Process For Concentration Of A Polypeptide

Abstract

The present invention comprises a method of concentrating a composition comprising a polypeptide of interest and the use of such a concentrated composition for the treatment of diseases in mammals, in particular by subcutaneous injection.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a method for concentrating a polypeptide of interest, to the use of a composition comprising a concentrated polypeptide of interest as a medicament for subcutaneous injection and to a composition comprising at least 10 mg/ml polypeptide of interest.

BACKGROUND OF THE INVENTION

[0002] Some polypeptides are useful as a medicament for the prevention and/or treatment of certain diseases. The ability to inject a medicament subcutaneously is an advantage as it makes it easy for the patients to administer the medication to themselves.

[0003] As there are physiological restrains on how large a volume it is possible to inject subcutaneously. Thus it is an advantage for medicaments which are to be administered subcutaneously that they are available in a high concentration so as to ensure that the patient receives an adequate amount of the medicament and/or to avoid multiple subcutaneous injections.

[0004] WO 99/37325 discloses methods of treating and preventing disease caused by absence or deficiency of the activity of enzymes belonging to the heme biosynthetic pathway. WO 03/002731 discloses a process for purification of recombinant porphobilinogen deaminase on an industrial scale and to the use of the purified product for the preparation of a medicament. Similarly, WO 02/099092 and WO 2005/094874 provides lysosomal alpha-mannosidase and therapeutic use hereof. Finally, WO 2005/073367 provides a process for purification of aryl sulfatase A and use of the enzyme in the treatment of metachromatic leukodystrophy.

[0005] The present invention relates to a method for concentrating a polypeptide of interest and to the use of a composition comprising a concentrated polypeptide of interest for the manufacture of a medicament for subcutaneous injection into mammal.

SUMMARY OF THE INVENTION

[0006] The present invention relates in one aspect to a method of concentrating a composition comprising a polypeptide of interest comprising: [0007] a) Centrifugation and/or filtration of a composition comprising a polypeptide of interest [0008] b) Concentrating the supernatant or retentate, respectively, obtained from step a).

[0009] In another aspect the present invention relates to a composition comprising at least 10 mg/ml polypeptide of interest.

[0010] In yet another aspect the present invention relates to use of a composition comprising 75-250 mg/ml polypeptide of interest for the manufacture of a medicament for subcutaneous injection into a mammal.

[0011] In yet another aspect the present invention relates to a method of treating a mammal for Acute Intermittent Porphyria comprising injecting subcutaneously a composition of 500-300 mg/ml PBGD.

[0012] In yet another aspect the present invention relates a method of treating a mammal for metachromatic leukodystrophy comprising subcutaneous injection of a composition of 50-300 mg/ml aryl sulfatase A.

[0013] In yet another aspect the present invention relates a method of treating a mammal for the lysosomal storage disorder alpha-mannosidosis comprising subcutaneous injection of a composition of 50-300 mg/ml lysosomal alpha-mannosidase.

[0014] In yet another aspect the present invention relates a method of treating a mammal for Krabbe disease comprising subcutaneous injection of a composition of 50-300 mg/ml galactosylcerebrosidase.

DEFINITIONS

[0015] For purposes of the present invention, alignments of sequences and calculation of homology scores may be done using a full Smith-Waterman alignment, useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA.

[0016] Alignment may be made with the FASTA package version v20u6 (W. R. Pearson and D. 3. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 183:63-98).

[0017] Multiple alignments of protein sequences may be made using "ClustalW" (Thompson, 1. D., Higgins, D. G. and Gibson, T J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). Multiple alignment of DNA sequences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence.

[0018] In the context of the present invention, the term "E. C." (Enzyme Class) refers to the internationally recognized enzyme classification system, Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press, Inc.

[0019] The term "origin" used in the context of amino acid sequences, e.g. proteins, or nucleic acid sequences is to be understood as referring to the organism from which it derives. Said sequence may be expressed by another organism using gene technology methods well known to a person skilled in the art. This also encompasses sequences which have been chemically synthesized. Furthermore, said sequences may comprise minor changes such as codon optimization, i.e. changes in the nucleic acid sequences which do not affect the amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptide of Interest

[0020] The polypeptide of the present invention may in particular be a hormone or hormone variant, an enzyme, a receptor or portion thereof, an antibody or portion thereof, an allergen or a reporter. The polypeptide of interest may in particular be an enzyme selected from one of six major enzyme groups, such as an oxidoreductase (E.C. 1), a transferase (E.C. 2), a hydrolase (E.C. 3), a lyase (E.C. 4), an isomerase (E.C. 5), or a ligase (E.C. 6). In a more particular aspect, the polypeptide of interest may be an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, cellobiohydrolase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, or beta-xylosidase.

[0021] The polypeptide of interest may in particular be a polypeptide which is useful as a medicament.

[0022] Examples of a suitable polypeptide of interest include but is not limited to one selected from the group consisting of a phorphobilinogen deaminase, an aryl sulfatase, an alpha-mannosidase and a galactocerebrosidase.

[0023] In principle a polypeptide of interest derivable from any source may be treated according to the methods of the present invention.

[0024] In a particular embodiment the polypeptide of interest may be of human origin. Especially in the context of using a polypeptide of interest for the manufacture of a medicament which is to be administered to humans may the polypeptide be of human origin as this may minimize the risk of unwanted allergic reactions. Natural variations of human polypeptide due to e.g. polymorphism are in the context of the present invention included in the term "human origin".

[0025] The polypeptide of interest may in particular be produced as a recombinant protein, i.e. a nucleotide sequence encoding the polypeptide of interest may be introduced into a cell for expression of the polypeptide of interest. The recombinant expression may be homologous or heterologous, i.e. the polypeptide of interest may be expressed in cell which it is naturally expressed by (homologous expression) or it may be expressed by a cell which it is not naturally expressed by (heterologous expression).

[0026] The recombinant polypeptide of interest may be expressed by any cell suitable for recombinant production of the particular polypeptide of interest. Examples of suitable cells include but are not limited to prokaryotic cells, such as an E. coli cell or a Bacillus cell. Examples of suitable eukaryotic cells include but are not limited to a yeast cell or a mammalian cell such as a Chinese Hamster Ovary (CHO). Alternatively, it may be a human cell.

[0027] Suitable host cells for the expression of glycosylated polypeptide are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. However, the host cell may also be a vertebrate cell, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure

[0028] The term "recombinant polypeptide" or "recombinant polypeptide of interest" denotes herein a recombinant produced polypeptide.

[0029] Reference to a particular polypeptide of interest includes in the context of the present invention also functionally equivalent parts or analogues of the polypeptide of interest. For example, if the polypeptide of interest is an enzyme a functionally equivalent part of the enzyme could be a domain or subsequence of the enzyme which includes the necessary catalytic site to enable the domain or subsequence to exert substantially the same enzymatic activity as the full-length enzyme or alternatively a gene coding for the catalyst. The term "substantially the same enzymatic activity" refers to an equivalent part or analogue having at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most preferably at least 97%, at least 98% or at least 99% of the activity of the natural enzyme. An example of an enzymatically equivalent analogue of the enzyme could be a fusion protein which includes the catalytic site of the enzyme in a functional form, but it can also be a homologous variant of the enzyme derived from another species. Also, completely synthetic molecules that mimic the specific enzymatic activity of the relevant enzyme would also constitute "enzymatic equivalent analogues".

[0030] Generally, the skilled person will be able to readily devise appropriate assays for the determination of enzymatic activity. For PBGD, however, a suitable assay is described in WO 03/002731, in example 2, as well as in the experimental sections of the present applications. Aryl sulfatase, in addition to its natural substrates, is also able to catalyze the hydrolysis of the synthetic, chromogenic substrate, para-Nitrocatechol sulfate (pNCS). The product, para-Nitrocatechol (pNC), absorbs light at 515 nm. An assay for determination of aryl sulfatase activity is described in details in WO 2005/073367 and in Fluharty et al. 1978, Meth. Enzymol. 50:537-47. For LAMAN, an appropriate enzyme activity assay is disclosed in WO 02/099092.

Porphobilinogen Deaminase

[0031] In one embodiment the polypeptide of interest of the invention may be porphobilinogen deaminase, (also known as porphobilinogen ammonia-lyase (polymerizing)), E.C. 4.3.1.8. (Waldenstrom 1937, 3. Acta. Med. Scand. Suppl. 8). Porphobilinogen deaminase is the third enzyme in the heme biosynthetic pathway. E.C. 4.3.1.8 has been transferred to E.C. 2.5.1.61, so porphobilinogen deaminase (PBGD) is now placed under this E.C. number.

[0032] Porphobilinogen deaminase catalyzes the reaction of 4 porphobilinogen+H.sub.2O hydroxymethylbilane+4 NH.sub.3.

[0033] PBDG is important in relation to Acute intermittent porphyria (AIP), which is an autosomal dominant disorder in man caused by a defect (50% reduction of activity) of PBDG (see WO01/07065 for further details in relation to this). Porphobilinogen deaminase is in short known as PBGD and in the context of the present invention these two terms may be used inter-changeably with one another.

[0034] For recombinant expression of PBGD a host cell may in particular be a yeast cell or an E. coli cell.

[0035] For a detailed example of construction of a recombinant E. coli cell reference is made to example 1 of WO01/07065 and for construction of recombinant HeLa cells and NIH 3T3 cells capable of expressing mouse PBGD reference is made to example 6 of WO01/07065.

[0036] The term "recombinant porphobilinogen deaminase (rPBGD)" denotes herein a recombinant produced PBGD. In the following, this enzyme and the recombinant human form will be termed "PBGD" and "rhPBGD", respectively. Within this term is also included an enzymatically equivalent part or analogue of PBGD. One example of an enzymatically equivalent part of the enzyme could be a domain or subsequence of the enzyme which includes the necessary catalytic site to enable the domain or subsequence to exert substantially the same enzymatic activity as the full-length enzyme or alternatively a gene coding for the catalyst. The term "substantially the same enzymatic activity" refers to an equivalent part or analogues enzyme having at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most preferably at least 97%, at least 98% or at least 99% of the activity of natural human rhPBGD measured in the rhPBGD activity assay described in example 2 of WO 03/002731. An example of an enzymatically equivalent analogue of the enzyme could be a fusion protein which includes the catalytic site of the enzyme in a functional form, but it can also be a homologous variant of the enzyme derived from another species. Also, completely synthetic molecules that mimic the specific enzymatic activity of the relevant enzyme would also constitute "enzymatic equivalent analogues".

[0037] An example of PBGD which may be used in the present invention includes any of those shown in Sequence 1-10 of the present application, or in Genebank no. X04217, X04808 or M95623.

Aryl Sulfatase

[0038] In another embodiment of the present invention the polypeptide of interest may be an arylsulfatase A.

[0039] Arylsulfatase A catalyzes the reaction of a cerebroside 3-sulfate+H2O=a cerebroside+sulphate.

[0040] ASA has been purified from a variety of sources including human liver, placenta, and urine. It is an acidic glucoprotein with a low isoelectric point. Above pH 6.5, the enzyme exists as a dimer with a molecular weight of approximately 110 kDa. ASA undergoes a pH-dependent polymerisation forming an octamer at pH 4.5. In human urine, the enzyme consists of two nonidentical subunits of 63 and 54 kDa. ASA purified from human liver, placenta, and fibroblasts also consist of two subunits of slightly different sizes varying between 55 and 64 kDa. As in the case of other lysosomal enzymes, ASA is synthesised on membrane-bound ribosomes as a glycosylated precursor. It then passes through the endoplasmic reticulum and Golgi, where its N-linked oligosaccharides are processed with the formation of phosphorylated and sulfated oligosaccharide of the complex type (Waheed A et al. Biochim Biophys Acta. 1985, 847, 53-61, Braulke T et al. Biochem Biophys Res Commun. 1987, 143, 178-185). In normal cultured fibroblasts, a precursor polypeptide of 62 kDa is produced, which translocates via mannose-6-phosphate receptor binding (Braulke T et al. 3 Biol. Chem. 1990, 265, 6650-6655) to an acidic prelysosomal endosome (Kelly B M et al. Eur J Cell Biol. 1989, 48, 71-78).

[0041] The arylsulfatase A may in particular be of human origin. The length (18 amino acids) of the human ASA signal peptide is based on the consensus sequence and a specific processing site for a signal sequence. Hence, from the deduced human ASA cDNA (EMBL GenBank accession numbers J04593 and X521151) the cleavage of the signal peptide should be done in all cells after residue number 18 (Ala), resulting in the mature form of the human ASA. In the following, recombinant arylsulfatase A will be abbreviated rASA, the mature form of arylsulfatase A including the mature form of human ASA will be termed "mASA" and the mature recombinant human ASA will be termed "mrhASA".

[0042] A protein modification has been identified in two eukaryotic sulfatases (ASA and arylsulfatase B (ASB)) and for one from the green alga Volvox carteri (Schmidt B et al. Cell. 1995, 82, 271-278, Selmer T et al. Eur J Biochem. 1996, 238, 341-345). This modification leads to the conversion of a cysteine residue, which is conserved among the known sulfatases, into a 2-amino-3-oxopropionic acid residue (Schmidt B et al. Cell. 1995, 82, 271-278). The novel amino acid derivative is also recognised as C*-formylglycin (FGly). In ASA and ASB derived from MSD cells, the Cys-69 residue is retained. Consequently, it is proposed that the conversion of the Cys-69 to FGly-69 is required for generating catalytically active ASA and ASB, and that deficiency of this protein modification is the cause of MSD. Cys-69 is referred to the precursor ASA which has an 18 residue signal peptide. In the mASA the mentioned cysteine residue is Cys-51. Further investigations have shown that a linear sequence of 16 residues surrounding the Cys-51 in the mASA is sufficient to direct the conversion and that the protein modification occurs after or at a late stage of co-translational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure (Dierks T et al. Proc Natl Acad. Sci. 1997, 94, 11963-1196, Wittke, D. et al. (2004), Acta Neuropathol. (Berl.), 108, 261-271).

