate the trend was not as strong as sodium chloride, sodium sulphate and sodium acetate. However, at 150 mM and above there is an increasing trend of stability with increasing sodium content. This trend was confirmed when the soluble aggregates were measured as shown by the strong trend of increasing monomer remaining with increasing sodium concentration. • The trend of increasing stability with increasing sodium concentration for the sodium phosphate samples continued through to 600 mM sodium and would probably continue to higher sodium concentrations. • For sodium citrate there was no obvious trend in albumin stability with sodium concentration as measured by the A350nm absorbance for the presence of insoluble aggregates. However, when the samples (after incubation at 65°C for 2 hours) were assessed for soluble aggregates through the measurement of % monomer content remaining by GP-HPLC then there was a trend. At 200 mM sodium and below, the monomer content was fairly flat but as the sodium content increased above 200 mM there was a definite trend of increasing monomer content and therefore albumin stability. Citrate is a chelating agent and therefore the sodium present is chelated to the citrate and is unlikely to be as available to stabilize the albumin as the sodium provided, for example, by NaCI. • Consequently, the inventors believe that any sodium salt (or any other mono or divalent anion based on the previous example) will impart stability on albumin, with a trend of increasing stability with increasing anion concentration.

Example 8: Effect of different buffers on albumin stability [0113] Method: A sample of purified albumin containing a low concentration of octanoate (-0.2 mM, 100 g/L albumin) was diluted initially to 50 mg/mL with water such that it contained 50 mg/mL albumin, 75 mM NaCI and no buffer constituent. The sample was pH adjusted with 0.5 M HCI to pH 6.43, the amount of HCI added was insignificant and would not have altered the albumin or constituent concentrations.

[0114] An unbuffered stock of 1 M NaCI together with the following buffers (Table 17) pH adjusted to pH 6.43 as per the albumin stock (so that when added to the albumin the pH would not change) was prepared. For phosphate the pH was adjusted with 27% NaOH, for citrate it was adjusted with citric acid (citric acid powder) and for acetate it was adjusted with acetic acid (glacial acetic acid):

Table 17:

The amount of acid added was insignificant and would not have altered the albumin or constituent concentrations.

[0115] The albumin and buffer stocks were used as detailed below, being made to a final volume of 1 mL in a polystyrene cuvette (Sarstedt 10x4x45 mm). The samples were gently mixed prior to the cuvettes being placed into a temperature controlled spectrophotometer that had been pre-equilibrated and controlled at 65°C. The absorbance at 350 nm, referenced against an empty cuvette, was then monitored over a 2 hour period with a reading taken every 30 seconds. The data was processed by taking the first 9 data points (~ the first 4 minutes), calculating the mean and then subtracting this from all data points in order to give a baseline absorbance. The time taken for the absorbance to increase by 0.1 AU above this baseline was recorded for that particular formulation sample. If the absorbance did not go above 0.1 AU in 2 hours (7200 seconds) then the data was extrapolated in order to get an approximate time.

[0116] A six cuvette holder in the spectrophotometer was used with the first sample always being a control and the other five samples using an increasing excipient concentration. The control was always a pH 6.5 sample containing 250 mM NaCI, this needed to remain in solution with no insoluble aggregates over the full 2 hour 65°C incubation for the test to be considered to be valid.

Results: All controls were valid. For the samples, the time taken for the A350 absorbance to increase to 0.1 AU above the base line was plotted against the sodium concentration. Figure 15 shows that for all buffers, albumin stability increases as sodium ion concentration increases.

[0117] For the citrate samples the trend appears offset relative to the other samples. Consequently, the data for no buffer (sodium provided only by NaCI) was plotted together with the samples buffered with sodium citrate, but with the sodium concentration coming from the sodium citrate ignored (Figure 16).

Conclusions: [0118] • For all buffers, and also with no buffer, there is a clear trend of increasing albumin stability with increasing sodium concentration. • Even with citrate, which did not appear to be as good a sodium donator for albumin stability as NaCI, there was a trend of increasing stability within increasing sodium concentration but it was offset slightly. The reason for this offset is that, like for all the buffers, the sodium from the buffer was used in the calculation of the total sodium content. Consequently, if this sodium is not as effective (e.g. available) as sodium from sodium chloride (as shown in the previous examples) then there will be an offset. This was confirmed by Figure 16. • Sodium phosphate is a good pH buffer, but as a donator for sodium for stabilization there are better donators and therefore it may be advantageous to combine sodium phosphate with another donator of cation (sodium or other cation) to stabilize albumin. • Consequently, the inventors believe that the buffer in the formulation is not particularly important and any buffer, or no buffer, can be used. However, if the buffer is chelating then the anion present from the buffer should not be included in the calculation of the required concentration of anion.

Example 9: Effect of high salt concentration on stability of albumin variants [0119] Method: Various albumins and variants (Table 19) were diluted with 0.5M sodium phosphate buffer (pH 6.5). The variants were mature HSA (SEQ ID No: 2) with point mutations (K573P, K500A, K573Y, K573W) and mouse serum albumin (MSA, SEQ ID No: 19). The rHSA concentration and sodium ion concentration of the stock solutions of the albumin variants are provided in Table 20.

[0120] As all the variants had been purified slightly differently to the wild type human albumin, the levels of octanoate present would have been slightly different for each variant. From previous results, it was estimated that the octanoate present in the variants would have been equivalent to ~4 mM at 100g/L albumin. As the wild-type stock had negligible levels of octanoate present, this stock was subsequently spiked with 5 pl_ of 2 M octanoate into the final volume to give an approximately equivalent concentration of octanoate, relative to the variant albumins.

[0121] The albumin stocks and a 1 M NaCI stock were used according to Table 21, each sample being made to a final volume of 1 mL in a polystyrene cuvette (Sarstedt 10x4x45 mm). The samples were gently mixed prior to the cuvettes being placed into a temperature controlled spectrophotometer that had been pre-equilibrated and controlled at 65°C. The absorbance at 350 nm, referenced against an empty cuvette, was then monitored over a 2 hour period with a reading taken every 30 seconds. The data was processed by taking the first 9 data points (~ the first 4 minutes), calculating the mean (average) and then subtracting this from all data points in order to give a baseline absorbance. The time taken for the absorbance to then increase by 0.1 AU above this baseline was recorded for that particular formulation sample. If the absorbance did not go above 0.1 AU in 2 hours (7200 seconds) then the data was extrapolated in order to get an approximate time.

[0122] A six cuvette holder in the spectrophotometer was used with the first sample always a control and the other five samples using an increasing excipient concentration. The control was always a pH 6.5 sample containing 250 mM NaCI, this needed to remain in solution with no insoluble aggregates over the full 2 hour 65°C incubation for the test to be considered to be valid.

Results: All controls were valid. Figure 17 and 18 shows the effect of sodium ion concentration on the time taken for the A350 absorbance to increase by 0.1 AU above the base line.

Conclusions: [0123] • All points follow a trend apart from the wild type albumin, 150 mM sodium sample. It is unclear why this point is out of trend but is likely to be an outlier and does not detract from the overall conclusions. • For all albumin variants there is a clear trend of increasing albumin stability with increasing sodium concentration. • Mature mouse serum albumin (SEQ ID No: 19) is 72.1% identical (using the algorithim described herein) to mature wild-type human serum albumin (SEQ ID NO: 2) and even though the overall stability was not as high as HSA, or HSA variants, there was still a clear trend of increasing stability with increasing sodium concentration at and above 200 mM. • It is difficult to say whether or not there is a significant difference in stability between the different variants because it is difficult to compare the stabilities of the variants to the stability of wild type albumin since the base formulation with respect to the level of octanoate present was not absolutely controlled to be the same between each variant. However, within the data sets for each variant the level of octanoate will be the same and therefore the increase in stability can only be due to the increasing level of sodium. • The one sample where the octanoate was known (wild type human serum albumin, equivalent to 4 mM at 100g/L albumin) shows that the observed stability increase with

sodium is also valid at this level of octanoate.

Example 10: Effect of pH on albumin stability [0124] Method: A sample of purified albumin containing low octanoate (-0.2 mM, 100 g/L albumin) was diluted initially to 50 mg/mL according to Table 22, using phosphate stocks according to Table 23.

Table 23:

[0125] The samples were pH adjusted with 0.5 M HCI (i.e. no added sodium) to give final pHs of 5.02 and 5.55 using the pH 5 stock, pHs of 6.00 and 6.49 using the pH 6.5 stock, pH of 7.04 using the pH 7 stock and pHs of 7.55 and 7.98 using the pH 8 stock. The amount of HCI added was insignificant and would not have altered the albumin or constituent concentrations.

[0126] The stocks were used as detailed below (Table 24), being made to a final volume of 1 mL in a polystyrene cuvette (Sarstedt 10x4x45 mm). The samples were gently mixed prior to the cuvettes being placed into a temperature controlled spectrophotometer that had been preequilibrated to and controlled at 65°C. The absorbance at 350 nm, referenced against an empty cuvette, was then monitored over a 2 hour period with a reading taken every 30 seconds. The data was processed by taking the first 9 data points (~ the first 4 minutes), calculating the mean (average) and then subtracting this from all data points in order to give a baseline absorbance. The time taken for the absorbance to then increase by 0.1 AU above this baseline was recorded for that particular formulation sample. If the absorbance did not go above 0.1 AU in 2 hours (7200 seconds) then the data was extrapolated in order to get an approximate time.

[0127] A six cuvette holder in the spectrophotometer was used with the first sample always being a control and the other five samples using an increasing excipient concentration. The control was always a pH 6.5 sample containing 250 mM NaCI, this needed to remain in solution with no insoluble aggregates over the full 2 hour 65°C incubation for the test to be considered to be valid.

[0128] For the pH 7 (measure pH 7.04), 7.5 (measured pH 7.55) and 8 (measure pH 7.98) samples the stabilizing effect of the sodium was inconclusive when measuring insoluble aggregates as detected by the A350nm absorbance increase. Therefore after the 2 hour incubation at 65°C incubation in the spectrophotometer the samples were removed, centrifuged to remove any large particles and the samples analyzed for soluble aggregates by GP-HPLC (as per Example 4). The data was expressed as % monomeric albumin remaining (the higher the value the more stable the formulation).

Results: All controls were valid. Figures 19, 20 and 21 show the stability of albumin relative to

sodium ion concentration. The results for the pH 6.5 controls run at the same time as the pH7, 7.5 and 8 samples gave a mean of 82% monomer content.