[0043] Multiple forms of ASA have been demonstrated on electrophoresis and isoelectric focusing of enzyme preparations from human urine, leukocytes, platelets, cultured fibroblasts and liver. Treatment with endoglycosidase H, sialidase, and alkaline phosphatase reduces the molecular size and complexity of the electrophoretic pattern, which suggests that much of the charge heterogeneity of ASA is due to variations in the carbohydrate content of the enzyme.

[0044] The arylsulfatase A may in particular be a form of arylsulfatase A, which is capable of crossing the blood brain barrier and/or a form of rASA, which possesses specific tags for entry into target cells within the brain. In particular, it may be a rASA, which is efficiently endocytosed in vivo via the mannose-6-phosphate pathway.

[0045] Thus the ASA may in particular be covalently bound to a so-called tag, peptides or proteins as vehicles or toxins as vehicles which are capable of increasing and/or facilitating transport of ASA over the blood-brain barrier and/or across cellular membranes in general (Schwarze et al., Trends Cell Biol. 2000; 10(7): 290-295; Lindgren et al., Trends Pharmacol. Sci. 2000; 21(3): 99-103). An ASA molecule containing such peptide sequences can be produced by expression techniques. The protein transduction process is not cell type specific and the mechanism by which it occurs is not fully elucidated, however, it is believed that it takes place by some sort of membrane perturbation and penetration process that is receptor independent. A partially unfolded state of the molecule may facilitate the process but is not essential.

[0046] An example of a suitable tag includes but is not limited to the mannose-6-phosphate tag.

[0047] Examples of peptides or proteins as vehicle include but are not limited to so-called protein-transducing domains. Examples of suitable protein-transducing domains include but are not limited to those mentioned in WO 2005/073367, which is incorporated herein by reference. Hence the protein-transducing domain may be the 11 residue basic peptide from the HIV TAT protein -YGRKKRRQRRR (Schwarze et al., Trends Cell Biol. 2000; 10(7): 290-295), a synthetic version of TAT-YARAAARQARA that confers more alpha-helicity and amphipathic nature to the sequence (Ho et al., Cancer Res. 2001; 61(2):474-477), a synthetic leader peptide composed of poly -R or a mixture of basic -R and -K residues in combination with other amino acids and peptides based on hydrophobic signal sequence moieties from either beta-3 integrin or Kaposi's sarcoma FGF (Dunican et al. Biopolymers 2001; 60(1): 45-60).

[0048] Examples of suitable toxins as vehicles include but are not limited to those described in WO 2005/073367, which is incorporated herein by reference.

[0049] The ASA may in particular comprise a nucleic acid sequence, which encodes: [0050] (a) the amino acid sequence of SEQ ID NO:2 in WO 2005/073367; [0051] (b) a portion of the sequence in (a), which is enzymatically equivalent to recombinant human arylsulfatase A [0052] (c) an amino acid sequence analogue having at least 75% sequence identity to any one of the sequences in (a) or (b) and at the same time comprising an amino acid sequence, which is enzymatically equivalent to recombinant human arylsulfatase A.

[0053] In the present context, an amino acid sequence or a portion of an amino acid sequence which is a polypeptide capable of hydrolysing an amount of the arylsulfatase A substrate pNCS at 37.degree. C. a rate corresponding to a specific activity of at least 20 U/mg polypeptide (preferably 50 U/mg polypeptide) when determined in an assay for measuring arylsulfatase A activity as described in example 1 of WO 2005/073367, and/or a polypeptide, which is capable of hydrolysing at least 40% of labelled arylsulfatase A substrate, fx. 14C palmitoyl sulfatide, loaded into MLD fibroblasts, when assayed by incubation at a dose level of 25 mU/ml in an assay as described in example 2 of WO 2005/073367.

[0054] The ASA may in another embodiment in particular comprise: [0055] (a) the nucleic acid sequence of SEQ ID NO: 1 in WO 2005/073367 [0056] (b) a portion of the sequence in (a), which encodes an amino acid sequence, which is enzymatically equivalent to recombinant human arylsulfatase A [0057] (c) a nucleic acid sequence analogue having at least 75% sequence identity to any one of the sequences in (a) or (b) and at the same time encoding an amino acid sequence, which is enzymatically equivalent to recombinant human arylsulfatase A

[0058] It may be preferred that the degree of sequence identity between the above mentioned nucleic acid sequence and SEQ ID NO: 1 of WO 2005/073367 is at least 80%, such as at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. It may be equally preferred that the degree of sequence identity between the amino acid sequence encoded by the above mentioned nucleic acid sequence and SEQ ID NO: 2 WO 2005/073367 is at least 80%, such as at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0059] For the purpose of the present invention it is preferred that the arylsulfatase A is a recombinant enzyme, particularly preferred is recombinant human arylsulfatase A (rhASA).

[0060] It is preferred that rASA is produced in a mammalian cell or cell line and that said mammalian cell or cell line produces a glycoform of rASA, which is efficiently endocytosed in vivo via the mannose-6-phosphate receptor pathway. Specifically, the preferred glycoform of rASA comprises an amount of exposed mannose-6-phosphate, which allows efficient endocytosis of rASA in vivo via the mannose-6-phosphate pathway.

[0061] In a particular embodiment at least one of the produced glycoforms of rASA is similar to a glycoform produced in CHO cells.

[0062] The post translational modification of the cysteine residue in position 51 in the mature human arylsulfatase A is relevant for the activity of the enzyme. Accordingly, in a preferred embodiment of the present invention production of the arylsulfatase A or its equivalent occurs at a rate and under conditions, which result in a product comprising an isoform of the enzyme in which the amino acid corresponding to Cys-69 in SEQ ID NO: 2 of WO 2005/073367 is converted to Formylglycine, corresponding to Fgly-51 in SEQ ID NO: 3 of WO 2005/073367. SEQ ID NO: 4 of WO 2005/073367 represents mature human arylsulfatase A after cleavage of the 18 amino acid signal peptide but prior to modification of C-51.

[0063] Thus in another embodiment of the present invention the ASA or its enzymatical equivalent may be selected from the group consisting of [0064] (a) the amino acid sequence of SEQ ID NO:3 of WO 2005/073367; [0065] (b) a portion of the sequence in (a), which is enzymatically equivalent to recombinant human arylsulfatase A [0066] (c) an amino acid sequence analogue having at least 75% sequence identity to any one of the sequences in (a) or (b) and at the same time being enzymatically equivalent to recombinant human arylsulfatase A.

[0067] It may be preferred that the degree of sequence identity between the enzyme produced according to the invention and SEQ ID NO: 3 of WO 2005/073367 or SEQ ID NO: 4 of WO 2005/073367 is at least 80%, such as at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0068] For the biological activity and the effects of the enzyme in vivo requires to be optimal it is an advantage if an adequate amount of the enzyme has acquired a glycosylation pattern as described above and has been modified post translationally at position 51. Thus at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the ASA of the present invention may be in the above described glycoform/isoform.

[0069] The ASA of the present invention may in terms of its structure be different from the rASA according to SEQ ID NO: 3 of 2005/073367. It may be an advantage that the sequence of amino acid residues surrounding the Cys-51 is identical or has a high degree of sequence identity to the corresponding sequence in SEQ ID NO: 3. Thus, it may be preferred that a linear sequence of 20 amino acids, such as 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acid residues surrounding the Cys-51 in the arylsulfatase A is identical or at least 90% identical, such as 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequence in SEQ ID NO: 3 of 2005/073367. As the active form of rASA within the lysosymes is an octamer the ASA of the present invention may in particular be a rASA which is an octamer or assembles into an octamer under physiological conditions.

[0070] The enzyme activity of ASA, which is to be understood as the catalytic activity of the rASA, may be measured in an enzyme assay based on the rASA mediated hydrolysis of either a detectable substrate or a substrate, which leads to a detectable end product. In a preferred aspect the assay is based on hydrolysis of the synthetic, chromogenic substrate, para-Nitrocatechol sulphate (pNCS) which has an end product, para-Nitrocatechol (pNC) that absorbs light at 515 nm.

Lysosomal Alpha-Mannosidase

[0071] In yet another embodiment the polypeptide of interest may be a lysosomal alpha-mannosidase (LAMAN). Lysomal alpha-mannosidase belongs to EC 3.2.1.24 and is an exoglycosidase which hydrolyses the terminal, non-reducing alpha-D-mannose residues in alpha-D-mannosides from the non-reducing end during the ordered degradation of N-linked glycoproteins (Aronson and Kuranda FASEB J 3:2615-2622. 1989). In the context of the present invention the term lysosomal alpha-mannosidase may be used interchangeably with the short term LAMAN.

[0072] The LAMAN of the present invention may in particular be of human origin. The human enzyme is synthesised as a single polypeptide of 1011 amino acids with a putative signal peptide of 49 residues that is processed into three main glycopeptides of 15, 42, and 70 kD (Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997).

[0073] The gene coding for LAMAN (MANB) is located at chromosome 19 (19cen-ql2), (Kaneda et al. Chromosoma 95:8-12. 1987). MANB consists of 24 exons, spanning 21.5 kb (GenBank accession numbers U60885-U60899; Riise et al. Genomics 42:200-207. 1997). The LAMAN transcript is >>3,500 nucleotides (nts) and contains an open reading frame encoding 1,011 amino acids (GenBank U60266.1).

[0074] The cloning and sequencing of the human cDNA encoding LAMAN has been published in three papers (Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997; Liao et all., Biol. Chem. 271, 28348-28358. 1996; Nebes et al. Biochem. Biophys. Res. Commun. 200, 239-245. 1994). Curiously, the three sequences are not identical. When compared to the sequence of Nilssen et al (accession # U60266.1) a TA to AT change at positions 1670 and 1671 resulting in a valine to aspartic acid substitution was found by Liao et al. and Nebes et al.

[0075] In a most preferred embodiment, the lysosomal alpha mannosidase comprises the amino acid sequence of SEQ ID NO.: 1 of WO 2005/094874.

[0076] For practical and economical reasons it is preferred that the LAMAN of the present invention is produced recombinant. By recombinant production it may also be possible to obtain a preparation of the enzyme wherein a large fraction contains mannose-6-phosphate. Recombinant production may be achieved after transfection of a cell using a nucleic acid sequence comprising the sequence of SEQ ID NO: 2 of WO 2005/094874.

[0077] The alpha-mannosidase is preferably made in a mammalian cell system as this will result in a glycosylation profile, which ensures efficient receptor mediated uptake in cells of for instance visceral organs of the body. In particular, it has been found that production of the enzyme in CHO, COS or BHK cells ensures adequate post-translational modification of the enzyme by addition of mannose-6-phosphate residues. In addition a correct sialylation profile is obtained. Correct sialylation is known to be important in order to prevent uptake by the liver, because of exposed galactose residues.

[0078] In even more preferred embodiments the mammalian cell system is therefore selected from the group comprising CHO, COS cells or BHK cells (Stein et al. 3 Biol. Chem. 1989, 264, 1252-1259). It may further be preferred that the mammalian cell system is a human fibroblast cell line.

[0079] In a most preferred embodiment, the mammalian cell system is a CHO cell line.

[0080] In another embodiment the lysosomal alpha-mannosidase may be a preparation of lysosomal alpha-mannosidase wherein a fraction of said preparation consists of lysosomal alpha mannosidase having one or more N-linked oligosaccharides carrying mannose 6-phosphate groups.

[0081] It is further preferred that a fraction of a preparation of said lysosomal alpha-mannosidase is capable of binding to mannose 6-phosphate receptors.

[0082] The ability of the enzyme to bind to mannose-6-phosphate receptors may be determined in an in vitro assay as described in example 1 of WO 2005/094874. Here, binding of the enzyme to a MPR affinity 300 Matrix provides a measure of its ability to bind to mannose-6-phosphate receptors. In a preferred embodiment of the invention binding of the enzyme to mannose-6-phosphate receptors occurs in vitro.

[0083] In more preferred embodiments of the invention this fraction corresponds to from 1 to 75% of the activity of a preparation of lysosomal alpha-mannosidase, such as from 2 to 70%, such as from 5 to 60%, such as from 10 to 50% such as from 15 to 45%, such as from 20 to 40%, such as from 30 to 35%.

[0084] Accordingly, it is preferred that the lysosomal alpha-mannosidase has a content of mannose 6-phosphate residues allowing mannose 6-phosphate dependent binding of from 2 to 100%, 5 to 95%, 10 to 90%, 20 to 80%, 30 to 70% or 40 to 60% of the amount of enzyme to a Man-6-P-receptor matrix. At present, the degree of phosphorylation has been analysed in several batches of enzyme and, typically, from 30 to 45% of the enzyme is phosphorylated and binds the affinity matrix.

[0085] It is further preferred that a fraction constituting from 2-100%, 5-90%, 10-80%, 20-75%, 30-70%, 35-65% or 40-60% of the amount of said lysosomal alpha-mannosidase binds to the Man-6-P-receptor with high affinity. Theoretically, two mannose 6-phosphate groups must be positioned close to each other in order for the enzyme to bind a Man-6-P-receptor with high affinity. Recent observations suggest that the distance between the phosphorylated mannose residues must be 40 .ANG. or less in order to obtain high affinity binding. In the human lysosomal alpha-mannosidase according to SEQ ID NO: 1 of WO 2005/094874 the two mannose 6-phosphate residues may be situated at the asparagines residues in positions 367 and 766. Accordingly, it is preferred that the medicament according to the present invention comprises lysosomal alpha-mannosidase, a fraction of which carries mannose 6-phosphate groups at both of these asparagine residues.

[0086] Preferably, the alpha-mannosidase is made by recombinant techniques. In a further embodiment, the alpha-mannosidase is of human origin (hLAMAN) and still more preferred a mature human alpha-mannosidase (mhLAMAN) or a fragment thereof. The fragment may be modified, however the active sites of the enzyme should be preserved.

[0087] It is to be expected that, in preparations of alpha-mannosidase according to the present invention, one fraction of the enzyme is represented by its precursor form, while other fractions represent the proteolytically processed forms of approximately 55 and 70 kDa.

Galactocerebrosidase

[0088] In another embodiment the polypeptide of interest may be a galactocerebrosidase, which may be shortended to GALC. Galactocerebrosidase belongs to E.C. 3.1.6.46 and are enzymes capable of catalysing the reaction of D-galactosyl-N-acylsphingosine+H.sub.2O=D-galactose+N-acylsphingosine, thus GALC catalyzes the degradation of galactolipids in for example myelin.

[0089] The GALC enzyme derived from humans is a glycosylated lysosomal enzyme comprising 643 amino acids and with a molecular weight of 72.8 kDa. The GALC of the present invention may in particular be of human origin. In a further embodiment the GALC may be expressed recombinant in one of the previously mentioned host cells. The host cell for recombinant expression of GALC may in particular be a CHO cell.

[0090] In the description and in the claims reference is made to the following amino acid and nucleic acid sequences:

TABLE-US-00001 Sequence Sequence description identifier PBGD coding sequence 1 SEQ ID NO.: 1 PBGD coding sequence 2 SEQ ID NO.: 2 PBGD coding sequence 3 SEQ ID NO.: 3 PBGD coding sequence 4 SEQ ID NO.: 4 PBGD coding sequence 5 SEQ ID NO.: 5 PBGD coding sequence 6 SEQ ID NO.: 6 PBGD coding sequence 7 SEQ ID NO.: 7 PBGD coding sequence 8 SEQ ID NO.: 8 PBGD coding sequence 9 SEQ ID NO.: 9 PBGD coding sequence 10 SEQ ID NO.: 10 PBGD coding sequence, GenBank Acc. No. X04217 SEQ ID NO.: 11 PBGD coding sequence, GenBank Acc. No. X04808 SEQ ID NO.: 12 PBGD coding sequence, GenBank Acc. No. M95623 SEQ ID NO.: 13 PBGD aa sequence from coding sequence, GenBank SEQ ID NO.: 14 Acc. No. M95623, Constitutive form PBGD aa sequence from coding sequence, GenBank SEQ ID NO.: 15 Acc. No. M95623, Erythropoietic form ASA coding sequence Genbank Acc. No. J04593 SEQ ID NO.: 16 ASA coding sequence SEQ ID NO.: SEQ ID NO.: 17 1 of WO 2005/073367 ASA aa sequence SEQ ID NO.: 2 of WO 2005/073367 SEQ ID NO.: 18 ASA aa sequence SEQ ID NO.: 3 of WO 2005/073367 SEQ ID NO.: 19 ASA aa sequence SEQ ID NO.: 4 of WO 2005/073367 SEQ ID NO.: 20 LAMAN aa sequence SEQ ID NO.: 1 of WO SEQ ID NO.: 21 2005/094874 LAMAN coding sequence SEQ ID NO.: SEQ ID NO.: 22 1 of WO 2005/094874 Galactocerebrosidase coding sequence SEQ ID NO.: 23 Galactocerebrosidase aa sequence SEQ ID NO.: 24

[0091] With reference to these sequences the polypeptide of interest, according to preferred embodiments of the invention, comprises an amino acid selected from the group consisting of: [0092] i) an amino acid sequence as defined by any of SEQ ID NOs.: 14, 15, 18, 19, 20, 21 and 24; [0093] ii) a functionally equivalent part of an amino acid sequence as defined in i); and [0094] iii) a functionally equivalent analogue of an amino acid sequence as defined in i) or ii), the amino acid sequence of said analogue being at least 75% identical to an amino acid sequence as defined in i) or ii).

[0095] In particular embodiments the analogue in iii) is at least 80% identical to a sequence as defined in i) or ii), such as at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or such as at least 99.5% identical to a sequence as defined in i) or ii).

[0096] Further, the polypeptide of interest may be obtained by recombinant expression using a nucleic acid sequence comprising a sequence selected from the group consisting of: [0097] i) a nucleic acid sequence as defined by any of SEQ ID NOs.: 1-13, 16, 17, 22 and 23; [0098] ii) a nucleic acid sequence which is at least 750% identical to a nucleic acid sequence as defined in i).

[0099] For recombinant production of the polypeptide it may further be preferred that the acid sequence in ii) is at least 80% identical to a sequence as defined in i), such as at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or such as at least 99.5% identical to a sequence as defined in i).

Composition Comprising a Polypeptide of Interest

[0100] The following description of a composition comprising a polypeptide of interest relates both to a composition comprising a polypeptide which is concentrated according to a method of the present invention and it also relates to a composition of the present invention comprising at least 10 mg/ml polypeptide of interest.

[0101] The present invention also relates to a composition comprising at least 10 mg/ml polypeptide of interest, wherein the polypeptide of interest may be any polypeptide according to the present invention, such as in particular rhPBGD, aryl sulfatase, alpha-mannosidase or galactocerebrosidase. Said composition may in particular comprise at least 25 mg/ml polypeptide of interest, such as at least 50 mg/ml or at least 75 mg/ml or at least 100 mg/ml polypeptide of interest. Thus said composition may in particular comprise between 10-1000 mg/ml polypeptide of interest, such as between 10-500 mg/ml or between 10-300 mg/ml or between 10-200 mg/ml or between 25-500 mg/ml or between 25-400 mg/ml or between 40-400 mg/ml or between 40-300 mg/ml or between 50-400 mg/ml or between 50-300 mg/ml or between 75-400 mg/ml or between 75-300 mg/ml or between 100-200 mg/ml or between 100-150 mg/ml polypeptide of interest.

[0102] The composition comprising a polypeptide of interest may in particular be an aqueous solution.

[0103] Besides comprising a high concentration of polypeptide of interest said composition may in particular further comprise no aggregates of the polypeptide of interest or at least only very few aggregates. Hence the amount of polypeptide of interest present as aggregates may in particular constitute less than 5 w/w % of the total amount of polypeptide of interest in the composition. In particular said aggregates may constitute less than 4 w/w %, such as less than 3 w/w %, or less than 2 w/w %, or less than 1 w/w %, or less than 0.5 w/w %, or less than 0.1 w/w % of the total amount of polypeptide of interest. In the present context the term "aggregates" means any form of the polypeptide of interest which is not monomeric. Thus the term encompasses any dimer or multimer of the polypeptide of interest.

[0104] Furthermore, it is an advantage if said composition comprises only the polypeptide of interest or at least only minor traces of other proteins, i.e. proteins different from polypeptide of interest. Hence in a particular embodiment said composition comprises less than 1 w/w %, such as less than 0.5 w/w %, or less than 0.1 w/w %, or less than 0.05 w/w %, or less than 0.01 w/w % other proteins than the polypeptide of interest.

[0105] A range of factors affect the stability and activity of polypeptides and the composition comprising a polypeptide of interest may therefore in particular be optimized to keep the polypeptide of interest as stable as possible.

[0106] The pH generally affects the stability of a polypeptide of interest, thus the pH of a composition comprising a polypeptide of interest may in particular be in the range of 7.5-8.5, such as in particular between pH 7.7-8.2, more particularly between pH 7.8-8.0 or between pH 7.85-7.95, such as pH 7.8 or pH 7.9. This may in particular be the case if the polypeptide of interest is PBGD.

[0107] Thus the composition comprising a polypeptide of interest may in particular comprise a buffer capable of keeping the composition within the described pH range. Examples of such buffers include but are not limited to TRIS-HCL, Na-Citrate and Na.sub.2HPO.sub.4. The concentration of such a buffer may depend on the choice of the particular buffer and the presence of other components in the composition. If the buffer is Na.sub.2HPO.sub.4 the concentration of Na.sub.2HPO.sub.4 may be in the range of 0.5-15 mM, such as in the range of 1-10 mM, or in the range of 1.5-7.5 mM, such as in the range of 1.83-7.4 mM, or in the range of 1.5-3 mM, such as in the range of 1.83-3.7 mM, or in the range of 1.83-2.45 mM, or in the range of 3.5-7.5 mM, such as in the range of 3.6-7.4 mM, or in the range of 5.4-7.4 mM, such as 1.84 mM, or 2.45 mM, or 3.67 mM or 5.51 mM or 7.34 mM.

[0108] If the buffer is TRIS-HCL the concentration of TRIS-HCL may in particular be in the range of 2-50 mM, such as 2-40 mM, or 2-30 mM, or 2-20 mM, or 2-10 mM, or 5-25 mM, or 5-20 mM, or 8-12 mM, or 9-11 mM, e.g. 10 mM.

[0109] Examples of other compounds which the composition comprising a polypeptide of interest may comprise include but are not limited to amino acids, sugars, alcohols and detergents. Examples of such suitable compounds include but are not limited to glycine, mannitol, sucrose, L-serine, Tween 80 or a combination of one or more of said compounds. The concentration of these compounds depend on the particular compound, but for glycine the concentration may in particular be in the range of 1-200 mM, such as in the range of 5-190 mM, or in the range of 10-180 mM, or in the range of 10-170 mM, or in the range of 20-160 mM, or in the range of 20-150 mM, or in the range of 25-125 mM, or in the range of 5-100 mM, or in the range of 5-90 mM, or in the range of 5-80 mM, or in the range of 5-70 mM, or in the range of 5-60 mM, or in the range of 10-100 mM, or in the range of 10-90 mM, or in the range of 10-80 mM, or in the range of 10-70 mM, or in the range of 10-60 mM, or in the range of 12-60 mM, or in the range of 12-55 mM, or in the range of 13.5-54 mM, or in the range of 10-30 mM, such as in the range of 13.5-27 mM, or in the range of 13.5-18 mM, or in the range of 25-55 mM, such as in the range of 27-54 mM, or in the range of 40-55, such as in the range of 40.5-54 mM, such as 12.5, 13, 13.5, 14, 14.5, 17, 17.5, 18, 18.5, 19, 25, 26, 27, 28, 29, 30, 39.5, 40, 40.5, 41, 41.5, or 53, 53.5, 53, 54.5 or 55 mM.

[0110] The concentration of mannitol may in particular be in the range of 50-1000 mM, such as in the range of 50-900 mM, or in the range of 50-800 mM, or in the range of 50-700 mM, or in the range of 50-600 mM, or in the range of 100-900 mM, or in the range of 100-800 mM, or in the range of 100-700 mM, or in the range of 100-600 mM, or in the range of 100-500 mM, or in the range of 120-525 mM, or in the range of 125-500 mM, or in the range of 100-300 mM, such as in the range of 120-275 mM, or in the range of 120-170 mM, or in the range of 200-600 mM, such as in the range of 225-550 mM, or in the range of 240-510 mM, or in the range of 370-525 mM, such as 120, 125, 130, 160, 165, 166.7, 170, 175, 200, 221, 225, 250, 275, 300, 365, 370, 375, 380, 385, 490, 495, 500, 505 or 510 mM.

[0111] The concentration of sucrose may in particular be in the range of 1-200 mM, such as in the range of 5-190 mM, or in the range of 10-180 mM, or in the range of 10-170 mM, or in the range of 20-160 mM, or in the range of 20-150 mM, or in the range of 25-125 mM, or in the range of 5-100 mM, or in the range of 5-90 mM, or in the range of 5-80 mM, or in the range of 5-70 mM, or in the range of 5-60 mM, or in the range of 10-100 mM, or in the range of 10-90 mM, or in the range of 10-80 mM, or in the range of 10-70 mM, or in the range of 10-60 mM, or in the range 35 of 12-60 mM, or in the range of 12-55 mM, or in the range of 13.5-54 mM, or in the range of 10-30 mM, such as in the range of 13.5-27 mM, or in the range of 13.5-18 mM, or in the range of 25-55 mM, such as in the range of 27-54 mM, or in the range of 40-55, such as in the range of 40.5-54 mM, such as 12.5, 13, 13.5, 14, 14.5, 17, 17.5, 18, 18.5, 19, 25, 26, 27, 28, 29, 30, 39.5, 40, 40.5, 41, 41.5, or 53, 53.5, 53, 54.5 or 55 mM. If sucrose is included in a composition which also comprises mannitol the concentration of mannitol may in particular be lowered corresponding to the concentration of sucrose; i.e. the concentration of mannitol and sucrose together may in particular be the same as the concentration of mannitol if this was to be used alone.

[0112] The concentration of Tween 80 may in particular be in the range of 0.001-1 w/v %, such as in the range of 0.005-1 w/v %, or in the range of 0.01-1 w/v %, or in the range of 0.001-0.5 w/v %, or in the range of 0.005-0.5 w/v %, or in the range of 0.01-0.5 w/v %, or in the range of 0.05-0.4 w/v %, or in the range of 0.05-0.3 w/v %, or in the range of 0.05-0.2 w/v %, or in the range of 0.075-0.4 w/v %, or in the range of 0.075-0.3 w/v %, or in the range of 0.075-0.2 w/v %, or in the range of 0.09-0.2 w/v %, such as 0.075, 0.08, 0.09, 0.1, 0.125, 0.15, 0.175 or 0.2 w/v %.

[0113] The composition comprising a polypeptide of interest, wherein the polypeptide in particular may be a PBGD, an aryl sulfatase, a lysosomal alpha-mannosidase or a galactocerebrosidase, may in particular comprise a combination of one or more of the above-mentioned compounds. A suitable example of such a composition may be one which besides the polypeptide of interest comprises Na.sub.2HPO.sub.4, glycine and mannitol. The pH of the composition and the concentration of the different compounds may be as described above. Hence said composition may in one embodiment comprise 0.5-15 mM Na.sub.2HPO.sub.4, 1-200 mM glycine, 50-1000 mM mannitol and a pH in the range of 7.5-8.5. Any combination of the above mentioned concentrations of compounds and pH are encompassed by the present invention. A specific example of a suitable combination of other compounds and pH in the composition comprising a polypeptide of interest is one which comprises 3.67 mM Na.sub.2HPO.sub.4, 27 mM glycine, 250 mM mannitol and has a pH in the range of 7.7 to 7.9.