Conclusions: [0129] • For all pHs from pH 5 to pH 6.5 it was clear that there was an increase in albumin stability with increasing albumin concentration as measured by the A350 absorbance increase (insoluble aggregates). • At pHs 7, 7.5 and 8 the trend was not clear (Figure 19) with a possible dip in stability around 150 mM Na. However, when these samples were analyzed by GP-HPLC for soluble aggregates and % monomer remaining (Figure 21) there was a clear trend of increasing stability with increasing sodium. The reason that this trend was not observed for the insoluble aggregates may be due to the fact that these pHs are the furthest from the pl of albumin (5.2 for albumin) and therefore they are less likely to precipitate with the aggregates coming out of solution. • The pH 6.5 controls had higher levels of monomer remaining at the same sodium ion concentration (250 mM) than any of the higher pHs showing that pH 6.5 is the more stable pH for albumin. • Combining both the insoluble and soluble aggregate data shows that increasing sodium concentration increases albumin stability from pH 5 to pH 8.

Example 11: Effect of Octanoate on Stem Cell Cultures [0130] Method: The effect of octanoate on stem cell culture was carried out by a contract research organization: Cellartis AB (Gothenburg, Sweden). Briefly, albumin at 100 g/L with varying levels of octanoate (0.2, 0.5, 1.0 and 8.0 mM) was used as the albumin supplement in standard stem cell culture media. Human embryonic stem cells (cell lines SA121 and SA181 (Cellartis AB, cell lines deposited in the European Human Embryonic Stem Cell Registry)) were transferred from their standard media and grown through 5 passages in 6 well plates in the media supplemented with the albumin containing varying levels of octanoate. The cell growth over the 5 passages was assessed by monitoring the cell doubling times during consecutive passages in cell production. The doubling times of cultures should be within a range of 28-40 hours, and can be seen as a trend indicator. In order to determine the undifferentiation state of the cells after 5 passages in albumin supplemented media, antibodies against four different accepted markers for the undifferentiated state, namely Oct-4, SSEA-3, Tra-1 60 and hES-Cellect, were used for immunostaining.

[0131] Results: Table 25 shows data for the doubling time. The initial doubling times are quite high, probably due to cells needing to adjust to the new culture medium composition. However, the doubling times between passages 2 and 4 are all within expected range except for the sample containing 8 mM octanoate which failed to maintain acceptable cell attachment and could not be continued past passage 2 even with modified medium and coating conditions. Doubling times for passage 5 were highly variable and for some samples lay considerably outside the standard range. However, it is difficult to draw conclusions as cultures were not expanded further.

Table 25: Doubling time in hours for each ofthe cell lines over five passages. Doubling time is presented in hours, and formula used is Td(h) = T * LOG2/LOG(cells harvested/cells seeded). T = time in hours between passages

[0132] Table 26 shows data for the immunocytochemical stainings for differentiation markers and shows that all the samples (except the sample containing 8 mM octanoate, since it did not reach 5 passages) supported cultures to maintain an undifferentiated state for the 5 passages tested.

Table 26: Summary of immunocytochemical staining performed on cells stained using antibodies against Oct-4, Tra-1 60, SSEA-3, hES-Cellect™ and SSEA-1. (+++) represents good staining and easy to detect, while (-) represents no staining detectable.

Conclusions:

[0133] • The octanoate level present in the albumin is important for stem cell attachment, maintenance of undifferentiated cell growth. At 8 mM octanoate in 100g/L albumin the octanoate is toxic to stem cells and does not allow their attachment to surfaces or cell growth.

SEQUENCE LISTING

[0134] <110> Novozymes Biopharma UK Limited

Novozymes Biopharma DK A/S Novozymes Biopharma AU Limited

Novozymes Biopharma US, Inc. <120> Albix formulation

<130> 12182-WO-PCT <150> US 61/504,406 <151> 2011-07-05 <150> EP 11174267.2 <151> 2011-07-15 <160> 19 <170> Patentin version 3.5 <210> 1 <211> 1758

<212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (1)..(1758)

<223> cDNA encoding HSA <400> 1 gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60 gecmggngu igatcgoEiu ugctqaguat ctncagcagic guccautuga agaucatgua.L^u aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa180 aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt240 cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa300 tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt360 gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat420 gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg480 tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca540 aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt600 gcaagtctcc aaaaatttgg agaaagagct ttcaaagaat gggcagtagc tcgcctgagc660 cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa720 gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt780 gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa840 aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct900 gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct960 gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat1020 tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc1080 tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt1140 gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag1200 tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact1260 ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaaaat1320 cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta1380 tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc1440 ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa1500 gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag1560 agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca1620 aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag1680 gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa1740 gctgccttag gcttataa1758 <210>2 <211 > 585

<212> PRT <213> Homo sapiens <400>2

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 15 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gin Tyr Leu Gin 20 25 30

Gin Cys Pro Phe Glu Asp His Val Lys Léu Val Asn Glu Val Thr Glu 35 4045

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 5560

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala ThrLeu 65 70 7580

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gin GluPro 85 9095

Glu Arg Asn Glu Cys Phe Leu Gin His Lys Asp Asp Asn Pro Asn Leu 100 105110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu lie Ala Arg 130 135140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala LysArg 145 150 155160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gin Ala Ala Asp Lys AlaAla 165 170175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185190

Ser Ala Lys Gin Arg Leu Lys Cys Ala Ser Leu Gin Lys Phe Gly Glu 195 200205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gin Arg Phe Pro 210 215220

Lys Ala Glu Phe Ala Glu Val Ser Lys Léu Val Thr Asp Leu ThrLys 225 230 235240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala AspAsp 245 250255

Arg Ala Asp Leu Ala Lys Tyr lie Cys Glu Asn Gin Asp Ser lie Ser 260 265270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280285

Cys lie Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp val Cys Lys Asn TyrAla 305 310 315320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg w -ΐΐη

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345350

Tyr Glu Thr Thr LeU Glu Lys Cys Cys Ala Ala Ala Asp Pro His G1U 355 360365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375380

Gin Asn Leu Ile Lys Gin Asn Cys Glu Leu Phe Glu Gin Leu GlyGlu 385 390 395400

Tyr Lys Phe Gin Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys ValPro 405 410415

Gin Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gin Leu Cys Val Leu His 450 455460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr GluSer 465 470 475480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp GluThr 485 490495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gin Ile Lys Lys Gin Thr Ala 515 520525

Leu Val Glu Leu Val Lys His LyS Pro Lys Ala Thr Lys Glu Gin Leu 530 535540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys CysLys 545 550 555560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys LeuVal 565 570575

Ala Ala Ser Gin Ala Ala Leu Gly Leu 580585 <210> 3 <211 > 609

<212> PRT <213> Homo sapiens <400> 3

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 1015

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 2530

His Arg Phe Lys Asp Leu Gly Glu Glu Ash Phe Lys Ala Leu Val Leu 35 4045

Ile Ala Phe Ala Gin Tyr Léu Gin Gin Cys Pro Phe Glu Asp His Val 50 5560

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val AlaAsp 65 70 7580

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105110

Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin 115 120125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe LeuLys 145 150 155160

Lys Tyr Leu Tyr Glu lie Ala Arg Arg His Pro Tyr Phe Tyr AlaPro 165 170175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185190

Cys Gin Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gin Arg Leu Lys Cys 210 215220

Ala Ser Leu Gin Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp AlaVal 225 230 235240

Ala Arg Leu Ser Gin Arg Phe Pro Lys Ala Glu Phe Ala Glu ValSer 245 250255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr lie 275 280285

Cys Glu Asn Gin Asp Ser lie Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295300

Lys Pro Leu Leu Glu Lys Ser His Cys lie Ala Glu Val Glu Ash Asp 305 310 315320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe AspGlu 385 390 395400

Phe Lys Pro Leu Val Glu Glu Pro Gin Asn Leu lie Lys Gin AsnCys 405 410415

Glu Leu Phe· Glu Gin Leu Gly Glu Tyr Lys Phe Gin Asn Ala Leu Leu 420 425430

Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val 435 440445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser ValVal 465 470 475480

Leu Asn Gin Leu Cys Val Leu His Glu Lys Thr Pro Val Ser AspArg 485 490495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Ash Ala 515 520525

Glu Thr Phe Thr Phe His Ala Asp He Cys Thr Leu Ser Glu Lys Glu 530 535540

Arg Gin lie Lys Lys Gin Thr Ala Leu Val Glu Leu Val Lys HisLys 545 550 555560

Pro Lys Ala Thr Lys Glu Gin Leu Lys Ala Val Met Asp Asp PheAla 565 570575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gin Ala Ala Leu Gly 595 600605

Leu <210>4 <211 > 621

<212> PRT <213> Pan troglodytes <400>4

Met Asn Glu Ser Ser Cys Cys Ser Thr Ser Leu Pro Ala Phe Gly Val 15 1015

Ser Val Leu Asp Ser Gly His Ser Ser Ser Ser Ala Tyr Ser Arg Gly 20 2530

Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys 35 4045

Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Val Ala Phe Ala 50 5560

Gin Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val Lys Leu ValAsn 65 70 7580

Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser AlaGlu 85 9095

Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr 100 105110

Val Ala Thr Leu Arg Glu Lys Tyr Gly Glu Met Ala Asp Cys Cys Ala 115 120125

Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin His Lys Asp Asp 130 135140

Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp val MetCys 145 150 155160

Thr Ala Phe His Asp Asn Glu Gly Thr Phe Leu Lys Lys Tyr LeuTyr 165 170175

Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe 180 185190

Phe Ala Glu Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gin Ala Ala 195 200205

Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu 210 215220

Gly Lys Ala Ser Ser Ala Lys Gin Arg Leu Lys Cys Ala Ser LeuGin 225 230 235240

Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg LeuSer 245 250255

Gin Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr 260 265270

Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu 275 280285

Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr He Cys Glu Asn Gin 290 295300

Asp Ser lie Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro LeuLeu 305 310 315320

Glu Lys Ser His Cys Leu Ala Glu Val Glu Asn Asp Glu Met ProAla 325 330335

Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Glu Val Cys 340 345350

Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr 355 360365

Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg 370 375380

Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu LyS Cys Cys Ala AlaAla 385 390 395400

Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys ProLeu 405 410415

Val Glu Glu Pro Gin Asn Leu lie Lys Gin Asn Cys Glu Leu Phe Glu 420 425430

Gin Leu Gly Glu Tyr Lys Phe Gin Asn Ala Leu Leu Val Arg Tyr Thr 435 440445

Lys Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 450 455460

Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu AlaLys 465 470 475480

Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn GinLeu 485 490495

Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys 500 505510

Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu 515 520525

Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr 530 535540

Phe His Ala Asp He Cys Thr Leu Ser Glu Lys Glu Arg Gin lieLys 545 550 555560

Lys Gin Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys AlaThr 565 570575

Lys Glu Gin Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu 580 585590

Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly 595 600605

Lys Lys Léu Val Ala Ala Ser Gin Ala Ala Leu Gly Leu 610 615620 <210> 5 <211 > 608

<212> PRT <213> Macaca mulatta <400> 5

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 15 1015

Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Val Ala 20 2530

His Arg Phe Lys Asp Leu Gly Glu Glu His Phe Lys Gly Léu Val Léu 35 4045

Val Ala Phe Ser Gin Tyr Leu Gin Gin Cys Pro Phe Glu Glu His Val 50 5560

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val AlaAsp 65 70 7580

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Lys Leu CyS Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105110

Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin 115 120125

His Lys Asp Asp Asn Pro Asn Leu Pro Pro Leu Val Arg Pro Glu Val 130 135140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Ala Thr Phe LeuLys 145 150 155160

Lys Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr AlaPro 165 170175

Glu Leu Leu Phe Phe Ala Ala Arg Tyr Lys Ala Ala Phe Ala Glu Cys 180 185190

Cys Gin Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200205 Léu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gin Arg Leu Lys Cys 210 215220

Ala Ser Leu Gin Lys Phe Gly Asp Arg Ala Phe Lys Ala Trp AlaVal 225 230 235240

Ala Arg Leu Ser Gin Lys Phe Pro Lys Ala Glu Phe Ala Glu ValSer 245 250255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Met 275 230285

Cys Glu Ash Gin Asp Ser lie Ser Ser Lys Leu Lys Glu Cys Cys Asp 290 295300

Lys Pro Leu Leu Glu Lys Ser His Cys Leu Ala Glu Val Glu AsnAsp 305 310 315320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Tyr Val GluSer 325 330335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Met 355 360365

Leu Leu Leu Arg Leu Ala Lys Ala Tyr Glu Ala Thr Leu Glu Lys Cys 370 375380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe AspGlu 385 390 395400

Phe Gin Pro Leu Val Glu Glu Pro Gin Asn Leu Val Lys Gin AsnCys 405 410415

Glu Leu Phe Glu Gin Leu Gly Glu Tyr Lys Phe Gin Asn Ala Leu Leu 420 425430

Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val 435 440445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ala Lys Cys Cys Lys Leu 450 455460

Pho Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser ValVal 465 470 475480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser GluLys 485 490495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505510

Ser Ala Leu Glu Leu Asp Glu Ala Tyr Val Pro Lys Ala Phe Asn Ala 515 520525

Glu Thr Phe Thr Phe His Ala Asp Met Cys Thr Leu Ser Glu Lys Glu 530 535540

Lys Gin Val Lys Lys Gin Thr Ala Leu Val Glu Leu Val Lys HisLys 545 550 555560

Pro Lys Ala Thr Lys Glu Gin Leu Lys Gly Val Met Asp Asn PheAla 565 570575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Ala Cys Phe 580 585590

Ala Glu Glu Gly Pro Lys Phe Val Ala Ala Ser Gin Ala Ala Leu Ala 595 600605 <210> 6 <211 > 608

<212> PRT <213> Mesocricetus auratus <400> 6

Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Asp Ser Ala 15 1015

Phe Ser Arg Gly Leu Phe Arg Arg Asp Ala His Lys Ser Glu Ile Ala 20 2530

His Arg Phe Lys Asp Leu Gly Glu Gin His Phe Lys Gly Leu Val Leu 35 4045

Ile Ala Phe Ser Gin Phe Leu Gin Lys Cys Pro Tyr Glu Glu His Val 50 5560

Lys Leu Val Asn Glu Val Thr Asp Phe Ala Lys Thr Cys Val AlaAsp 65 70 7580

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Lys Leu Cys Ala Ile Pro Thr Leu Arg Asp Ser Tyr Gly Glu Leu Ala 100 105110

Asp Cys Cys Ala Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe Leu Lys 115 120125

His Lys Asp Asp His Pro Asn Leu Pro Pro Phe Val Arg Pro Asp Ala 130 135140

Glu Ala Met Cys Thr Ser Phe Gin Glu Asn Ala Val Thr Phe Met Gly 145 150 155160

His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170175

Glu Leu Leu Tyr Tyr Ala Glu Lys Tyr Ser Ala Ile Met Thr Glu Cys 180 185190

Cys Gly Glu Ala Asp Lys Ala Ala Cys lie Thr Pro Lys Leu Asp Ala 195 200205

Leu Lys Glu Lys Ala Leu Ala Ser Ser Val Asn Gin Arg Leu Lys Cys 210 215220

Ser Ser Leu Gin Arg Phe Gly Gin Arg Ala Phe Lys Ala Trp Ala Val 225 230 235240

Ala Arg Met Ser Gin Lys Phe Pro Lys Ala Asp Phe Ala Glu lie Thr 245 250255

Lys Leu Ala Thr Asp Leu Thr Lys Leu Thr Glu Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280285

Cys Glu Asn Gin Ala Ser lie Ser Ser Lys Leu Gin Ala Cys Cys Asp 290 295300

Lys Pro Val Leu Lys Lys Ser His Cys Leu Ser Glu Val Glu Asn Asp 305 310 315320

Asp Leu Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Asp 325 330335

Lys Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345350

Thr Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Ala 355 360365

Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375380

Cys Ala Glu Ala Asp Pro Ser Ala Cys Tyr Gly Lys Val Leu AspGlu 385 390 395400

Phe Gin Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Ala AsnCys 405 410415

Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gin Asn Ala Leu lie 420 425430

Val Arg Tyr Thr Gin Lys Ala Pro Gin Val Ser Thr Pro Thr Leu Val 435 440445

Glu Ala Ala Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Val Leu 450 455460

Pro Glu Ala Gin Arg Leu Pro Cys Val Glu Asp Tyr He Ser Alalie 465 470 475480

Leu Asn Arg Val Cys Val Leu His Glu Lys Thr Pro Val Ser GluGin 485 490495

Val Thr Lys Cys Cys Thr Gly Ser Val Val Glu Arg Arg Pro Cys Phe 500 505510

Ser Ala Leu Pro Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Ser Leu Pro Glu Lys Glu 530 535540

Lys Gin. Met Lys Lys Gin Ala Ala Leu Val Glu Leu Val Lys HisLys 545 550 555560

Pro Lys Ala Thr Gly Pro Gin Leu Arg Thr Val Leu Gly Glu PheThr 565 570575

Ala Phe Leu Asp Lys Cys Cys Lys Ala Glu Asp Lys Glu Ala Cys Phe 580 585590

Ser Glu Asp Gly Pro Lys Leu Val Ala Ser Ser Gin Ala Ala Leu Ala 595 600605 <210> 7 <211 > 608

<212> PRT <213> Cavia porcellus <400> 7

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Val 15 1015

Tyr Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 2530

His Arg Phe Asn Asp Leu Gly Glu Gly His Phe Lys Gly Leu Val Leu 35 4045

Ile Thr Leu Ser Gin His Leu Gin Lys Ser Pro Phe Glu Glu His Val 50 5560

Lys Leu Val Asn Glu Val Thr Asp Phe Ala Lys Ala Cys Val AlaAsp 65 70 7580

Glu Ser Ala Gin Asn Cys Gly Lys Ala Ile Ala Thr Leu Phe GlyAsp 85 9095

Lys Val Cys Ala Ile Pro Ser Leu Arg Glu Thr Tyr Gly Glu Leu Ala 100 105110

Asp Cys Cys Ala Lys Glu Asp Pro Asp Arg Val Glu Cys Phe Leu Gin 115 120125

His Lys Asp Asp Asn Pro Asn Leu Pro Pro Phe Glu Arg Pro Glu Pro 130 135140

Glu Ala Leu Cys Thr Ala Phe Lys Glu Asn Asn Asp Arg Phe lie Gly 145 150 155160

His Tyr Leu Tyr Glu Val Ser Arg Arg His Pro Tyr Phe Tyr AlaPro 165 170175

Glu Leu Leu Tyr Tyr Ala Glu Lys Tyr Lys Asn Ala Leu Thr Glu Cys 180 185190

Cys Glu Ala Ala Asp Lys Ala Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200205 lie Lys Glu Lys Ala Leu Val Ser Ser Ala Gin Gin Arg Leu Lys Cys 210 215220

Ala Ser Leu Gin Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Val 225 230 235240

Ala Arg Leu Ser Giri Lys Phe Pro Lys Ala Glu Phe Ala Glu He Ser 245 250255

Thr lie Val Thr Ser Leu Thr Lys Val Thr Lys Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Gin Glu Leu Ala Lys Tyr Met 275 280285

Cys Glu His Gin Asp Ser He Ser Ser Lys Leu Lys Glu Cys Cys Val 290 295300

Lys Pro Thr Leu Gin Lys Ala His Cys He Leu Glu He Gin Arg Asp 305 310 315320

Glu Leu Pro Thr Glu Leu Pro Asp Leu Ala Val Asp Phe Val Glu Asp 325 330335

Lys Glu Val Cys Lys Asn Phe Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345350

Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ser He Gly 355 360365

Met Leu Leu Arg He Ala Lys Gly Tyr Glu Ala Lys Leu Glu Lys Cys 370 375380

Cys Ala Glu Ala Asp Pro His Ala Cys Tyr Ala Lys Val Phe AspGlu 385 390 395400

Leu Gin Pro Leu He Asp Glu Pro Lys Lys Leu Val Gin Gin AsnCys 405 410415

Glu Leu Phe Asp Lys Leu Gly Glu Tyr Gly Phe Gin Asn Ala Leu Ala 420 425430

Val Arg Tyr Thr Gin Lys Ala Pro Gin Val Ser Thr Pro Thr Leu Val 435 440445 βΤπ Twr Ma Arrr T.vr T.c*n (ΖΊ vz Val (ΖΊ xz Thr Τ·νβ Ρχζ« Πνζ» Rpf T.pii — — — -J'- ·**—** “ J ~ ’-—J' — · W— — —J —J — —<JT—« — --- — — 450 455460

Pro Glu Thr Glu Arg Leu Ser Cys Thr Glu Asn Tyr Leu Ala Leulie 465 470 475480

Leu Asn Arg Leu Cys He Leu His Glu Lys Thr Pro Val Ser GluArg 485 490495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505510

Ser Ala Leu His Val Asp Glu Thr Tyr Val Pro Lys Pro Phe His Ala 515 520525

Asp Ser Phe Thr Phe His Ala Asp lie Cys Thr Leu Pro Glu Lys Glu 530 535540

Lys Gin Val Lys Lys Gin Met Ala Leu Val Glu Leu Val Lys His Lys 545 550 555560

Pro Lys Ala Ser Glu Glu Gin Met Lys Thr Val Met Gly Asp Phe Ala 565 570575

Ala Phe Leu Lys Lys Cys Cys Asp Ala Asp Asn Lys Glu Ala Cys Phe 580 585590

Thr Glu Asp Gly Pro Lys Leu Val Ala Lys Cys Gin Ala Thr Leu Ala 595 600605 <210> 8 <211 > 608

<212> PRT <213> Mus musculus <400> 8

Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala 15 1015

Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu lie Ala 20 2530

His Arg Tyr Asn Asp Leu Gly Glu Gin His Phe Lys Gly Leu Val Leu 35 4045 lie Ala Phe Ser Gin Tyr Leu Gin Lys Cys Ser Tyr Asp Glu His Ala 50 5560

Lys Leu Val Gin Glu Val Thr Asp Phe Ala Lys Thr Cys Val AlaAsp 65 70 7580

Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Lys Leu Cys Ala He Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala 100 105110

Asp Cys Cys Thr Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin 115 120125

His Lys Asp Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala 130 135140

Glu Ala Met Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe MetGly 145 150 155160

His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr AlaPro 165 170175

Glu Leu Leu Tyr Tyr Ala Glu Gin Tyr Asn Glu lie Leu Thr Gin Cys 180 185190

Cys Ala Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly 195 200205

Val Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gin Arg Met Lys Cys 210 215220

Ser Ser Met Gin Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp AlaVal 225 230 235240

Ala Arg Leu Ser Gin Thr Phe Pro Asn Ala Asp Phe Ala Glu lieThr 245 250255

Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280285

Cys Glu Asn Gin Ala Thr He Ser Ser Lys Leu Gin Thr Cys Cys Asp 290 295300

Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu val Glu HisAsp 305 310 315320

Thr Met Pro Ala Asp Leu Pro Ala lie Ala Ala Asp Phe Val GluAsp 325 330335

Gin Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345350

Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser 355 360365

Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375380

Cys Ala Glu Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu AlaGlu 385 390 395400

Phe Gin Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr AsnCys 405 410415

Asp leu Tyr Glu Lys leu Gly Glu Tyr Gly Phe Gin Asn Ala He leu 420 425430

Val Arg Tyr Thr Gin Lys Ala Pro Gin Val Ser Thr pro Thr Leu Val 435 440445

Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu 450 455460

Pro Glu Asp Gin Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Alalie 465 470 475480

Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser GluHis 485 490495

Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe 500 505510

Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520525

Glu Thr Phe Thr Phe His Ser Asp He Cys Thr Leu Pro Glu Lys Glu 530 535540

Lys Gin lie Lys Lys Gin Thr Ala Leu Ala Glu Leu Val Lys HisLys 545 550 555560

Pro Lys Ala Thr Ala Glu Gin Leu Lys Thr Val Met Asp Asp PheAla 565 570575

Gin Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe 580 585590

Ser Thr Glu Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala 595 600605 <210> 9 <211 > 608

<212> PRT <213> Rattus norvegicus <400> 9

Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe He Ser Gly Ser Ala 15 1015

Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu He Ala 20 2530

His Arg Phe Lys Asp Leu Gly Glu Gin His Phe Lys Gly Leu Val Leu 35 4045

He Ala Phe Ser Gin Tyr Leu Gin Lys Cys Pro Tyr Glu Glu His He 50 5560

Lvs Τ.ΑΊ Val Gin Glu Val Thr Asn Phe Ala T.vs Thr CvS Val Ala Asti —J — — — — . —— — — -------— —J —· ---- —J—· . — — _ — r 65 70 7580

Glu Asn Ala Glu Asn Cys Asp Lys Ser lie His Thr Leu Phe Gly Asp 85 9095

Lys Leu Cys Ala lie Pro Lys Leu Arg Asp Asn Tyr Gly Glu Leu Ala 100 105110

Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin 115 120125

His Lys Asp Asp Asn Pro Asn Leu Pro Pro Phe Gin Arg Pro Glu Ala 130 135140

Glu Ala Met Cys Thr Ser Phe Gin Glu Asn Pro Thr Ser Phe LeuGly 145 150 155160

His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr AlaPro 165 170175

Glu Leu Leu Tyr Tyr Ala Glu Lys Tyr Asn Glu Val Leu Thr Gin Cys 180 185190

Cys Thr Glu Ser Asp Lys Ala Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200205

Val Lys Glu Lys Ala Leu Val Ala Ala Val Arg Gin Arg Met Lys Cys 210 215220

Ser Ser Met Gin Arg Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235240

Ala Arg Met Ser Gin Arg Phe Pro Asn Ala Glu Phe Ala Glu lie Thr 245 250255

Lys Leu Ala Thr Asp Val Thr Lys He Asn Lys Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280285

Cys Glu Asn Gin Ala Thr lie Ser Ser Lys Leu Gin Ala Cys Cys Asp 290 295300

Lys Pro Val Leu Gin Lys Ser Gin Cys Leu Ala Glu He Glu His Asp 305 310 315320

Asn He Pro Ala Asp Leu Pro Ser He Ala Ala Asp Phe Val Glu Asp 325 330335

Lys Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345350

Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser 355 360365

Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375380

Cys Ala Glu Gly Asp Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu 385 390 395400

Phe Gin Pro Leu Val Glu Glu Pro Lys Asn Leu val Lys Thr AsnCys 405 410415

Glu Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gin Asn Ala Val Leu 420 425430

Val Arg Tyr Thr Gin Lys Ala Pro Gin Val Ser Thr Pro Thr Leu Val 435 440445

Glu Ala Ala Arg Asn Leu Gly Arg val Gly Thr Lys Cys Cys Thr Leu 450 455460

Pro Glu Ala Gin Arg Léu Pro Cys Val Glu Asp Tyr Leu Ser Alalie 465 470 475480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser GluLys 485 490495

Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg pro Cys Phe 500 505510

Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520525

Glu Thr Phe Thr Phe His Ser Asp He Cys Thr Leu Pro Asp Lys Glu 530 535540

Lys Gin He Lys Lys Gin Thr Ala Leu Ala Glu Leu Val Lys His Lys 545 550 555560

Pro Lys Ala Thr Glu Asp Gin Leu Lys Thr Val Met Gly Asp Phe Ala 565 570575

Gin Phe Val Asp Lys Cys Cys Lys Ala Ala Asp Lys Asp Asn Cys Phe 580 585590

Ala Thr Glu Gly Pro Asn Leu Val Ala Arg Ser Lys Glu Ala Leu Ala 595 600605 <210> 10 <211 > 607

<212> PRT <213> Bos taurus <400> 10

Met Lys Trp Val Thr Phe He Ser Leu Leu Leu Leu Phe Ser Ser Ala 15 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu He Ala 20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu His Phe Lys Gly Leu Val Leu 35 4U40

Ile Ala Phe Ser Gin Tyr Leu Gin Gin Cys Pro Phe Asp Glu His Val 50 5560

Lys Leu Val Asri Glu Leu Thr Glu Phe Ala Lys Thr Cys Val AlaAsp 65 70 7580

Glu Ser His Ala Gly Cys Glu Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Glu Leu Cys Lys Val Ala Ser Leu Arg Glu Thr Tyr Gly Asp Met Ala 100 105110

Asp CyS Cys Glu Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Ser 115 120125

His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Asp Pro Asn 130 135140

Thr Leu Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp GlyLys 145 150 155160

Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala ProGlu 165 170175

Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gin Glu Cys Cys 180 185190

Gin Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Glu Thr Met 195 200205

Arg Glu Lys val Leu Ala Ser Ser Ala Arg Gin Arg Leu Arg Cys Ala 210 215220

Ser Ile Gin Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser ValAla 225 230 235240

Arg Leu Ser Gin Lys Phe Pro Lys Ala Glu Phe Val Glu Val ThrLys 245 250255

Leu Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280285

Asp Asn Gin Asp Thr Ile Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys 290 295300

Pro Leu Leu Glu Lys Ser His Cys ile Ala Glu Val Glu Lys AspAla 305 310 315320

Ile Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu AspLys 325 330335

Asp Val Cys Lys Asn Tyr Gin Glu Ala Lys Asp Ala Phe Leu Gly Ser 340 345350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val 355 360365

Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Glu Cys Cys 370 375330

Ala Lys Asp Asp Pro His Ala CyS Tyr Ser Thr Val Phe Asp LysLeu 385 390 395400

Lys His Leu Val Asp Glu Pro Gin Asn Leu lie Lys Gin Asn CysAsp 405 410415

Gin Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gin Asn Ala Leu He Val 420 425430

Arg Tyr Thr Arg Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val Glu 435 440445

Val Ser Arg Ser Leu Gly Lys Val Gly Thr Arg Cys Cys Thr Lys Pro 450 455460

Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu He Leu 465 470 475480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val 485 490495

Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505510

Ala Leu Thr Pro Asp Glu Thr Tyr Val Pro Lys Ala Phe Asp Glu Lys 515 520525

Leu Phe Thr Phe His Ala Asp lie Cys Thr Leu Pro Asp Thr Glu Lys 530 535540

Gin lie Lys Lys Gin Thr Ala Leu Val Glu Leu Leu Lys His LysPro 545 550 555560

Lys Ala Thr Glu Glu Gin Leu Lys Thr Val Met Glu Asn Phe ValAla 565 570575

Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Ala Cys Phe Ala 580 585590

Val Glu Gly Pro Lys Leu Val Val Ser Thr Gin Thr Ala Leu Ala 595 600605 <210> 11 <211 > 607