[0114] Other examples of suitable compositions include, but are not limited to any of the following: [0115] 1.84 mM Na.sub.2HPO.sub.4, 13.5 mM glycine, 125 mM mannitol and pH in the range of 7.7 to 7.9. [0116] 2.45 mM Na.sub.2HPO.sub.4, 18 mM glycine, 167 mM mannitol and pH in the range of 7.7 to 7.9. [0117] 5.51 mM Na.sub.2HPO.sub.4, 40.5 mM glycine, 375 mM mannitol and pH in the range of 7.7 to 7.9. [0118] 7.34 mM Na.sub.2HPO.sub.4, 54 mM glycine, 500 mM mannitol and pH in the range of 7.7 to 7.9. [0119] 3.67 mM Na.sub.2HPO.sub.4, 27 mM glycine, 220 mM mannitol, 30 mM sucrose and pH in the range of 7.7 to 7.9. [0120] 3.67 mM Na.sub.2HPO.sub.4, 245 mM mannitol, 32 mM sucrose and pH in the range of 7.7 to 7.9. [0121] 3.67 mM Na.sub.2HPO.sub.4, 27 mM L-serine, 250 mM mannitol and pH in the range of 7.7 to 7.9. [0122] 10 mM TRIS-HCl, 27 mM glycine, 250 mM mannitol and pH in the range of 7.7 to 7.9. [0123] 3.67 mM NaCitrat, 27 mM glycine, 250 mM mannitol and pH in the range of 7.7 to 7.9. [0124] 3.67 mM Na.sub.2HPO.sub.4, 27 mM glycine, 220 mM mannitol, 29 mM sucrose, 0.1% (w/v) Tween 80 and pH in the range of 7.7 to 7.9. [0125] 3.67 mM Na.sub.2HPO.sub.4, 27 mM glycine, 220 mM mannitol, 29 mM sucrose, 0.1% (w/v) Tween 80 and pH in the range of 7.7 to 7.9.

[0126] The composition comprising a polypeptide of interest may in particular be used for therapeutic applications in mammals. Thus the composition comprising a polypeptide of interest may in particular be isotonic with regard to the tissue of mammals, e.g. it may in particular have an osmolality in the range of 200-400 mOsm/kg, such as in the range of 250-350 mOsm/kg or in the range of 275-325 mOsm/kg or in the range of 295-305 mOsm/kg, such as 295 mOsm/kg or 300 mOsm/kg or 305 mOsm/kg.

Method of Concentrating a Polypeptide of Interest

[0127] The method of the present invention comprises the steps of a) centrifugation and/or filtration of a composition comprising a polypeptide of interest and b) concentrating the composition from step a). The inventors of the present invention have found that by centrifugation and/or filtrating a composition comprising a polypeptide of interest prior to concentrating said composition it is possible to obtain a composition comprising a highly concentrated polypeptide of interest without any or with at least only few aggregates of the polypeptide of interest. Furthermore, it is generally an advantage for therapeutic applications of a polypeptide that the amount of polypeptide aggregates is reduced, e.g. as they may increase the risk of eliciting an immune response towards the polypeptide.

[0128] For administration of a polypeptide subcutaneously it is an advantage that the polypeptide composition has a high activity in a small volume as only small volumes can be injected subcutaneously.

[0129] Proteins or polypeptides may in general form aggregates when they are concentrated. Thus it is an advantage that when the method of the present invention is used to concentrate a polypeptide of interest it does not cause a high rate of polypeptide aggregate formation. As shown in the examples the amount of PBGD aggregates in the composition obtained by the concentration method of the present invention is similar to that of a non-concentrated PBGD composition.

[0130] In a particular embodiment step a) of the method is performed prior to step b).

Step a) Centrifugation and/or Filtration

[0131] The inventors of the present invention have found that prior to concentrating a composition comprising a polypeptide of interest it is an advantage to pre-treat the composition by centrifugation and/or filtration of the composition as by this pre-treatment many or most of the polypeptide aggregates are removed.

[0132] When the concentration of the composition in step b) is performed by a method which relies on the use of a filter or membrane, such as ultrafiltration, the presence of aggregates may block the filter or membrane so that small molecules and liquid are not able to cross the filter or membrane. This may decrease the speed by which the composition is concentrated and/or completely block any further concentration.

[0133] Hence for this type of concentration the pre-treatment according to step a) is an advantage as removal of the aggregates makes it possible to obtain compositions of a polypeptide of interest which are more concentrated than if said composition were not been pre-treated.

[0134] When the concentration of the composition in step b) is performed by a method which is based on the removal of water, such as freeze-drying or evaporation, the pre-treatment in step a) has the advantage that it reduces the amount of aggregates present in the concentrated composition.

[0135] Step a) may be performed by one of the following three alternatives: [0136] Centrifugation, [0137] Filtration, or [0138] Centrifugation and filtration.

[0139] If step a) comprises both centrifugation and filtration it is an advantage to perform the centrifugation prior to the filtration as the inventors of the present invention have found that the centrifugation removes most of large aggregates and the filtration subsequently removes the remaining smaller aggregates.

Centrifugation

[0140] To be able to remove the aggregates the composition comprising a polypeptide of interest may be centrifuged at a force in the range of 1500-3000 g, such as in the range of 1800-2500 g, or in the range of 2000-2300 g.

[0141] Typically the composition may be centrifuged for 10-60 minutes, such as for 15-50 minutes or for 20-40 minutes.

[0142] As the temperature may affect the stability of the polypeptide of interest the centrifugation may be performed at a temperature in the range of 2-20.degree. C., such as from 3-15.degree. C. or in the range of 3-10.degree. C., or in the range of 3-8.degree. C., such as at 4.degree. C. or 5.degree. C. or 6.degree. C.

[0143] The centrifugation results in that the polypeptide of interest aggregates sediment, i.e. they form a pellet, while the individual polypeptide of interest molecules stays in the solution. So it is the supernatant of the centrifuged composition which is subsequently used in the method of the present invention.

Filtration

[0144] The composition comprising a polypeptide of interest may be filtered through a filter having a pore-size in the range of 0.20-5 .mu.m, such as in the range of 0.2-2.5 .mu.m.

[0145] Besides the pore-size of the filter also the material of which the filter is made of may affect filtration of polypeptide of interest. Examples of suitable membrane filters include but are not limited to polyethersulfone (PES), cellulose acetate, regenerated cellulose and polyvinylidene flouride (PVDF).

[0146] When molecules such as proteins are filtered it is usually the small molecules which are removed thus after filtration the polypeptide of interest may generally be present in the retentate. Hence it is generally the retentate from the filtration which is used in the subsequent steps of the present invention.

Step b) Concentrating

[0147] In principle any method of concentrating the polypeptide of interest composition may be used in step b) of the present invention.

[0148] Examples of such suitable methods include but are not limited to ultrafiltration and concentration by removal of water.

Ultrafiltration

[0149] Ultrafiltration is a separation method in which hydraulic pressure is used to force molecules and solvent across a membrane comprising pores of a particular size, also known as the cut-off size of value. Only molecules which have a molecular weight smaller than the cut-off value of the membrane are able to cross the membrane while those with a larger molecular weight do not cross the membrane and form the so called retentate. The molecules present in the retentate are thereby concentrated as the solvent flows across the membrane.

[0150] In a particular embodiment the concentration of the solution or composition comprising a polypeptide of interest may be performed by Tangential flow filtration (TFF). This method is in particular useful for large-scale concentration, i.e. for concentration of solutions with a volume from one litre to several hundreds of litres. Thus this method is in particular useful for production of concentrated solutions of a polypeptide of interests on an industrial scale.

[0151] The TFF technique is based on the use of a particular apparatus which causes the solution which is to be filtrated to flow across a semi-permeable membrane; only molecules which are smaller than the membrane pores will pass through the membrane, forming the filtrate, leaving larger matter to be collected (retentate). With the TFF method two different pressures are applied; one to pump the solution into the system and to circulate it in the system (inlet pressure), and another pressure is applied over the membrane (membrane pressure) to force the small molecules and the solvent across the membrane. The inlet pressure may typically be in the range of 1-3 bar, such as between 1.5-2 bar. The membrane pressure may typically be larger than 1 bar.

[0152] The concentrated composition of a polypeptide of interest may be collected as the retentate when TFF is used to concentrate the composition.

[0153] Membranes useful for TFF may typically be made of regenerated cellulose or polyethersolufone (PES).

[0154] The pore-size of the membrane may typically have a molecular weight cut-off which is smaller than 10.000 Mw, such as in the range of 10-10.000 Mw.

[0155] In another embodiment the concentration of the composition comprising a polypeptide of interest may be performed by the use of a centrifugal device. The principle of this method is that the solution is filtrated over a membrane by the application of a centrifugal force over the membrane. Such membranes are often characterized by a molecular weight (Mw) cut-off, i.e. this is the maximum molecular size of compounds which are able to cross the membrane and compound with a molecular size larger than this will not cross the membrane. The Mw cut-off of the membranes used in the present invention may in particular be smaller than 30.000 Mw, such as between 10-30.000 Mw.

[0156] The membrane may in particular be made of polyethersulfone (PES) or regenerated cellulose.

[0157] Examples of such suitable commercial filter devices may be Centricon Plus-80 or Centricon Plus-15.

[0158] The concentration may typically be performed by centrifugation at 2000-4500 g, such as between 2500-4000 g, or between 2750-3500 g, or between 3000-3500 g, such as at 3000 g or 3100 g or 3200 g or 3300 g or 3400 g or 3500 g.

[0159] Typically the centrifugation may be run for several hours, e.g. for more than one hour, such as for 1-10 hours.

[0160] To minimize any negative effects on the stability of the polypeptide of interest the centrifugation may in particular be performed at a temperature in the range of 2-20.degree. C., such as in the range of 3-15.degree. C. or in the range of 3-10.degree. C. or in the range of 3-6.degree. C.

Concentrating by Removal of Water

[0161] The principle of concentration by removal of water is usually that all, or most, of the water is removed to obtain a solid, and then subsequently diluting or dissolving this solid in a volume of water which is less than what it was previously diluted or dissolved in. However, it may in principle be performed by just removing the necessary amount of water to obtain the desired concentration without subsequently re-diluting or re-dissolving the compound.

[0162] Examples of suitable methods of concentrating by removal of water include freeze-drying and evaporation.

[0163] Both for freeze-drying and evaporation the three most relevant parameters is the temperature, pressure and the time.

[0164] The method of freeze-drying may be comprise the following three or four steps; a freezing-phase, a primary drying phase and a secondary drying phase and optionally a step of annealing after the freezing phase. Freeze-drying may in particular be performed as described with regard to freeze-drying included as a further step of the method of the present invention.

Further Steps

[0165] The polypeptide of interest may derive from a natural source, i.e. from cells naturally expressing the polypeptide of interest, or it may in particular be expressed recombinant.

[0166] Independent of where the polypeptide of interest derives from it may have been purified before being subjected to a method of the present invention.

[0167] Such "purification" may in particular include but is not limited to removal of cell debris, removal of other proteins than polypeptide of interest and removal of other components which may be present in the source from which the polypeptide of interest is derived. Thus in a particular embodiment of the present invention the composition comprising a polypeptide of interest comprises less than 5 w/w %, or less than 1 w/w % or less 0.5 w/w % or less than 0.1 w/w % or less than 0.05 w/w % or less than 0.01 w/w % other proteins than the polypeptide of interest.

[0168] Thus other proteins which are expressed by e.g. a host cell may be removed from the composition comprising a polypeptide of interest before it is used in a method of the present invention.

[0169] Thus in a particular embodiment the method of the present invention may comprise one or more of following steps prior to step a): [0170] i) recombinant expression of a polypeptide of interest [0171] ii) purification of polypeptide of interest composition by one or more steps of chromatography [0172] iii) exchange of the formulation buffer

[0173] Recombinant expression of a polypeptide of interest may in particular be performed as described previously with regard to the polypeptide of interest.

[0174] If the polypeptide of interest is PBGD examples of suitable types of chromatography include but are not limited to affinity chromatography, Ion Exchange Chromatography (IEC) and chromatography on a hydroxyapatite column. In principle any combination of these chromatography methods may be used. The inventors of the present invention have previously found for PBGD that it is an advantage to perform at least the step of affinity chromatography and if this is combined with any of the other methods of chromatography it is an advantage to perform the step of affinity chromatography prior to the other chromatography steps (see e.g. WO 03/002731).

[0175] For the embodiment where the polypeptide of interest is PBGD examples of commercially available affinity chromatography columns include affinity coupling, group specific affinity, and metal chelate affinity columns.

[0176] The product catalogue 2001 of the company Amersham Pharmacia Biotech gives examples of affinity coupling columns such as columns comprising immobilising ligands containing --NH.sub.2 and columns comprising ligands containing primary amino groups.

[0177] Metal chelate affinity columns are specially preferred for purifying proteins via metal ion complex formation with exposed histidine groups. Example 3 of WO01/07065 describes construction of a recombinant human Porphobilinogen deaminase with a "His-Tag" (rhPBGD-His). In order to purify rhPBGD-His it is preferred to use a metal chelate affinity column, such as a column having a cobalt metal affinity resin.

[0178] Examples of other suitable methods of affinity chromatography include but are not limited to columns having porcine heparin as ligand or columns having 1-Amino-4-[[4-[[4-chloro-6-[[3 (or 4)-sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]-9,10- -dihydro-9,10-dioxo-2-anthracenesulfonic acid, also known as Cibracon Blue 3G, as ligand and using Triazine coupling as the ligand coupling method. A commercially available example of the latter is Blue Sepharose 6 Fast Flow (FF) from Amersham Pharmacia Biotech. Accordingly, a preferred embodiment of the invention relates to the process, as described herein, wherein the affinity chromatography column of step (i) is a column using a triazine coupling as ligand coupling method, and more preferably wherein the ligand is Cibacron Blue 3G.