<212> PRT <213> Equus caballus <400> 11

Met Lys Trp Val Thr Phe Val Ser Leu Leu Phe LeU Phe Ser Ser Ala 15 1015

Tyr Ser Arg Gly Val Leu Arg Arg Asp Thr His Lys Ser Glu lie Ala 20 2530

His Arg Phe Asn Asp Leu Gly Glu Lys His Phe Lys Gly Leu Val Leu 35 4045

Val Ala Phe Ser Gin Tyr Leu Gin Gin Gys Pro Phe Glu Asp His Val 50 5560

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Lys Cys Ala AlaAsp 65 70 7580

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Lys Leu Cys Thr Val Ala Thr Leu Arg Ala Thr Tyr Gly Glu Leu Ala 100 105110

Asp Cys Cys Glu Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Thr 115 120125

His Lys Asp Asp His Pro Asn Leu Pro Lys Leu Lys Pro Glu Pro Asp 130 135140

Ala Gin Cys Ala Ala Phe Gin Glu Asp Pro Asp Lys Phe Leu GlyLys 145 150 155160

Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Gly ProGlu 165 170175

Leu Leu Phe His Ala Glu Glu Tyr Lys Ala Asp Phe Thr Glu Cys Cys 180 185190

Pro Ala Asp Asp Lys Léu Ala Cys Leu He Pro Lys Leu Asp Ala Leu 195 200205

Lys Glu Arg He Leu Leu Ser Ser Ala Lys Glu Arg Leu Lys Cys Ser 210 215220

Ser Phe Gin Asn phe Gly Glu Arg Ala Val Lys Ala Trp Ser ValAla 225 230 235240

Arg Leu Ser Gin Lys Phe Pro Lys Ala Asp Phe Ala Glu val SerLys 245 250255 lie Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr He Cys 275 280285

Glu His Gin Asp Ser lie Ser Gly Lys Leu Lys Ala Cys Cys Asp Lys 290 295300

Pro Leu Leu Gin Lys Ser His Cys He Ala Glu Val Lys Glu Asp Asp 305 310 315320

Leu Pro Ser Asp Leu Pro Ala Leu Ala Ala Asp Phe Ala Glu Asp Lys 325 330335

Glu lie CyS Lys His Tyr Lys Asp Ala Lys Asp Val Phe Leu Gly Thr 340 345350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu 355 360365

Leu Leu Arg lie Ala Lys Thr Tyr Glu Ala Thr Leu Glu Lys Cys Cys 370 375380

Ala Glu Ala Asp Pro Pro Ala Cys Tyr Arg Thr Val Phe Asp GinPhe 385 390 395400

Thr Pro Léu Val Glu Glu Pro Lys Ser Leu Val Lys Lys Asn CysAsp 405 410415

Leu Phe Glu Glu Val Gly Glu Tyr Asp Phe Gin Asn Ala Leu lie Val 420 425430

Arg Tyr Thr Lys Lys Ala Pro Gin Val Ser Thr Pro Thr Leu Val Glu 435 440445

He Gly Arg Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro 450 455460

Glu Ser Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu AlaLeu 465 470 475480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu LysHe 485 490495

Thr Lys Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro Cys Phe Ser 500 505510

Ala Leu Glu Leu Asp Glu Gly Tyr Val Pro Lys Glu Phe Lys Ala Glu 515 520525

Thr Phe Thr Phe His Ala Asp He Cys Thr Leu Pro Glu Asp Glu Lys 530 535540

Gin He Lys Lys Gin Ser Ala Leu Ala Glu Leu Val Lys His LysPro 545 550 555560

Lys Ala Thr Lys Glu Gin Leu Lys Thr Val Leu Gly Asn Phe SerAla 565 570575

Phe Val Ala Lys Cys Cys Gly Arg Glu Asp Lys Glu Ala Cys Phe Ala 580 585590

Glu Glu Gly Pro Lys Leu Val Ala Ser Ser Gin Leu Ala Leu Ala 595 600605 <210> 12 <211 > 607

<212> PRT <213> Equus asinus <400> 12

Met Lys Trp Val Thr Phe Val Ser Leu Leu Phe Leu Phe Ser Ser Ala 15 1015

Tyr Phe Arg Gly Val Leu Arg Arg Asp Thr His Lys Ser Glu Ile Ala 20 2530

His Arg Phe Asn Asp Leu Gly Glu Lys His Phe Lys Gly Leu Val Leu 35 4045

Val Ala Phe Ser Gin Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val 50 5560

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Lys Cys Ala AlaAsp 65 70 7580

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Lys Leu Cys Thr Val Ala Thr Leu Arg Ala Thr Tyr Gly Glu Leu Ala 100 105110

Asp Cys Cys Glu Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Thr 115 120125

His Lys Asp Asp His Pro Asn Leu Pro Lys Leu Lys Pro Glu Pro ASp 130 135140

Ala Gin Cys Ala Ala Phe Gin Glu Asp Pro Asp Lys Phe Leu GlyLys 145 150 155160

Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Gly ProGlu 165 170175

Leu Leu Phe His Ala Glu Glu Tyr Lys Ala Asp Phe Thr Glu Cys Cys 180 185190

Pro Ala Asp Asp Lys Ala Gly Cys Leu Ile Pro Lys Leu Asp Ala Leu 195 200205

Lys Glu Arg Ile Leu Leu Ser Ser Ala Lys Glu Arg Leu Lys Cys Ser 210 215220

Ser Phe Gin Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Val Ala 225 230 235240

Arg LéU Ser Gin Lys Phe Pro Lys Ala Asp Phe Ala Glu Val Ser Lys 245 250255

Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp

26U 26527U

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Thr Lys Tyr lie Cys 275 280285

Glu His Gin Asp Ser lie Ser Gly Lys Leu Lys Ala Cys Cys Asp Lys 290 295300

Pro Leu Leu Gin Lys Ser His Cys He Ala Glu Val Lys Glu AspAsp 305 310 315320

Leu Pro Ser Asp Leu Pro Ala Leu Ala Ala Asp Phe Ala Glu AspLys 325 330335

Glu Tie Cys Lys His Tyr Lys Asp Ala Lys Asp Val Phe Leu Gly Thr 340 345350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu 355 360365

Leu Leu Arg He Ala Lys Thr Tyr Glu Ala Thr Leu Glu Lys Cys Cys 370 375380

Ala Glu Ala Asp Pro Pro Ala Cys Tyr Ala Thr Val Phe Asp GinPhe 385 390 395400

Thr Pro Leu Val Glu Glu Pro Lys Ser Leu Val Lys Lys Asn CysAsp 405 410415

Leu Phe Glu Glu Val Gly Glu Tyr Asp Phe Giri Asn Ala Leu lie Val 420 425430

Arg Tyr Thr Lys Lys Ala Pro Gin Val Ser Thr Pro Thr Leu Val Glu 435 440445

He Gly Arg Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro 450 455460

Glu Ser Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu AlaLeu 465 470 475480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu LysHe 485 490495

Thr Lys Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro Cys Phe Ser 500 505510

Ala Leu Glu Leu Asp Glu Gly Tyr lie Pro Lys Glu Phe Lys Ala Glu 515 520525

Thr Phe Thr Phe His Ala Asp He Cys Thr Leu Pro Glu Asp Glu Lys 530 535540

Gin He Lys Lys Gin Ser Ala Leu Ala Glu Leu Val Lys His LysPro 545 550 555560

Lys Ala Thr Lys Glu Gin Leu Lys Thr Val Leu Gly Asn Phe SerAla 565 570575

Phe Val Ala Lys Cys Cys Gly Ala Glu Asp Lys Glu Ala Cys Phe Ala 580 585 590

Glu Glu Gly Pro Lys Leu Val Ala Ser Ser Gin Leu Ala Leu Ala 595 600 605 <210> 13 <211 > 608

<212> PRT <213> Oryctolagus cuniculus <400> 13

Met Lys Trp Val Thr Phe lie Ser Leu Leu Phe LeU Phe Ser Ser Ala 15 1015

Tyr Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu He Ala 20 2530

His Arg Phe Asn Asp Val Gly Glu Glu His Phe lie Gly Leu Val Leu 35 4045 lie Thr Phe Ser Gin Tyr Leu Gin Lys Cys Pro Tyr Glu Glu His Ala 50 5560

Lys Leu Val Lys Glu Val Thr Asp Leu Ala Lys Ala Cys Val AlaAsp 65 70 7580

Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Asp He Phe GlyAsp 85 9095

Lys He Cys Ala Leu Pro Ser Leu Arg Asp Thr Tyr Gly Asp Val Ala

100 105HO

Asp Cys Cys Glu Lys Lys G1U Pro Glu Arg Asn Glu Cys Phe Leu His 115 120125

His Lys Asp Asp Lys Pro Asp Leu Pro Pro Phe Ala Arg Pro Glu Ala 130 135140

Asp Val Leu Cys Lys Ala Phe His Asp Asp Glu Lys Ala Phe PheGly 145 150 155160

His Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr AlaPro 165 170175

Glu Leu Leu Tyr Tyr Ala Gin Lys Tyr Lys Ala He Leu Thr Glu Cys 180 185190

Cys Glu Ala Ala Asp Lys Gly Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200205

Leu Glu Gly Lys Ser Leu lie Ser Ala Ala Gin Glu Arg Leu Arg Cys 210 215220 A±a ser lie Gin Lys Phe Gly Asp Arg Ala Tyr Lys Ala Trp A±a Leu 225 230 235240

Val Arg Leu Ser Gin Arg Phe Pro Lys Ala Asp Phe Thr Asp IleSer 245 250255

Lys Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Met 275 280285

Cys Glu His Gin Glu Thr Ile Ser Ser His Leu Lys Glu Cys Cys Asp 290 295300

Lys Pro Ile Leu Glu Lys Ala His Cys Ile Tyr Gly Leu His AsnAsp 305 310 315320

Glu Thr Pro Ala Gly Leu Pro Ala Val Ala Glu Glu Phe Val GluAsp 325 330335

Lys Asp Val Cys Lys Asn Tyr Glu Glu Ala Lys Asp Leu Phe Leu Gly 340 345350

Lys Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Val 355 360365

Leu Leu Leu Arg Leu Gly Lys Ala Tyr Glu Ala Thr Leu Lys Lys Cys 370 375380

Cys Ala Thr Asp Asp Pro His Ala CyS Tyr Ala Lys Val Leu ASpGlu 385 390 395400

Phe Gin Pro Leu Val Asp Glu Pro Lys Asn Leu Val Lys Gin AsnCys 405 410415

Glu Leu Tyr Glu Gin Leu Gly Asp Tyr Asn Phe Gin Asn Ala Leu Leu 420 425430

Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val 435 440445

Glu Ile Ser Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455460

Pro Glu Ala Glu Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser ValVal 465 470 475480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser GluLys 485 490495

Val Thr Lys Cys Cys Ser Glu Ser Leu Val Asp Arg Arg Pro Cys Phe 500 505510

Ser Ala Leu Gly Pro Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520525

Glu Thr Phe Thr Phe His Ala Asp lie Cys Thr Leu Pro Glu Thr Glu 530 535540

Arg Lys He Lys Lys Gin Thr Ala Leu Val Glu Leu Val Lys HisLys 545 550 555560

Pro His Ala Thr Asn Asp Gin Leu Lys Thr Val Val Gly Glu PheThr 565 570575

Ala Leu Leu Asp Lys Cys Cys Ser Ala Glu Asp Lys Glu Ala Cys Phe 580 585590

Ala Val Glu Gly Pro Lys Leu Val Glu Ser Ser Lys Ala Thr Leu Gly 595 600605 <210> 14 <211 > 583

<212> PRT <213> Capra hircus <400> 14

Asp Thr His Lys Ser Glu He Ala His Arg Phe Asn Asp Leu Gly Glu 1 5 1015

Glu Asn Phe Gin Gly Leu Val Leu lie Ala Phe Ser Gin Tyr Leu Gin 20 2530

Gin Cys Pro Phe Asp Glu His Val Lys Leu Val Lys Glu Leu Thr Glu 35 4045

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser His Ala Gly Cys Asp Lys 50 5560

Ser Leu His Thr Leu Phe Gly Asp Glu Leu Cys Lys Val Ala ThrLeu 65 70 7580

Arg Glu Thr Tyr Gly Asp Met Ala Asp Cys Cys Glu Lys Gin GluPro 85 9095

Glu Arg Asn Glu Cys Phe Leu Lys His Lys Asp Asp Ser Pro Asp Leu 100 105110

Pro Lys Leu Lys Pro Glu Pro Asp Thr Leu Cys Ala Glu Phe Lys Ala 115 120125

Asp Glu Lys Lys Phe Trp Gly Lys Tyr Leu Tyr Glu Val Ala Arg Arg 130 135140

His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala Asn LysTyr 145 150 155160

Asn Gly Val Phe Gin Glu Cys Cys Gin Ala Glu Asp Lys Gly AlaCys 165 170175

Leu Leu Pro Lys lie Glu Thr Met Arg Glu Lys Val Leu Ala Ser Ser 180 185190

Ala Arg Gin Arg Leu Arg Cys Ala Ser lie Gin Lys Phe Gly Glu Arg 195 200205

Ala Leu Lys Ala Trp Ser Val Ala Arg Leu Ser Gin Lys Phe Pro Lys 210 215220

Ala Asp Phe Thr Asp Val Thr Lys He Val Thr Asp Leu Thr LysVal 225 230 235240

His Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp AspArg 245 250255

Ala Asp Leu Ala Lys Tyr lie Cys Asp His Gin Asp Thr Leu Ser Ser 260 265270

Lys Leu Lys Glu Cys Cys Asp Lys Pro Val LeU Glu Lys Ser His Cys 275 280285 lie Ala Glu lie Asp Lys Asp Ala Val Pro Glu Asn Leu Pro Pro Leu 290 295300

Thr Ala Asp Phe Ala Glu Asp Lys Glu Val Cys Lys Asn Tyr Gin Glu 305 310 315320

Ala Lys Asp Val Phe Leu Gly Ser Phe Leu Tyr Glu Tyr Ser Arg Arg 325 330335

His Pro Glu Tyr Ala Val Ser Val Leu Leu Arg Leu Ala Lys Glu Tyr 340 345350

Glu Ala Thr Leu Glu Asp Cys Cys Ala Lys Glu Asp Pro His Ala Cys 355 360365

Tyr Ala Thr Val Phe Asp Lys Leu Lys His Leu Val Asp Glu Pro Gin 370 375380

Asn Leu He Lys Lys Asn Cys Glu Leu Phe Glu Lys His Gly GluTyr 385 390 395400

Gly Phe Gin Asn Ala Leu lie Val Arg Tyr Thr Arg Lys Ala ProGin 405 410415

Val Ser Thr Pro Thr Leu Val Glu lie Ser Arg Ser Leu Gly Lys Val 420 425430

Gly Thr Lys Cys Cys Ala Lys Pro Glu Ser Glu Arg Met Pro Cys Thr 435 440445

Glu Asp Tyr Leu Ser Leu He Leu Asn Arg Leu Cys Val Leu His Glu 450 455460

Lys Thr Pro Val Ser Glu Lys Val Thr Lys Cys Cys Thr Glu SerLeu 465 470 475480

Val Asn Arg Arg Pro Cys Phe Ser Asp Leu Thr Leu Asp Glu ThrTyr 485 490495

Val Pro Lys Pro Phe Asp Gly Glu Ser Phe Thr Phe His Ala Asp Ile 500 505510

Cys Thr Leu Pro Asp Thr Glu Lys Gin Ile Lys Lys Gin Thr Ala Leu 515 520525

Val Glu Leu Leu Lys His Lys Pro Lys Ala Thr Asp Glu Gin Leu Lys 530 535540

Thr Val Met Glu Asn Phe Val Ala Phe Val Asp Lys Cys Cys AlaAla 545 550 555560

Asp Asp Lys Glu Gly Cys Phe Leu Leu Glu Gly Pro Lys Léu ValAla 565 570575

Ser Thr Gin Ala Ala Leu Ala 580 <210> 15 <211 > 607

<212> PRT <213> Ovis aries <400> 15

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala 15 1015

Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu ile Ala 20 2530

His Arg Phe Asn Asp Leu Gly Glu Glu Asn Phe Gin Gly Leu Val Leu 35 4045

Ile Ala Phe Ser Gin Tyr Leu Gin Gin Cys Pro Phe Asp Glu His Val 50 5560

Lys Leu Val Lys Glu Leu Thr Glu Phe Ala Lys Thr Cys Val AlaAsp 65 70 7580

Glu Ser His Ala Gly Cys Asp LyS Ser Léu His Thr LeU Phe GlyAsp 85 9095

Glu Leu Cys Lys Val Ala Thr Leu Arg Glu Thr Tyr Gly Asp Met Ala 100 105110

Asp Cys Cys Glu Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Asn 115 120125

His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Glu Pro Asp 130 135140

Thr Leu Cys Ala Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys 145 150 155160 xyx lieu xyr u>iu vui λι<± Axy Hiy rus r-xo xyr fiie iyr hiu rru uiu 165 170175

Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gin Glu Cys Cys 180 185190

Gin Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Asp Ala Met 195 200205

Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gin Arg Leu Arg Cys Ala 210 215220

Ser Ile Gin Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser ValAla 225 230 235240

Arg Leu Ser Gin Lys Phe Pro Lys Ala Asp Phe Thr Asp Val ThrLys 245 250255

Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280285

Asp His Gin Asp Ala Leu Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys 290 295300

Pro Val Leu Glu Lys Ser His Cys Ile Ala Glu Val Asp Lys Asp Ala 305 310 315320

Val Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys 325 330335

Glu Val Cys Lys Asn Tyr Gin Glu Ala Lys Asp Val Phe Leu Gly Ser 340 345350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val 355 360365

Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Asp Cys Cys 370 375380

Ala Lys Glu Asp Pro His Ala Cys Tyr Ala Thr Val Phe Asp LysLeu 385 390 395400

Lys His Leu Val Asp Glu Pro Gin Asn Leu Ile Lys Lys Asn CysGlu 405 410415

Leu Phe Glu Lys His Gly Glu Tyr Gly Phe Gin Asn Ala Leu Ile Val 420 425430

Arg Tyr Thr Arg Lys Ala Pro Gin Val Ser Thr Pro Thr Leu Val Glu 435 440445

Ile Ser Arg Ser Leu Gly Lys Val Gly Thr Lys Cys Cys Ala Lys Pro 450 455460

Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu 465 470 475480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val 485 490495

Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505510

Asp Leu Thr Leu Asp Glu Thr Tyr Val Pro Lys Pro Phe Asp Glu Lys 515 520525

Phe Phe Thr Phe His Ala Asp lie Cys Thr Leu Pro Asp Thr Glu Lys 530 535540

Gin lie Lys Lys Gin Thr Ala Leu Val Glu Leu Leu Lys His LysPro 545 550 555560

Lys Ala Thr Asp Glu Gin Leu Lys Thr Val Met Glu Asn Phe ValAla 565 570575 phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Gly Cys Phe Val 580 585590

Leu Glu Gly Pro Lys Leu Val Ala Ser Thr Gin Ala Ala Leu Ala 595 600605 <210> 16 <211 > 608

<212> PRT <213> canis lupus familiaris <400> 16

Met Lys Trp Val Thr Phe lie Ser Leu Phe Phe Leu Phe Ser Ser Ala 15 1015

Tyr Ser Arg Gly Leu Val Arg Arg Glu Ala Tyr Lys Ser Glu lie Ala 20 2530

His Arg Tyr Asn Asp Leu Gly Glu Glu His Phe Arg Gly Leu Val Leu 35 4045

Val Ala Phe Ser Gin Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val 50 5560

Lys Leu Ala Lys Glu Val Thr Glu Phe Ala Lys Ala Cys Ala AlaGlu 65 70 7580

Glu Ser Gly Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe GlyAsp 85 9095

Lys Leu Cys Thr Val Ala Ser Leu Arg Asp Lys Tyr Gly Asp Met Ala 100 105110

Asp Cys Cys Glu Lys Gin Glu Pro Asp Arg Asn Glu Cys Phe Leu Ala

1t r 1 on1 OR

His Lys Asp Asp Asn Pro Gly Phe Pro Pro Leu Val Ala Pro Glu Pro 130 135140

Asp Ala Leu Cys Ala Ala Phe Gin Asp Asn Glu Gin Leu Phe LeuGly

145 150 155ISO

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr AlaPro 165 170175

Glu Leu Leu Tyr Tyr Ala Gin Gin Tyr Lys Gly Val Phe Ala Glu Cys 180 185190

Cys Gin Ala Ala Asp Lys Ala Ala Cys Leu Gly Pro Lys Ile Glu Ala 195 200205

Leu Arg Glu Lys Val Leu Leu Ser Ser Ala Lys Glu Arg Phe Lys Cys 210 215220

Ala Ser Leu Gin Lys Phe Gly Asp Arg Ala Phe Lys Ala Trp Ser Val 225 230 235240

Ala Arg Leu Ser Gin Arg Phe Pro Lys Ala Asp Phe Ala Glu Ile Ser 245 250255

Lys Val Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly 260 265270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Met 275 280285