[0179] The term "Ion Exchange Chromatography (IEC)" should herein be understood according to the art as a column separating molecules such as proteins on the basis of their net charge at a certain pH by electrostatic binding to a charged group on the column. Ion exchange denotes the absorption of ions of one type onto a column in exchange for others which are lost into solution.

[0180] Examples of suitable IEC columns are columns such as a Q Sepharose column, a Q SP Sepharose column, or a CM Sepharose column, it may in particular be a DEAE Sepharose column.

[0181] An example of a suitable hydroxyapatite column is a ceramic hydroxyapatite column. Hydroxyapatite (Ca.sub.5(PO.sub.4).sub.3OH).sub.2 is a form of calcium phosphate that can be used for the separation and purification of proteins, enzymes, nucleic acids, viruses, and other macromolecules. Ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. CHT Type I (Bio-Rad) is an example of a suitable commercially available ceramic hydroxyapatite chromatography column.

[0182] In one embodiment the method of the present invention may comprise the following steps prior to step a): [0183] i) recombinant expression of PBGD [0184] ii) subjecting the PBGD composition from step i) to affinity chromatography [0185] iii) subjecting the PBGD composition of step ii) to ion exchange chromatography

[0186] In a further embodiment the method of the present invention may comprise the following steps prior to step a): [0187] i) recombinant expression of PBGD [0188] ii) subjecting the PBGD composition from step i) to affinity chromatography [0189] iii) subjecting the PBGD composition from step ii) to ion exchange chromatography [0190] iv) subjecting the PBGD composition from step iii) to a hydroxyapatite column

[0191] Both of these methods may optionally include a further step of dilution of diafiltration of the PBGD composition obtained from step ii). Thus said step should be after step ii) and before iii), i.e. a step iia). Step iia) has the purpose of reducing the concentration of salts to suitable conductivity, e.g. <10 mS/cm. This may in particular be relevant if DEAE Sepharose is used as resin in the ion exchange chromatography step, i.e. step iii), as this may facilitate binding of the captured PBGD to the DEAE Sepharose resin. Dilution may be obtained by addition of purified water directly or by ultrafiltration against purified water.

[0192] The recombinant expression of PBGD, step i) may be performed by any of the methods described above.

[0193] Examples of suitable affinity chromatography columns in step ii) may be any of the above mentioned.

[0194] Examples of suitable methods of performing ion exchange chromatography in step iii) may be any of the above mentioned.

[0195] Examples of suitable hydroxyapatite chromatography columns in step iv) may be any of the above mentioned.

[0196] In a particular embodiment the affinity chromatography column may be a column using a triazine coupling as ligand coupling method, and in particular such a method wherein the ligand is Cibracon Blue 3G. This may in particular be a Blue Sepharose 6 Fast Flow column, and the ion exchange chromatography column may be DEAE Sepharose column, and in the embodiment wherein the method also comprises a step iv) this column may in particular be a ceramic hydroxyapatite column.

[0197] The method of the present invention may also comprise further steps after step b) of the method. Such steps include but are not limited to one or more of the following: [0198] freeze-drying the composition comprising a concentrated polypeptide of interest, [0199] changing the buffer of the composition comprising a concentrated polypeptide of interest, [0200] sterile filtration of the composition comprising a concentrated polypeptide of interest [0201] evaporation

[0202] Different freeze-driers, volume of solutions to be freeze-dried and other parameters may be used in the method of the present invention. An example of a suitable freeze-dryer includes but is not limited to a Lyostar (FTM-systems) freeze-drier as used the examples of the present invention, where the solutions comprising a concentrated polypeptide of interest, i.e. in this case PBGD, were filled in 2 and 6 ml injection glass vials (type 1) and stoppered with rubber stoppers (chlorobutyl). The freeze-drying may be performed by the following three steps; [0203] i) freezing, [0204] ii) primary drying, and [0205] iii) secondary drying.

[0206] Step i) freezing may in particular be performed by first loading a sample in ambient temperature and cooling it to 0.degree. C. and keeping it at 0.degree. C. for 30 minutes, before lowering the temperature by 1.degree. C. per minute to -40.degree. C. and keeping it at -40.degree. C. for 30 minutes.

[0207] Step ii) primary drying may in particular be performed by drawing the vacuum pressure 126 mTorr, raising the temperature by 1.degree. C. per minute to 0.degree. C. and keeping the sample at 0.degree. C. for 360 minutes

[0208] Step iii) secondary drying may in particular be performed by drawing the full vacuum simultaneously with raising the temperature by 0.5.degree. C. per minute to +30.degree. C. and keeping the sample at +30.degree. C. for 360 minutes. After the secondary drying the sample may further be closed under vacuum or closed after filling with nitrogen.

[0209] An example of a suitable freeze-drying method includes the one described in the examples of the present invention.

[0210] The freeze-drying may in further embodiment comprise an annealing step prior to the primary drying phase. The inventors of the present invention have found that inclusion of an annealing step in the freeze-drying method improves the visual appearance, as visualised by fewer cracks, and/or results in a shorter reconstitution time of the freeze-dried product compared to when the same method of freeze-drying is used but without the annealing step. It is an advantage that the time for reconstitution of a freeze-dried product is reduced, especially if it is to be used as a pharmaceutical which is administered as a solution. An improved visual appearance is usually also regarded as an advantage for most products.

[0211] Thus the freeze-drying may comprise the following steps: [0212] i) freezing [0213] ii) annealing [0214] iii) primary drying [0215] iv) secondary drying.

[0216] The freezing, primary drying and secondary drying steps may in particular be performed as described above. The annealing step, i.e. step ii) may in particular be performed by after 30 minutes of freezing, raising the temperature at e.g. a rate of 2.degree. C. per minute to -10.degree. C. or -20.degree. C. and keeping this temperature for 120 or 420 minutes and then lowering the temperature e.g. a rate of 2.degree. C. per minute to -40.degree. C. at which temperature the sample may be kept at 60-90 minutes before start of the step of primary drying.

[0217] Changing the buffer of the composition comprising a concentrated polypeptide of interest may in particular be performed by a) diluting, e.g. 5-15 times, the composition comprising a concentrated polypeptide of interest in a buffer or formulation, b) concentrating the diluted composition again and performing the steps a) and b) a sufficient number of times so that amount of the excipients in the buffer or formulation present in the composition before these steps constitute less than e.g. 5 v/v % or less than 1 v/v % of excipients in the buffer or formulation present in said composition after said steps were performed.

[0218] In particular the composition comprising a polypeptide of interest obtained from step b) of the present invention may in particular further comprise a step of sterile filtration of said composition and/or a step of freeze-drying the composition.

[0219] Sterile filtration is generally performed by filtration of the composition through a filter with a pore-size of 0.22 .mu.m or 0.20 .mu.m. Freeze-drying may in particular be performed as described above.

[0220] The present invention also relates to a freeze-dried composition obtained by a method of the present invention.

Subcutaneous Injection

[0221] The present invention also relates to the use of a composition comprising in the range of 50-300 mg/ml polypeptide of interest for the manufacture of a medicament for subcutaneous injection into a mammal.

[0222] The polypeptide of interest may be any polypeptide of interest according to the present invention, including but not limited to PBGD, aryl sulfatase A, lysosomal alpha-mannosidase and galactocerebrosidase.

[0223] The term subcutaneous is often shortened to s.c. and the two terms may be used interchangeably in the context of the present invention.

[0224] When injection is performed subcutaneously it is usually not possible to inject more than 1.5 mL due to physiologically restraints.

[0225] As the patient usually needs a certain amount of the particular polypeptide of interest there is a correlation between the volume of the composition comprising a polypeptide of interest which needs to be administered to the patient and of the concentration of polypeptide of interest in said composition.

[0226] It is therefore an advantage of the present invention that the composition comprising a polypeptide of interest comprises a high concentration of the polypeptide of interest and that this high concentration of the polypeptide of interest can be obtained without the formation of large amounts of polypeptide aggregates. The use of such concentrated polypeptide of interest compositions makes it possible to inject a smaller volume of said composition and at the same time ensure that the patient receives an adequate amount of the polypeptide of interest; thus making it easier to administer the polypeptide of interest subcutaneously.

[0227] The above-mentioned composition comprising a polypeptide of interest may in particular comprise between 75-250 mg/ml, such as between 75-200 mg/ml or between 75-150 mg/ml or between 100-150 mg/ml or between 100-125 mg/ml or between 125-150 mg/ml of polypeptide of interest.

[0228] As described above the volume of composition comprising a polypeptide of interest which it is necessary to inject into the patient to ensure that the patient receives an adequate amount of the polypeptide of interest correlates with the concentration of the polypeptide of interest in said composition.

[0229] Thus the volume of such a composition will generally be adjusted according to the concentration of the polypeptide of interest in the composition. However, the volume may generally be in the range of 0.1-1.5 ml, such as in the range of 0.1-1.5 ml or in the range of 0.5-1.5 ml or in the range of 0.5-1.5 ml or in the range of 0.75-1.5 ml or in the range of 0.75-1.5 ml or in the range of 1-1.5 ml or in the range of 1-1.5 ml.

[0230] The amount of polypeptide of interest which it is relevant to administer to a patient generally depends on the weight of the individual and the particular polypeptide of interest.

[0231] In one embodiment the present invention relates to a method of treating a mammal for Acute Intermittent Porphyria comprising subcutaneous injection of a composition of 50-300 mg/ml PBGD.

[0232] Administration of PBGD may in particular be useful for the treatment of Acute Intermittent Porphyria. However, it is contemplated that administration of PBGD also may be useful for the treatment of other porphyrias, such as Hereditary coproporphyria or Variegata porphyria. Porphyria is a term used to collectively describe a number of diseases caused by different deficiencies in the heme biosynthetic pathway. Hence it is contemplated that administration of PBGD, e.g. in combination with other therapeutics, to a patient suffering from any type of porphyria may help to increase the overall turnover of the different intermediates in the pathway. For example Meissner P N et al., 1986, European Journal of Clinical Investigation, vol. 16, 257-261; Hift R J et al., 1997, S. Afr. Med. J., vol. 87, 718-27 and Meissner P et al., 1993, J. Clin. Invest., vol. 91, 1436-44 describe accumulation of ALA and PBG in Hereditary coproporhyria and Variegata porphyria. In theses diseases the accumulation of ALA and PBG results from enzymatic defects that are located four and five steps downstream form the conversion of ALA to PBG, respectively. In the two most recent papers it is described how the porphyrinogen which accumulates in patients with Variegata porphyria is capable of inhibiting PBG-deaminase.

[0233] In a further embodiment the present invention relates to a method of treating a mammal for metachromatic leukodystrophy comprising subcutaneous injection of a composition of 50-300 mg/ml aryl sulfatase A.

[0234] Metachromatic leukodystrophy (MLD) is caused by an autosomal recessive genetic defect in the lysosomal enzyme Arylsulfatase A (ASA), resulting in a progressive breakdown of membranes of the myelin sheath (demyelination) and accumulation of galactosyl sulphatide (cerebroside sulphate) in the white matter of both the central nervous system (CNS) and the peripheral nervous system. In histologic preparations, galactosyl sulphatide forms spherical granular masses that stain metachromatically. Galactosyl sulphatide also accumulates within the kidney, gallbladder, and certain other visceral organs and is excreted in excessive amounts in the urine.

[0235] Galactosyl sulfatide is normally metabolised by the hydrolysis of 3-O-sulphate linkage to form galactocerebroside through the combined action of the lysosomal enzyme arylsulfatase A (EC 3.1.6.8) (Austin et al. Biochem J. 1964, 93, 15C-17C) and a sphingolipid activator protein called saposin B. A profound deficiency of arylsulfatase A occurs in all tissues from patients with the late infantile, juvenile, and adult forms of MLD (see below). In the following, the arylsulfatase A protein will be termed "ASA". A profound deficiency of ASA occurs in all tissues from patients with MLD.

[0236] In yet another embodiment the present invention relates to a method of treating a mammal for the lysosomal storage disorder alpha-mannosidosis comprising subcutaneous injection of a composition of 50-300 mg/ml lysosomal alpha-mannosidase.

[0237] Alpha-mannosidosis is a recessive, autosomal disease that occurs world wide with a frequency of between 1/1.000.000 and 1/500.000. Mannosidosis is found in all ethnic groups in Europe, America, Africa and also Asia. It is detected in all countries with a good diagnostic service for lysosomal storage disorders, at a similar frequency. They are born apparently healthy; however the symptoms of the diseases are progressive. Alpha-mannosidosis displays clinical heterogeneity, ranging from very serious to very mild forms. Typical clinical symptoms are: mental retardation, skeletal changes, impaired immune system resulting in recurrent infections, hearing impairment and often the disease is associated with a typical facial characteristics such as a coarse face, a prominent forehead, a flattened nasal bridge, a small nose, and a broad mouth. In the most severe cases (mannosidosis type I) the children suffer from hepatosplenomegaly, and they die during the first years of life. Possibly this early death is caused by severe infections due to the immunodeficiency caused by the disease. In milder cases (mannosidosis type 2) the patients usually reach adult age. The skeletal weaknesses of the patients result in the needs of wheeling chairs at age 20 to 40. The disease causes a diffuse dysfunction of the brain often resulting in weak mental performances that excludes anything but the most basic skills of simple reading and writing. These problems associated with hearing inabilities and other clinical manifestations preclude the patient from an independent life, the consequence being that life long caretaking is needed.

[0238] In yet another embodiment the present invention relates to a method of treating a mammal for Krabbe disease comprising subcutaneous injection of a composition of 50-300 mg/ml galactosylcerebrosidase.