Cys Glu Asn Gin Asp Ser Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp 290 295300

Lys Pro Val Leu Glu Lys Ser Gin Cys Leu Ala Glu Val Glu Arg Asp 305 310 315320

Glu Leu Pro Gly Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Asp 325 330335

Lys Glu Val Cys Lys Asn Tyr Gin Glu Ala Lys Asp Val Phe Leu Gly 340 345350

Thr Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Glu Tyr Ser Val Ser 355 360365

Leu Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Lys Cys 370 375380

Cys Ala Thr Asp Asp Pro Pro Thr Cys Tyr Ala Lys Val Leu AspGlu 385 390 395400

Phe Lys Pro Leu Val Asp Glu Pro Gin Asn Leu Val Lys Thr AsnCys 405 410415

Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gin Asn Ala Leu Leu 420 425430

Val Arg Tyr Thr Lys Lys Ala Pro Gin Val Ser Thr Pro Thr Leu Val 435 440445

Glu Val Ser Arg Lys Leu Gly Lys Val Gly Thr Lys Cys Cys Lys Lys 450 455460

Pro Glu Ser Glu Arg Met Ser Cys Ala Glu Asp Phe Leu Ser ValVal 465 470 475480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser GluArg 485 490495

Val Thr Lys Cys Cys Ser Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505510

Ser Gly Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520525

Glu Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Ala Glu 530 535540

Lys Gin Val Lys Lys Gin Thr Ala Leu Val Glu Leu LeU Lys HisLys 545 550 555560

Pro Lys Ala Thr Asp Glu Gin Leu Lys Thr Val Met Gly Asp PheGly 565 570575

Ala Phe Val Glu Lys Cys Cys Ala Ala Glu Asn Lys Glu Gly Cys Phe 580 585590

Ser Glu Glu Gly Pro Lys Leu Val Ala Ala Ala Gin Ala Ala Leu Val 595 600605 <210> 17 <211> 615

<212> PRT <213> Gallus gallus <400> 17 Mét Lys Trp Val Thr Leu Ile Ser Phe Ilé Phe Léu Phe Ser Ser Ala 15 1015

Thr Ser Arg Asn Leu Gin Arg Phe Ala Arg Asp Ala Glu His Lys Ser 20 2530

Glu Ile Ala His Arg Tyr Asn Asp Leu Lys Glu Glu Thr Phe Lys Ala 35 4045

Val Ala Met Ile Thr Phe Ala Gin Tyr Leu Gin Arg Cys Ser Tyr Glu 50 5560

Gly Leu Ser Lys Leu Val Lys Asp Val Val ASp Leu Ala Gin LysCys 65 70 7580

Val Ala Asn Glu Asp Ala Pro Glu Cys Ser Lys Pro Leu Pro SerIle 85 9095

Ile Leu Asp Glu Ile Cys Gin Val Glu Lys Leu Arg Asp Ser Tyr Gly 100 105110

Ala Met Ala Asp Cys Cys Ser Lys Ala Asp Pro Glu Arg Asn Glu Cys 115 120125

Phe Leu Ser Phe Lys Val Ser Gin Pro Asp Phe Val Gin Pro Tyr Gin 130 135140

Arg Pro Ala Ser Asp Val Ile Cys Gin Glu Tyr Gin Asp Asn ArgVal 145 150 155160

Ser Phe Leu Gly His Phe Ile Tyr Ser Val Ala Arg Arg His ProPhe 165 170175

Leu Tyr Ala Pro Ala Ile Leu Ser Phe Ala Val Asp Phe Glu His Ala 180 185190

Leu Gin Ser Cys Cys Lys Glu Ser Asp Val Gly Ala Cys Leu Asp Thr 195 200205

Lys Glu Ile Val Met Arg Glu Lys Ala Lys Gly Val Ser Val Lys Gin 210 215220

Gin Tyr Phe Cys Gly Ile Leu Lys Gin Phe Gly Asp Arg Val PheGin 225 230 235240

Ala Arg Gin Leu Ile Tyr Leu Ser Gin Lys Tyr Pro Lys Ala ProPhe 245 250255

Ser Glu Val Ser Lys Phe Val His Asp Ser Ile Gly Val His Lys Glu 260 265270

Cys Cys Glu Gly Asp Met Val Glu Cys Met Asp Asp Met Ala Arg Met 275 280285

Met Ser Asn Léu Cys Ser Gin Gin Asp Val Phe Ser Gly Lys- Ile Lys 290 295300

Asp Cys Cys Glu Lys Pro Ile Val Glu Arg Ser Gin Cys Ile MetGlu 305 310 315320

Ala Glu phe Asp Glu Lys Pro Ala Asp Leu Pro Ser Leu Val GluLys 325 330335

Tyr Ile Glu Asp Lys Glu Val Cys Lys Ser Phe Glu Ala Gly His Asp 340 345350

Ala Phe Met Ala Glu Phe Val Tyr Glu Tyr Ser Arg Arg His Pro Glu 355 360365

Phe Ser Ile Gin Leu Ile Met Arg Ile Ala Lys Gly Tyr Glu Ser Leu 370 375380

Leu Glu Lys Cys Gys Lys Thr Asp Asn Pro Ala Glu Cys Tyr Ala Asn 385 390 395400

Ala Gin Glu Gin Leu Asn Gin His He Lys Glu Thr Gin Asp Val Val 405 410415

Lys Thr Asn Cys Asp Leu Leu His Asp His Gly Glu Ala Asp Phe Leu 420 425430

Lys Ser lie Leu lie Arg Tyr Thr Lys Lys Met Pro Gin Val Pro Thr 435 440445

Asp Leu Leu Leu Glu Thr Gly Lys Lys Met Thr Thr lie Gly Thr Lys 450 455460

Cys Cys Gin Leu Gly Glu Asp Arg Arg Met Ala Cys Ser Glu GlyTyr 465 470 475480

Leu Ser lie Val lie His Asp Thr Cys Arg Lys Gin Glu Thr ThrPro 485 490495 lie Asn Asp Asn Val Ser Gin Cys Cys Ser Gin Leu Tyr Ala Asn Arg 500 505510

Arg Pro Cys Phe Thr Ala Met Gly Val Asp Thr Lys Tyr Val Pro Pro 515 520525

Pro Phe Asn Pro Asp Met Phe Ser Phe Asp Glu Lys Leu Cys Ser Ala 530 535540

Pro Ala Glu Glu Arg Glu Val Gly Gin Met Lys Leu Leu lie AsnLeu 545 550 555560 lie Lys Arg Lys Pro Gin Met Thr Glu Glu Gin lie Lys Thr lieAla 565 570575

Asp Gly Phe Thr Ala Met Val Asp Lys Cys Cys Lys Gin Ser Asp He 580 585590

Asn Thr Cys Phe Gly Glu Glu Gly Ala Asn Leu lie Val Gin Ser Arg 595 600605

Ala Thr Leu Gly He Gly Ala 610615 <210> 18 <211 > 607

<212> PRT <213> Sus scrofa <400> 18

Met Lys Trp Val Thr Phe He Ser Leu Leu Phe Leu Phe Ser Ser Ala 15 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr Tyr Lys Ser Glu He Ala 20 25 30

His Arg Phe Lys Asp Leu Gly Glu Gin Tyr Phe Lys Gly Leu Val Leu 35 4045

Ile Ala Phe Ser Gin His Leu Gin Gin Cys Pro Tyr Glu Glu His Val 50 5560

Lys Leu Val Arg Glu Val Thr G1U Phe Ala Lys Thr Cys Val AlaAsp 65 70 7580

Glu Ser Ala Glu Asn Cys Asp Lys Ser Ilé His Thr Leu Phe GlyAsp 85 9095

Lys Leu Cys Ala Ile Pro Ser Leu Arg Glu His Tyr Gly Asp Leu Ala 100 105110

Asp Cys Cys Glu Lys Glu Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin 115 120125

His Lys Asn Asp Asn Pro Asp Ile Pro Lys Leu Lys Pro Asp Pro Val 130 135140

Ala Leu Cys Ala Asp Phe Gin Glu Asp Glu Gin Lys Phe Trp GlyLys 145 150 155160

Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala ProGlu 165 170175

Leu Leu Tyr Tyr Ala Ile Ile Tyr Lys Asp Val Phe Ser Glu Cys Cys 180 185190

Gin Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Ile Glu His Leu 195 200205

Arg Glu Lys Val Leu Thr Ser Ala Ala Lys Gin Arg Leu Lys Cys Ala 210 215220

Ser Ile Gin Lys phe Gly Glu Arg Ala phe Lys Ala Trp Ser LeuAla 225 230 235240

Arg Leu Ser Gin Arg Phe Pro Lys Ala Asp Phe Thr Glu ile SerLys 245 250255

Ile Val Thr ASp Leu Ala Lys Val His Lys Glu Cys Cys His Gly Asp 260 265270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280285

Glu Asn Gin Asp Thr Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp Lys 290 295300

Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Ala Lys Arg AspGlu 305 310 315320

Leu Pro Ala Asp Leu Asn Pro Leu Glu His Asp Phe Val Glu AspLys 325 330335

Glu Val Cys Lys Asn Tyr Lys Glu Ala Lys His val Phe Leu Gly Thr 340 345350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu 355 360365

Leu Leu Arg lie Ala Lys lie Tyr Glu Ala Thr Leu Glu Asp Cys Cys 370 375380

Ala Lys Glu Asp Pro Pro Ala Cys Tyr Ala Thr Val Phe Asp LysPhe 385 390 395400

Gin Pro Leu Val Asp Glu Pro Lys Asn Leu He Lys Gin Asn CysGlu 405 410415

Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gin Asn Ala Leu He val 420 425430

Arg Tyr Thr Lys Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val Glu 435 440445

Val Ala Arg Lys Leu Gly Leu Val Gly Ser Arg Cys Cys Lys Arg Pro 450 455460

Glu Glu Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Leu ValLeu 465 470 475480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro val Ser Glu Lysval 485 490495