[0239] In humans a deficiency in the GALC enzyme results in an autosomal inherited genetic Lysosomal Storage disease known as Krabbe disease or Globoid Cell Leukodystrophy. The enzyme is generally expressed in the testis, kidneys, placenta, liver and brain of human beings and a deficiency in the GALC enzyme generally results in a disorder in the myelin metabolism and in the central and peripheral nervous systems (the CNS and PNS, respectively).

[0240] Krabbe disease has been observed in humans of any age, nationality and sex.

[0241] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. In particular, all of the embodiments described for the composition comprising a polypeptide of interest, such as the presence of further compounds, buffers and pH also apply to the composition comprising a polypeptide of interest used in the present applications.

[0242] When an object according to the present invention or one of its features or characteristics is referred to in singular this also refers to the object or its features or characteristics in plural. As an example, when referring to "a polypeptide" it is to be understood as referring to one or more polypeptides.

[0243] Throughout the present specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0244] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0245] The invention will now be described in further details in the following non-limiting Experimental sections.

EXPERIMENTAL

Materials

[0246] rhPBGD

[0247] The rhPBGD used in the following experiments were obtained according to process 2 in example 1 of WO 03/002731, where process 2 is the process which includes step 1V, i.e. the ceramic hydroxyapatite chromatography step.

Formulation Buffer

[0248] The recombinant and purified rhPBGD was present in the following aqueous formulation buffer:

3.67 mM Na.sub.2HPO.sub.4

27 mM Glycine

250 mM Mannitol

[0249] and a pH of 7.9

[0250] The formulation buffer was then sterile-filtered trough a 0.22 .mu.m filter.

Methods

Freeze-Drying

[0251] The freeze-drying of the purified rhPBGD solutions were performed in a Lyostar (FTM-systems) freeze-drier according to the following schedule:

TABLE-US-00002 Freezing phase 0.degree. C. 30 min 760 Torr 0.degree. C. to -40.degree. C. 1.degree. C./min 760 Torr -40.degree. C. 30 min 760 Torr Primary drying -40.degree. C. to 0.degree. C. 1.degree. C./min 169 mTorr 0.degree. C. 240 min 169 mTorr Secondary drying 0.degree. C. to 30.degree. C. 10.degree. C./60 min, 20 mTorr 180 min 30.degree. C. 720 min 20 mTorr

Visual Observation (Clarity and Colour)

[0252] The liquid was visually studied with respect to colour, clarity and precipitates according to the scheme below.

Colour: 1: No colour; 2: Slightly yellow; 3: Yellow Clarity: 1: Clear; 2: Slightly turbid; 3: Turbid

[0253] Other remarks: Other observations from the operator were in some instances included here (e.g. precipitates, undissolved material etc)

pH-Measurement

[0254] The pH-meter (Metrohm 691 pH Meter) and electrode (combined LL pH electrode) were calibrated with 3 standard reference solutions (Merck) in the range 4.00 to 9.00. The liquid was finally analysed.

Protein Concentration

[0255] Protein concentration in extract, in-process samples, bulk drug substance and final product was determined by a method that utilizes principles of the reduction of Cu2+ to Cu+ by protein in an alkaline medium (the Biuret reaction). The Cu+ ions were then reacted with a reagent containing bicinchoninic acid resulting in a highly sensitive and selective colorimetric detection.

Purity

[0256] Recombinant human Porphobilinogen Deaminase (rhPBGD) and rhPBGD variants were separated according to their ability to adsorb and desorb to silica based stationary media depending on the percentage of organic modifier (acetonitrile) in the mobile phase.

rhPBGD Activity

[0257] Porphobilinogen deaminase (PBGD) catalyzes the addition of 4 molecules of porphobilinogen (PBG) to form a linear tetramer, preuroporphyrinogen, which is released from the enzyme and in vivo circularized to uroporphyrinogen III by the action of Uroporphyrinogen III synthase. Preuroporphyrinogen can be chemically oxidized with benzoquinone to form uroporphyrin, which absorbs light at 405 nm.

[0258] The analyses were performed on one single vial on each test occasion. For the determination of rhPBGD activity and protein concentration the tests were performed in duplicate and triplicate respectively, for each vial.

Osmolality

[0259] One vial of freeze-dried rhPBGD was resuspended in 1.00 ml MilliQ-water. The vial of frozen aqueous solution of rhPBGD was thawed. The osmometer (Vapro osmometer) was calibrated with 3 standard solutions in the range 100-1000 mOsm/kg (100, 290, 1000 mOsm/kg). The liquid was then analyzed.

Example 1

Concentrating with Centrifugal Filter Devices

[0260] Frozen PBGD-bulk solution (7 mg/mL rhPBGD, 3.67 mM Na.sub.2HPO.sub.4, 27 mM glycine, 250 mM Mannitol, pH 7.9) was thawed in a water-bath at 20.degree. C., centrifuged at 3200 g for 10 min and thereafter sterile-filtrated by 0.20 .mu.m-PES filters (Nalgene Polyethersulfone filters). The PBGD-bulk solution was concentrated to 100 mg/ml by running the Centrifugal Filter Devices Centricon Plus-80 (Mw cut-off 30000) and Centricon Plus-15 (Mw cut-off 30000) at 3200 g for several hours. The concentrated solution, i.e. the retentate, was sterile-filtrated by 0.22 .mu.m-filters (Millex GV) and finally a part of this solution was diluted with sterile formulation buffer to get 50 mg/ml. The 5 mg/ml-solution was prepared by directly diluting the recombinant and purified hPBGD with sterile formulation buffer.

[0261] The 5 mg/mL, 50 mg/mL and 100 mg/mL rhPBGD were then freeze-dried as described above. Several vials of each the above-mentioned freeze-dried rhPBGD solutions with 5, 50 and 100 mg/mL rhPBGD and of the aqueous 5 mg/mL rhPBGD solution were stored at 40.degree. C..+-.2.degree. C., 75%.+-.5% relative humidity (RH). The vials were stored protected from light in a well sealed secondary package (paper box).

[0262] At the indicated time points (i.e. time of storage) a vial of each freeze-dried samples were resuspended in 1.00 mL Millipore water.

[0263] Each of the resuspended vials and the aqueous vial of rhPBGd were then visually observed with regard to colour, clarity and precipitates, and the pH, protein concentration, purity and rhPBGD activity were measured as described above.

[0264] The results are given in the following tables 1-4:

TABLE-US-00003 TABLE 1 Freeze-dried product, 5 mg/mL Specific Time-point Activity Concentration activity Purity Visual (month) (U/ml) (mg/ML) (U/mg) (%) observation 0 93.2 4.3 21.5 99.6 Colour: 1, clarity: 1 0.5 81.0 5.2 15.6 ND Colour: 1, clarity: 1 1 76.6 5.9 13.1 99.9 Colour: 1, clarity: 1 1.5 87.0 5.5 15.9 99.7 Colour: 1, clarity: 1 2 53.3 4.7 11.4 99.6 Colour: 1, clarity: 1 3 50.8 4.8 10.7 99.6 Colour: 1, clarity: 1 6 34.3 5.3 6.5 99.6 Colour: 1, clarity: 1

TABLE-US-00004 TABLE 2 freeze-dried product; 50 mg/ml Specific Time-point Activity Concentration activity Purity Visual (month) (U/ml) (mg/ML) (U/mg) (%) observation 0 888 41.4 21.5 99.1 Colour: 2, clarity: 1 0.5 842 50.6 16.6 ND Colour: 2, clarity: 1 1 746 50.6 14.8 100 Colour: 2, clarity: 1 2 640 52.9 12.1 100 Colour: 2, clarity: 1 3 634 49.0 12.9 100 Colour: 2, clarity: 1 6 422 43.0 9.8 100 Colour: 2, clarity: 1

TABLE-US-00005 TABLE 3 Freeze-dried product; 100 mg/ml Specific Time-point Activity Concentration activity Purity Visual (month) (U/ml) (mg/ML) (U/mg) (%) observation 0 1944 83.7 23.2 99.1 Colour: 3, clarity: 1 1 1470 98.7 14.9 100 Colour: 3, clarity: 1 2 1282 94.8 13.5 100 Colour: 3, clarity: 1 3 1253 82.6 15.2 100 Colour: 3, clarity: 1 6 739 75.5 9.8 100 Colour: 3, clarity: 1

TABLE-US-00006 TABLE 4 Aqueous product; 5 mg/ml Specific Time-point Activity Concentration activity Purity Visual (month) (U/ml) (mg/ML) (U/mg) (%) observation 0 95.6 4.0 23.7 99.1 Colour: 1, clarity: 1 0.5 48.1 5.4 8.9 ND Colour: 1, clarity: 1 1 28.6 5.9 4.8 96.1 Colour: 1, clarity: 1 1.5 12.3 5.6 2.2 91.4 Colour: 1, clarity: 1 2 4.5 4.4 1.0 90.7 Colour: 1, clarity: 1 3 7.1 3.1 2.3 87.3 Colour: 2, clarity: 2 6 4.4 2.1 2.1 58.1 Colour: 2, clarity: 2

Example 2

Concentrating a rhPBGD Composition by Centrifugal Filter Devices

[0265] Frozen PBGD-bulk solution (7 mg/mL rhPBGD, 3.67 mM Na.sub.2HPO.sub.4, 27 mM glycine, 250 mM Mannitol, pH 7.9) was thawed in a water-bath at 20.degree. C., centrifuged at 3200 g for 10 min and thereafter sterile-filtrated by 0.20 .mu.m-PES filters (Nalgene Polyethersulfone filters). The PBGD-bulk solution was concentrated to 100 mg/ml by running the Centrifugal Filter Devices Centricon Plus-80 (Mw cut-off 30000) and Centricon Plus-15 (Mw cut-off 30000) at 3200 g for several hours. The concentrated solution, i.e. the retentate, was sterile-filtered by 0.22 .mu.m-filters (Millex GV) and diluted with sterile filtered formulation buffer (see above) to get solutions of lower concentrations. A fraction in volume of each concentration was freeze-dried as described above.

[0266] The different concentrations of freeze-dried rhPBGD and aqueous solution of rhPBGD were stored at 5.degree. C..+-.3.degree. C. or at -20.degree. C..+-.5.degree. C. (ambient relative humidity (RH)). All vials were stored protected from light in a well-sealed secondary package (paper box).

[0267] At the indicated time points (i.e. time of storage) a vial of each freeze-dried samples were resuspended in 1.00 mL Millipore water and then tested together with the aqueous solution of rhPBGD by visually observing the colour, clarity and precipitates, and by measuring pH, protein concentration, purity, osmolality and rhPBGD activity.

[0268] The results are given in the following tables 5-19:

TABLE-US-00007 TABLE 5 Aqueous product; 11 mg/ml; Storage temp.: +5.degree. C. Visual observation Colour 1-3 Protein Specific Clarity 1-3 Time-point conc. Activity activity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) Purity (%) PH (mOsm/kg) Aggregates 0 10.9 255.0 23.4 100.0 7.80 290 Colour: 2 Clarity: 1 Clear None/few 1 9.5 216.8 22.8 100.0 7.81 305 Colour: 2 Clarity: 1 Clear None/few 2 10.9 230.2 21.1 98.0 7.80 300 Colour: 2 Clarity: 1 Clear None/few 3 11.2 226.6 20.2 100.0 7.76 290 Colour: 2 Clarity: 1 Clear Few 6 14.7 271.1 18.4 100.0 7.77 300 Colour: 2 Clarity: 1 Clear Several

TABLE-US-00008 TABLE 6 Aqueous product: 11 mg/ml; Storage temp: -20.degree. C. Visual observation Colour 1-3 Protein Specific Clarity 1-3 Time-point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) PH (mOsm/kg) Aggregates 0 10.4 236.1 22.6 100 7.80 290 Colour: 2 Clarity: 1 Clear None 1 11.7 270.3 23.1 100 7.81 302 Colour: 2 Clarity: 1 Clear None 2 ND ND ND ND ND ND ND 3 12.4 247.7 20.0 100 7.77 288 Colour: 2 Clarity: 1 Clear None 6 13.4 291.5 21.8 100 7.77 301 Colour: 2 Clarity: 1 Clear None

TABLE-US-00009 TABLE 7 Freeze-dried product, 11 mg/ml; Storage temp.: +5.degree. C. Visual observation Colour 1-3; Time- Protein Specific Clarity 1-3; point conc. Activity activity Purity Osmolality Solution; (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 10.9 230.0 21.2 100.0 7.80 290 Colour: 2 Clarity: 1 Clear None 1 ND ND ND ND ND ND ND 2 ND ND ND ND ND ND ND 3 13.3 269.3 20.2 100.0 7.74 282 Colour: 2 Clarity: 1 Clear None 6 14.7 237.9 16.2 100.0 7.76 290 Colour: 2 Clarity: 1 Clear None

TABLE-US-00010 TABLE 8 Aqueous product, 17 mg/ml; Storage temp.: +5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 18.0 471.0 26.1 100.0 7.80 298 Colour: 2 Clarity: 1 Clear None/few 1 17.5 360.4 20.6 100.0 7.81 311 Colour: 2 Clarity: 1 Clear None/few 2 18.3 397.0 21.7 100.0 7.83 302 Colour: 2 Clarity: 1 Clear None/few 3 16.6 376.5 22.7 100.0 7.77 294 Colour: 2 Clarity: 1 Clear Few 6 16.0 257.3 16.1 100.0 7.76 305 Colour: 2 Clarity: 1 Clear Several

TABLE-US-00011 TABLE 9 Aqueous product, 17 mg/ml; Storage temp.: -20.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) PH (mOsm/kg) Aggregates 0 17.9 411.6 23.0 100.0 7.80 298 Colour: 2 Clarity: 1 Clear None 1 17.4 439.5 25.3 100.0 7.80 310 Colour: 2 Clarity: 1 Clear None 2 ND ND ND ND ND ND ND 3 16.4 389.4 23.7 100.0 7.77 292 Colour: 2 Clarity: 1 Clear None 6 18.0 373.8 20.8 100.0 7.76 305 Colour: 2 Clarity: 1 Clear None