Thr Lys Cys Cys Thr Glu Ser Leu Val Ash Arg Arg Pro Cys Phe Ser 500 505510

Ala Leu Thr Pro Asp Glu Thr Tyr Lys Pro Lys Glu Phe Val Glu Gly 515 520525

Thr Phe Thr Phe His Ala Asp Leu CyS Thr Leu Pro Glu Asp Glu Lys 530 535540

Gin He Lys Lys Gin Thr Ala Leu Val Glu Leu Leu Lys His LysPro 545 550 555560

His Ala Thr Glu Glu Gin Leu Arg Thr val Leu Gly Asn Phe AlaAla 565 570575

Phe Val Gin Lys Cys Cys Ala Ala Pro Asp His Glu Ala Cys Phe Ala 580 585590

Val Glu Gly Pro Lys Phe Val lie Glu lie Arg Gly lie Leu Ala 595 600605 <210> 19 <211 > 584

<212> PRT <213> Mus musculus <400> 19

Glu Ala His Lys Ser Glu lie Ala His Arg Tyr Asn Asp Leu Gly Glu 15 10 15

Gin His Phe Lys Gly Leu Val Leu He Ala Phe Ser Giri Tyr Leu Gin 20 2530

Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gin Glu Val Thr Asp 35 4045

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Gys Asp Lys 50 5560

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro AsnLeu 65 70 7580

Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gin GluPro 85 9095

Glu Arg Asn Glu Cys Phe Leu Gin His Lys Asp Asp Asn Pro Ser Leu 100 105110

Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys 115 120125

Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala Arg 130 135140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala GluGin 145 150 155160

Tyr Asn Glu Ile Leu Thr Gin Cys Cys Ala Glu Ala Asp Lys GluSer 165 170175

Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys Ala Leu Val Ser 180 185190

Ser Val Arg Gin Arg Met Lys Cys Ser Ser Met Gin Lys Phe Gly Glu 195 200205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gin Thr Phe Pro 210 215220

Asn Ala Asp Phe Ala Glu Ile Thr Lys Leu Ala Thr Asp Leu Thr Lys 225 230 235240

Val Asn Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250255

Arg Ala Glu Leu Ala Lys Tyr Met Cys Glu Asn Gin Ala Thr Ile Ser 260 265270

Ser Lys Leu Gin Thr Cys Cys Asp Lys Pro Leu Leu Lys Lys Ala His 275 280285

Cys Leu Ser Glu Val Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala 290 295300

Ile Ala Ala Asp Phe Val Glu Asp Gin Glu Val Cys Lys Asn TyrAla 305 310 315320

Glu Ala Lys Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg 3^3 33U335

Arg His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys 340 345350

Tyr Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala 355 360365

Cys Tyr Gly Thr Val Leu Ala Glu Phe Gin Pro Leu Val Glu Glu Pro 370 375380

Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu GlyGlu 385 390 395400

Tyr Gly Phe Gin Asn Ala Ile Leu Val Arg Tyr Thr Gin Lys AlaPro 405 410415

Gin Val Ser Thr Pro Thr Leu val Glu Ala Ala Arg Asn Leu Gly Arg 420 425430

Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp Gin Arg Leu Pro Cys 435 440445

Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn Arg Val Cys Leu Leu His 450 455460

Glu Lys Thr Pro Val Ser Glu His Val Thr Lys Cys Cys Ser Gly Ser 465 470 475480

Leu Val Glu Arg Arg Pro Cys Phe Ser Ala Leu Thr Val Asp Glu Thr 485 490495

Tyr Val Pro Lys Glu Phe Lys Ala Glu Thr Phe Thr Phe His Ser Asp 500 505510

Ile Cys Thr Leu Pro Glu Lys Glu Lys Gin Ile Lys Lys Gin Thr Ala 515 520525

Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala Thr Ala Glu Gin Leu 530 535540

Lys Thr Val Met Asp Asp Phe Ala Gin Phe Leu Asp Thr CyS CysLys 545 550 555560

Ala Ala Asp Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn LeuVal 565 570575

Thr Arg Cys Lys Asp Ala Leu Ala 580

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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Claims (21)

1. Sammensætning omfattende fra 25 til 400 g.L-1 albumin, et opløsningsmiddel, fra 200 mM til 1000 mM kationer, med en pH fra ca. 5,0 til ca. 8,0 og hvor sammensætningen omfatter mindre end eller lig med 5 mM octanoat, og hvor albuminen er humant serumalbumin (HSA) med aminosyresekvens af SEQ ID NO: 2 eller en variant deraf med mindst 80% sekvensidentitet med SEQ ID NO: 2.A composition comprising from 25 to 400 g of L-1 albumin, a solvent, from 200 mM to 1000 mM cations, with a pH of ca. 5.0 to approx. And wherein the composition comprises less than or equal to 5 mM octanoate and wherein the albumin is human serum albumin (HSA) having amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 80% sequence identity with SEQ ID NO: 2. 2. Sammensætningen ifølge krav 1, der omfatter mindre end eller lig med 1 mM octanoat.The composition of claim 1 comprising less than or equal to 1 mM octanoate. 3. Sammensætningen ifølge krav 2, hvor kationerne er til stede ved fra 200 til 350 mM.The composition of claim 2, wherein the cations are present at from 200 to 350 mM. 4. Sammensætningen ifølge et hvilket som helst af kravene 1 til 3, hvor pH er fra ca. 6,0 til ca. 7,0.The composition of any one of claims 1 to 3, wherein the pH is from ca. 6.0 to approx. 7.0. 5. Sammensætningen ifølge et hvilket som helst af kravene 1 til 4, hvor pH er ca. pH 6,5.The composition of any one of claims 1 to 4, wherein the pH is approx. pH 6.5. 6. Sammensætningen ifølge et hvilket som helst af kravene 1 til 5, hvor kationerne er valgt fra: natrium, kalium, calcium, magnesium, og ammonium.The composition of any one of claims 1 to 5, wherein the cations are selected from: sodium, potassium, calcium, magnesium, and ammonium. 7. Sammensætningen ifølge et hvilket som helst af kravene 1 til 6, hvor kationerne er natriumioner.The composition of any one of claims 1 to 6, wherein the cations are sodium ions. 8. Sammensætningen ifølge et hvilket som helst af kravene 1 til 7 der omfatter: (a) mindre end 5 mM aminosyrer (såsom N-acetyltryptophan), fortrinsvis mindre end 1 mM aminosyrer; og/eller (b) mindre end 20 mg.L'1 detergent (såsom polysorbat 80), fortrinsvis mindre end 5 mg.L'1 detergent.The composition of any one of claims 1 to 7 comprising: (a) less than 5 mM amino acids (such as N-acetyltryptophan), preferably less than 1 mM amino acids; and / or (b) less than 20 mg.L'1 detergent (such as polysorbate 80), preferably less than 5 mg.L'1 detergent. 9. Sammensætningen ifølge krav 7, der er i det væsentlige fri for aminosyrer (såsom N-acetyltryptophan) og/eller i det væsentlige fri for detergent (såsom polysorbat 80).The composition of claim 7 which is substantially free of amino acids (such as N-acetyltryptophan) and / or substantially free of detergent (such as polysorbate 80). 10. Sammensætning ifølge et hvilket som helst af kravene 1 til 9, hvor albuminet er HSA med aminosyresekvensen af SEQ ID NO: 2.The composition of any one of claims 1 to 9, wherein the albumin is HSA having the amino acid sequence of SEQ ID NO: 2. 11. Sammensætningen ifølge et hvilket som helst af kravene 1 til 10, hvor albuminet er i en koncentration på fra 50 g.L-1 til 400 g.L'1.The composition of any one of claims 1 to 10, wherein the albumin is in a concentration of from 50 g.L-1 to 400 g.L'1. 12. Sammensætningen ifølge et hvilket som helst af kravene 1 til 11, hvor albuminet er et rekombinant albumin.The composition of any one of claims 1 to 11, wherein the albumin is a recombinant albumin. 13. Sammensætningen ifølge krav 1, omfattende: (a) 50 til 250 g.L'1 albumin; (b) 225 til 275 mM Na+; (c) 20 til 30 mM phosphat; og med en pH på ca. 6,5, og hvor sammensætningen omfatter mindre end 2 mM octanoat.The composition of claim 1, comprising: (a) 50 to 250 g of L1 albumin; (b) 225 to 275 mM Na +; (c) 20 to 30 mM phosphate; and with a pH of approx. 6.5, and wherein the composition comprises less than 2 mM octanoate. 14. Sammensætningen ifølge et hvilket som helst af kravene 1 til 13 der er i det væsentlige fri for octanoat.The composition of any one of claims 1 to 13 which is substantially octanoate free. 15. Sammensætningen ifølge et hvilket som helst af kravene 1 til 14 der er mere stabil end en kontrolopløsning af albumin hvor kontrolopløsningen er den samme koncentration i 150 mM Na eller vand.The composition of any one of claims 1 to 14 which is more stable than a control solution of albumin wherein the control solution is the same concentration in 150 mM Na or water. 16. Fremgangsmåde til fremstilling af et celledyrkningsmedium omfattende at supplere et basalmedium med sammensætningen ifølge et hvilket som helst af de foregående krav.A method of preparing a cell culture medium comprising supplementing a basal medium with the composition of any one of the preceding claims. 17. Fremgangsmåden ifølge krav 16, hvor celledyrkningsmediet er et stamcelledyrkningsmedium.The method of claim 16, wherein the cell culture medium is a stem cell culture medium. 18. Fremgangsmåden ifølge krav 16 eller 17, hvor celledyrkningsmediet er i det væsentlige fri for dyre-afledte komponenter.The method of claim 16 or 17, wherein the cell culture medium is substantially free of animal-derived components. 19. Anvendelse af en sammensætning ifølge et hvilket som helst af kravene 1 til 15 til dyrkning af celler.Use of a composition according to any one of claims 1 to 15 for culture of cells. 20. Fremgangsmåde ifølge et hvilket som helst af kravene 16 til 18, yderligere omfattende inkubering af celler i celledyrkningsmediet.The method of any one of claims 16 to 18, further comprising incubating cells in the cell culture medium. 21. Anvendelse eller fremgangsmåde ifølge krav 19 eller 20, i hvilken cellerne omfatter stamceller.The use or method of claim 19 or 20, wherein the cells comprise stem cells.