TABLE-US-00012 TABLE 10 Freeze-dried product, 17 mg/ml; Storage temp.: 5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) PH (mOsm/kg) Aggregates 0 16.9 380.1 22.5 100.0 7.80 298 Colour: 2 Clarity: 1 Clear None 1 ND ND ND ND ND ND ND 2 ND ND ND ND ND ND ND 3 15.6 391.9 25.1 100.0 7.76 285 Colour: 2 Clarity: 1 Clear None 6 16.6 341.3 20.6 100.0 7.75 297 Colour: 2 Clarity: 1 Clear None

TABLE-US-00013 TABLE 11 Aqueous product; 36 mg/ml; Storage temp.: +5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 36.0 844.4 23.4 100.0 7.81 305 Colour: 2 Clarity: 1 Clear None/few 1 35.5 778.1 21.9 100.0 7.82 314 Colour: 2 Clarity: 1 Clear None/few 2 35.4 798.5 22.6 100.0 7.81 310 Colour: 2 Clarity: 1 Clear None/few 3 28.9 687.9 23.8 100.0 7.77 303 Colour: 2 Clarity: 1 Clear Few 6 37.2 537.3 14.4 100.0 7.77 312 Colour: 2 Clarity: 1 Clear Several

TABLE-US-00014 TABLE 12 Aqueous product, 36 mg/ml; Storage temp.: -20.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 34.0 853.4 25.1 100.0 7.81 305 Colour: 2 Clarity: 1 Clear None 1 38.0 853.6 22.5 100.0 7.83 321 Colour: 2 Clarity: 1 Clear None 2 ND ND ND ND ND ND ND 3 31.6 776.3 24.6 100.0 7.76 299 Colour: 2 Clarity: 1 Clear None 6 30.6 543.8 17.8 100.0 7.75 311 Colour: 2 Clarity: 1 Clear None

TABLE-US-00015 TABLE 13 Freeze-dried product, 36 mg/ml; Storage temp.: 5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 29.5 657.0 22.3 100.0 7.81 305 Colour: 2 Clarity: 1 Clear None 1 ND ND ND ND ND ND ND 2 ND ND ND ND ND ND ND 3 28.7 747.6 26.0 100.0 7.75 290 Colour: 2 Clarity: 1 Clear None 6 29.8 579.3 19.4 100.0 7.76 300 Colour: 2 Clarity: 1 Clear None

TABLE-US-00016 TABLE 14 Aqueous product, 50 mg/ml; Storage temp.: 5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 46.2 780.9 16.9 96.3 7.59 317 Colour: 3 Clarity: 1 Slightly opalescent None 1 47.9 915 19.1 90 7.58 305 Colour: 3 Clarity: 1 Slightly opalescent None 2 47.2 898.3 19.0 100 7.60 318 Colour: 3 Clarity: 1 Slightly opalescent None 3 60.8 1102.6 18.1 100 7.72 314 Colour: 3 Clarity: 1 Clear None 6 62.5 902.8 14.4 100 7.60 331 Colour: 3 Clarity: 2 Clear None 9 41.7 618.5 14.8 100 7.60 336 Colour: 3 Clarity: 2 Clear None 12 50.2 540.8 10.8 97.5 7.60 329 Colour: 3 Clarity: 2 Clear None

TABLE-US-00017 TABLE 15 Aqueous product, 50 mg/ml; Storage temp.: -20.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) PH (mOsm/kg) Aggregates 0 46.2 780.9 16.9 96.3 7.59 317 Colour: 3 Clarity: 1 Slightly opalescent None 1 47.2 899.1 19.0 93.7 7.58 313 Colour: 3 Clarity: 1 Slightly opalescent None 2 53 1222.7 23.1 100.0 7.60 315 Colour: 3 Clarity: 1 Slightly opalescent None 3 61.2 1336.2 21.8 100.0 7.75 320 Colour: 3 Clarity: 1 Slightly opalescent None 6 52.2 1001.3 19.2 100.0 7.60 321 Colour: 3 Clarity: 1 Slightly opalescent None 12 50.4 887.9 17.6 100.0 7.60 320 Colour: 3 Clarity: 1 Slightly opalescent None

TABLE-US-00018 TABLE 16 Freeze-dried product, 50 mg/ml; Storage temp.: 5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Cake/solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 42.7 759.4 17.8 100.0 7.58 292 Colour: 3 Clarity: 1 Cake: yellow, some cracks Solution: Clear None 1 42.6 840.4 19.7 63.1 7.58 293 Colour: 3 Clarity: 1 Cake: yellow, some cracks Solution: Clear None 2 42.1 937.0 22.3 100.0 7.60 292 Colour: 3 Clarity: 1 Cake: yellow, some cracks Solution: Clear None 3 47.4 1014.7 21.4 100.0 7.75 291 Colour: 3 Clarity: 1 Cake: yellow, some cracks Solution: Clear None 6 49.0 876.5 17.9 100.0 7.60 304 Colour: 3 Clarity: 1 Cake: yellow, some cracks Solution: Clear None 12 51.3 945.0 18.4 100.0 7.60 308 Colour: 3 Clarity: 1 Cake: yellow, some cracks Solution: Clear None

TABLE-US-00019 TABLE 17 Aqueous product, 100 mg/ml; Storage temp.: 5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) PH (mOsm/kg) Aggregates 0 81.8 1705.7 20.9 99.9 7.60 350 Colour: 3 Clarity: 1 Slightly opalescent None 1 85.9 1942.4 22.6 96.9 7.55 352 Colour: 3 Clarity: 1 Slightly opalescent None 2 95.7 1690.8 17.7 96.9 7.65 357 Colour: 3 Clarity: 1 Slightly opalescent None 3 104.3 1671.2 16.0 100.0 7.65 350 Colour: 3 Clarity: 1 Slightly opalescent None 6 96.0 1642.6 17.1 100.0 7.62 360 Colour: 3 Clarity: 1 Slightly opalescent None 9 102.8 1270.8 12.4 100.0 7.63 352 Colour: 3 Clarity: 2 Slightly opalescent None 11 86.2 1140.2 13.2 100.0 7.60 353 Colour: 3 Clarity: 2 Slightly opalescent None 12 113.9 1550.6 13.6 100.0 7.58 350 Colour: 3 Clarity: 2 Slightly opalescent None 15 114.7 1160.6 10.1 98.3 7.61 350 Colour: 3 Clarity: 2 Slightly opalescent None 18 86.2 907.4 10.5 100.0 7.67 340 Colour: 3 Clarity: 2 Slightly opalescent None

TABLE-US-00020 TABLE 18 Aqueous product, 100 mg/ml; Storage temp.: -20.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 81.8 1705.7 20.9 99.9 7.60 316 Colour: 3 Clarity: 1 Slightly opalescent None 1 89.3 2108.8 23.6 100.0 7.56 350 Colour: 3 Clarity: 1 Slightly opalescent None 2 112.0 2066.5 18.5 100.0 7.65 353 Colour: 3 Clarity: 1 Slightly opalescent None 3 100.2 2172.4 21.7 96.7 7.65 352 Colour: 3 Clarity: 1 Clear None 6 87.5 2672.3 30.6 100.0 7.62 352 Colour: 3 Clarity: 1 Clear None 9 97.1 2040.3 21.0 100.0 7.62 353 Colour: 3 Clarity: 1 Clear None 11 104.6 2234.0 21.4 100.0 7.60 353 Colour: 3 Clarity: 1 Clear None 12 94.5 1608.8 17.0 100.0 7.57 350 Colour: 3 Clarity: 1 Slightly opalescent None 15 118.0 2015.9 17.1 100.0 7.62 351 Colour: 3 Clarity: 1 Slightly opalescent None 18 90.6 1736.4 19.2 100.0 7.69 338 Colour: 3 Clarity: 1 Slightly opalescent None

TABLE-US-00021 TABLE 19 Freeze-dried product, 100 mg/ml; Storage temp.: 5.degree. C. Visual observation Colour 1-3 Time- Protein Specific Clarity 1-3 point conc. Activity activity Purity Osmolality Cake/solution (month) (mg/ml) (U/ml) (U/mg) (%) pH (mOsm/kg) Aggregates 0 76.0 1638.3 21.5 100.0 7.60 316 Colour: 3 Clarity: 1 Cake: Yellow, some cracks Solution: Clear None 1 71.6 1747.6 24.4 100.0 7.55 318 Colour: 3 Clarity: 1 Cake: Yellow, some cracks Solution: Clear None 2 81.6 1769.9 21.7 100.0 7.63 313 Colour: 3 Clarity: 1 Cake: Yellow, some cracks Solution: Clear None 3 84.1 1616.6 19.2 98.2 7.65 320 Colour: 3 Clarity: 1 Cake: Yellow, some cracks Solution: Clear None 6 96.7 2197.6 22.7 100.0 7.60 324 Colour: 3 Clarity: 1 Cake: Yellow, some cracks Solution: Clear None 9 ND ND ND ND ND ND ND 12 96.0 1978.4 20.6 100.0 7.57 322 Colour: 3 Clarity: 1 Cake: Yellow, some cracks Solution: Clear None 15 ND ND ND ND ND ND ND 18 80.6 1602.6 19.9 100.0 7.75 310 Colour: 3 Clarity: 1 Cake: Yellow, some cracks Solution: Clear None

Example 3

[0269] Concentrating a rhPBGD composition by tangential flow filtration (TFF)

[0270] The bulk solution of rhPBGD was then thawed for a minimum of three days at 5.degree. C. and in darkness.

[0271] The thawed solution was then centrifuged with 200 mL conical centrifuge tubes for approximately 10 minutes at 2200 g.

[0272] The solution was then filtered through a series of filters with the following pore-sizes: 5.0 .mu.m; 0.65 .mu.m; 0.45 .mu.m and 0.20 .mu.m before it was concentrated by tangential flow filtration (TFF).

[0273] The concentration by TFF was performed with a Millipore Labscale TFF System and Millipore Pellicon.RTM. XL Filter with a pump inlet pressure of approximately 20-25 psi and a pressure over the Pellicon.RTM. XL Filter of approximately 4-6 psi. The rhPBGD was protected from light during the procedure by covering the sample container of the TFF System by sheets of aluminium foil.

[0274] The concentrated rhPBGD solution obtained from the TFF procedure was then buffer-changed against a formulation buffer containing 3.67 mM Na.sub.2HPO.sub.4.times.2H.sub.2O, 27 mM glycin and 220 mM Mannitol prepared in sterile water. This was performed by continuously adding said buffer to the TFF-system and pressing it across the membrane until said buffer has replaced the previous buffer.

[0275] The concentrated and buffer-changed rhPBGD solution was then sterile filtered by passing it through a filter with a pore-size of 0.22 .mu.m. This sterile filtration was performed twice with a new filter each time.

[0276] The sterile concentrated rhPBGD solution was then placed in vials before it was freeze-dried as described in the method section.

Example 4

The Effect of Different Modes of Freeze-Drying and/or the Amount of Excipients on the Reconstitution Time

[0277] PBGD was concentrated as described in example 3 and after the exchange of the buffer was the concentration of PBGD determined.

[0278] The concentrated PBGD solution was then freeze-dried in a Lyostar (FTM-systems) freeze-dryer. The solutions were filled in 2 and 6 ml injection glass vials (type 1) and stoppered with rubber stoppers (chlorobutyl).

Original Freeze-Drying Cycle:

[0279] The samples were loaded in ambient temperature and the shelves were cooled down to 0.degree. C. for 30 minutes. The temperature were lowered to -40.degree. C. (1.degree. C. per minute) and held there for 30 minutes and then the vacuum pressure was drawn to 126 mTorr and the primary drying began by raising the temperature to 0.degree. C. (1.degree. C. per minute). After 360 minutes of primary drying the temperature was raised to +30.degree. C. (0.5.degree. C. per minute) and full vacuum was drawn simultaneously (start of secondary drying). The temperature was held at +30.degree. C. for 360 minutes and the vials were then stoppered under vacuum.

Freeze-Drying with Inclusion of an Annealing Step:

[0280] After 30 minutes at -40.degree. C. the temperature was raised with a rate of 2.degree. C. per minute to -10.degree. C. or -20.degree. C. at which temperature they were kept for 120 or 420 minutes before the temperature was lowered again with 2.degree. C. per minute to -40.degree. C. were the samples were kept for 60-90 minutes before start of primary drying.

[0281] The results are shown in Table 20 where the short terms used with regard to the excipients and the freeze-drying cycle mean the following:

[0282] 1.times. amount of excipients refers to that the PBGD solution comprises 3.67 mM Na.sub.2HPO.sub.4.times.2H.sub.2O, 27 mM glycin and 220 mM Mannitol prepared in sterile water.

[0283] 1.5.times. amount excipients refers to that the PBGD solution comprises 5.51 mM Na.sub.2HPO.sub.4.times.2H.sub.2O, 40.5 mM glycin and 375 mM Mannitol prepared in sterile water, i.e. 1.5.times. of each of the components present in the 1.times. buffer.

[0284] 2.times. excipients refers to that the PBGD solution comprises 7.34 mM Na.sub.2HPO.sub.4.times.2H.sub.2O, 54 mM glycin and 500 mM Mannitol prepared in sterile water, i.e. 2.times. of each of the components present in the 1.times. buffer.

[0285] The original freeze-drying cycle is as described above.

[0286] The annealing freeze-drying cycle is as described above where the annealing step comprises raising the temperature to -10.degree. C. at keeping the sample at this temperature for 120 minutes before lowering it to -40.degree. C. again.

[0287] The extended annealing freeze-drying cycle is as described above where the annealing step comprises raising the temperature to -20.degree. C. at keeping the sample at this temperature for 420 minutes before lowering it to -40.degree. C. again.

TABLE-US-00022 TABLE 20 Reconstitution time for Amount Protein different free-drying cycles of concentration Extended excipients (mg/ml) Original Annealing annealing 1x 198 600 550 480 1x 175 540 500 450 1x 150 450 480 180 1x 125 330 100 10 1x 100 40 10 10 1x 80 25 10 10 1.5x 200 480 40 60 1.5x 175 220 10 10 1.5x 150 60 10 10 1.5x 125 15 10 10 1.5x 100 10 10 10 2x 200 120 20 2x 175 40 20 2x 150 20 10 2x 100 10 10

Example 5

The Effect of Different Modes of Freeze-Drying and/or the Amount of Excipients on the Appearance of the Freeze-Dried Product

[0288] Concentrated and freeze-dried solutions of PBGD were prepared as described in example 4 and references to the amount of excipients and the type of freeze-drying cycle has the same meaning as in example 4.

[0289] The following results were obtained by visual inspection of the freeze-dried products:

[0290] A: Comparison of three products prepared from solutions comprising respectively, 4.6 mg/ml 66.6 mg/ml and 109.4 mg/ml rhPBGD showed that the number of cracks in the freeze-dried product increased as concentration of rhPBGD increased.

[0291] B: Comparison of two products, prepared from a solution comprising 150 mg/ml rhPBGD, and comprising 1.times. and 1.5.times. amount of excipients showed that the number of cracks in the freeze-dried product was lower for the product which comprised 1.5.times. amount of excipients than the product comprising 1.times. amount of excipients.

[0292] C: Comparison of two freeze-dried products prepared from a 150 mg/ml rhPBGD solution, comprising 1.times. and 2.times. amount of excipients showed that the number of cracks in the freeze-dried product with 2.times. amount of excipients was lower than the product comprising the 1.times. amount of excipients.

[0293] D: Comparison of three freeze-dried products prepared from a 150 mg/ml rhPBGD solution by using the original, the annealing and the extended annealing freeze-drying cycle showed that the number of cracks in the freeze-dried product was lower in the product which was prepared according to the annealing freeze-drying cycle than in the product prepared according to the original freeze-drying cycle. Furthermore, the number of cracks in the product prepared according to the extended annealing freeze-drying cycle was lower than in the product prepared according to the annealing freeze-drying cycle.

[0294] E: Three freeze-dried products were prepared from a 150, 175 and 200 mg/ml, respectively, rhPBGD solution. The freeze-dried products each comprised 1.5.times. amount of excipients and they were freeze-dried with the annealing cycle. None of the freeze-dried products comprised any cracks.

[0295] F: Two freeze-dried rhPBGD products were prepared from a 150 mg/ml rhPBGD solution. One of them comprised 1.times. amount of excipients and was prepared according to the original freeze-drying cycle, while the other comprised 1.5.times. amount of excipients and was prepared according to the extended annealing free-drying cycle. The product comprising 1.5.times. amount of excipients and prepared according to the extended annealing freeze-drying cycle comprised fewer cracks than the product comprising 1.times. amount of excipients and prepared according to the original freeze-drying cycle.

[0296] G: Two freeze-dried rhPBGD products were prepared from a 150 mg/ml rhPBGD solution. One of them comprised 1.times. amount of excipients and was prepared according to the original freeze-drying cycle, while the other comprised 0.1% Tween 80 in combination with the 1.times. amount of excipients and was prepared according to the extended annealing freeze-drying cycle. The product comprising the 0.1% Tween 80 in combination with the 1.times. amount of excipients and which was prepared according to the extended annealing freeze-drying cycle comprised fewer cracks than the product which comprised 1.times. amount of excipients and which was prepared according to the original freeze-drying cycle.

Example 6

The Effect of Recovery Volume, the Amount of Excipients and the Mode of Freeze-Drying on the Stability of Freeze-Dried rhPBGD

[0297] Concentrated rhPBGD solutions freeze-dried samples were prepared as described in example 4.

[0298] The "bulk solution" is a concentrated solution of PBGD before freeze-drying.

[0299] Table 21 shows the results of rhPBGD solutions having the following characteristics with regard to the concentration of rhPBGD, amount of excipients (were the same definitions as in example 4 are used), the mode of freeze-drying (were the same definitions as in example 4 are used) and the ratio of the filling volume (fill. Vol which is the volume of the composition before it is freeze-dried) versus the recovery volume (Rec. vol which is the volume in which the freeze-dried product is resuspended):

Solution 1:

[0300] Approximately 5 mg/ml rhPBGD [0301] 1.times. amount of excipient [0302] Original freeze-drying cycle [0303] Fill. vol=Rec. vol

Solution 2:

[0303] [0304] Approximately 70 mg/ml rhPBGD [0305] 1.times. amount of excipient [0306] Original freeze-drying cycle [0307] Fill. vol=2.times.Rec. vol

Solution 3:

[0307] [0308] Approximately 110 mg/ml rhPBGD [0309] 1.times. amount of excipient [0310] Original freeze-drying cycle [0311] Fill. vol=Rec. vol

Solution 4:

[0311] [0312] Approximately 70 mg/ml rhPBGD [0313] 1.times. amount of excipient [0314] Original freeze-drying cycle [0315] Fill. vol=1.5.times.Rec. vol

Solution 5:

[0315] [0316] Approximately 90 mg/ml rhPBGD [0317] 2/3.times. amount of excipient [0318] Original freeze-drying cycle [0319] Fill. vol=1.5.times.Rec. vol

Solution 6:

[0319] [0320] Approximately 60 mg/ml rhPBGD [0321] 1/2.times. amount of excipient [0322] Original freeze-drying cycle [0323] Fill. vol=2.times.Rec. vol

Solution 7:

[0323] [0324] Approximately 110 mg/ml rhPBGD [0325] 1.times. amount of excipient [0326] Annealing freeze-drying cycle [0327] Fill. vol=Rec. vol

Solution 8:

[0327] [0328] Approximately 60 mg/ml rhPBGD [0329] 1.times. amount of excipient [0330] Annealing freeze-drying cycle [0331] Fill. vol=2.times.Rec. vol

Solution 9:

[0331] [0332] Approximately 150 mg/ml rhPBGD [0333] 1.times. amount of excipient [0334] Annealing freeze-drying cycle [0335] Fill. vol=Rec. vol

Solution 10:

[0335] [0336] Approximately 150 mg/ml rhPBGD [0337] 1.times. amount of excipient [0338] Original freeze-drying cycle [0339] Fill. vol=Rec. vol

[0340] Although not shown in Table 21 the purity was also tested for each time point as was found to 1000% in all cases.

[0341] For solution 2 at the week 4 and 9 time point and for solution 4 the week 9 time point a wrong recovery volume was used.

TABLE-US-00023 TABLE 21 Measuring Protein Specific point Fill. Vol Rec. Vol Osmolality concentration Activity activity Solution (week) (ml) (ml) pH (mosmol/kg) (mg/ml) (U/ml) (U/mg) 1 bulk 4.6 78 17.1 0 0.67 0.67 7.54 274 4.8 85 17.8 2 0.67 0.67 7.22 274 4.6 87 19.4 4 0.67 0.67 7.78 279 5.1 75 14.5 7 0.67 0.67 7.87 284 5.1 68 13.3 9 0.67 0.67 7.67 403 7.0 93 13.2 2 bulk 66.6 1129 16.9 0 0.67 0.335 7.64 525 113 1915 16.9 2 0.67 0.335 7.63 459 93.6 1593 17.0 4 0.67 0.67 7.75 264 64.6 1104 17.1 7 0.67 0.335 7.95 451 106.4 2106 19.8 9 0.67 0.67 7.59 247 51.4 859 16.7 3 bulk 109.4 1491 13.6 0 0.67 0.67 7.75 274 99.9 1598 16.0 2 0.67 0.67 7.64 269 91.4 1543 16.9 4 0.67 0.67 7.68 274 101.2 1825 18.0 7 0.67 0.67 7.71 278 103.4 2045 19.8 9 0.67 0.67 7.67 274 88.3 1656 18.8 4 bulk 71.5 1244 17.4 0 0.67 0.45 7.64 448 113.8 1748 15.4 2 0.67 0.45 7.63 411 86.4 1806 20.9 4 0.67 0.45 7.77 362 109.9 1897 17.3 7 0.67 0.45 7.90 379 95.2 686 (7.2) 9 0.67 0.67 7.63 273 59.7 1090 18.3 5 bulk 91.0 1610 17.7 0 0.67 0.45 7.65 296 119.4 2014 16.9 2 0.67 0.45 7.61 285 112.3 2093 18.6 4 0.67 0.45 7.90 292 125.1 2409 19.3 7 0.67 0.45 7.88 297 116.4 1928 16.6 9 0.67 0.45 7.34 278 102.5 1490 14.5 6 bulk 60.7 992 16.3 0 0.67 0.335 7.63 295 112.6 1753 15.6 2 0.67 0.335 7.60 288 86.9 1787 20.6 4 0.67 0.335 7.83 287 116.4 2106 18.1 7 0.67 0.335 8.20 299 109.7 695 (6.3) 9 0.67 0.335 7.44 287 95.2 1636 17.2 7 bulk 116.4 1926 16.5 0 0.67 0.67 7.56 275 101.1 1750 17.3 2 0.67 0.67 7.51 276 93.4 1831 19.6 4 0.67 0.67 7.60 270 101.6 1774 17.5 7 0.67 0.67 7.53 283 102.2 1639 16.0 9 0.67 0.67 7.46 274 89.9 960 10.7 8 bulk 64.5 1119 17.4 0 0.67 0.335 7.52 511 100.7 1718 17.1 2 0.67 0.335 7.51 459 99.3 1900 19.1 4 0.67 0.335 7.70 482 114.5 1913 16.7 9 0.67 0.335 7.29 425 102.3 1650 16.1 9 bulk 165 3587 21.7 0 0.60 0.60 7.71 309 121.4 2819 23.2 4 0.60 0.60 7.74 -- 140.3 2014 14.4 7.5 0.60 0.60 7.61 292 135.9 1640 12.1 10 bulk 165 3587 21.7 0 0.60 0.60 7.86 276 142.1 2397 16.9 3 0.40 0.40 8.20 314 141.9 2381 16.8 5 0.60 0.60 7.60 302 131.8 2304 17.5

Example 7

Effect of Different Excipients on the Stability of rhPBGD

[0342] rhPBGD was concentrated as described in example 4 and then the buffer was changed as to one of the four buffers described below. The products were then freeze-dried as described in example 4 with an original annealing step included and the stability of the samples were tested as described in example 6.

[0343] The effect of the following four formulations on the stability of rhPBGD was tested:

[0344] Formulation A (corresponds to solution 9 in example 6): 250 mM mannitol, 27 mM glycine and 3.67 mM Na.sub.2HPO.sub.4.

[0345] Formulation B: 250 mM mannitol, 27 mM glycine and 10 mM TRIS-HCL.

[0346] Formulation C: 250 mM mannitol, 27 mM glycine, 3.67 mM Na.sub.2HPO.sub.4 and 0.1% Tween 80.

[0347] Formulation D: 221 mM mannitol, 29 mM sucrose, 27 mM glycine, 3.67 mM Na.sub.2HPO.sub.4 and 0.1% Tween 80.

[0348] The results are shown in Table 22.

TABLE-US-00024 TABLE 22 Measuring Protein Specific point Fill. Vol Rec. Vol Osmolality concentration Activity activity Formulation (week) (ml) (ml) pH (mosmol/kg) (mg/ml) (U/ml) (U/mg) A Bulk 7.69 366 165 3587 21.7 0 0.60 0.60 7.71 309 121.4 2819 23.2 4 0.60 0.60 7.74 -- 140.3 2014 14.4 7.5 0.60 0.60 7.61 292 135.9 1640 12.1 B Bulk 7.54 320 173 3595 20.8 0 0.60 0.60 7.58 284 148.1 3726 25.2 3 0.60 0.60 7.57 280 165.4 2947 17.8 4 0.60 0.60 7.69 -- 167.5 2367 14.1 7.5 0.60 0.60 7.60 283 150.4 2235 14.9 C Bulk 7.40 338 178 3606 20.2 0 0.60 0.60 7.76 290 142.9 2662 18.6 3 0.60 0.60 7.43 285 181.7 2332 12.8 4 0.60 0.60 7.42 -- 173.1 1436 8.3 6 0.60 0.60 7.55 274 156.6 1254 7.4 7.5 0.60 0.60 7.34 274 141.5 1252 8.9 D Bulk 7.41 337 175 3869 22.1 0 0.60 0.60 7.80 292 127.5 2355 18.5 3 0.60 0.60 7.35 288 143.9 1988 13.8 4 0.60 0.60 7.26 -- 159.3 1644 10.3 6 0.60 0.60 7.30 281 135.7 1236 9.1 7.5 0.60 0.60 7.28 282 125.7 1146 9.1

Sequence CWU 1

1

2611035DNAHomo sapiens 1atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540tggcacaacc gggttgggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020aacgatgccc attaa 103521035DNAHomo sapiens 2atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020aacgatgccc attaa 103531035DNAHomo sapiens 3atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020aacgatgccc attaa 103541034DNAHomo sapiens 4atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt cttcacccaa aatttgttgg gaagacccta gaaaccctgc 360cagagaagag tgtggtggga accagctccc tgcgaagagc agcccagctg cagagaaagt 420tcccgcatct ggagttcagg agtattcggg gaaacctcaa cacccggctt cggaagctgg 480acgagcagca ggagttcagt gccatcatcc tggcaacagc tggcctgcag cgcatgggct 540ggcacaaccg ggtggggcag atcctgcacc ctgaggaatg catgtatgct gtgggccagg 600gggccttggg cgtggaagtg cgagccaagg accaggacat cttggatctg gtgggtgtgc 660tgcacgatcc cgagactctg cttcgctgca tcgctgaaag ggccttcctg aggcacctgg 720aaggaggctg cagtgtgcca gtagccgtgc atacagctat gaaggatggg caactgtacc 780tgactggagg agtctggagt ctagacggct cagatagcat acaagagacc atgcaggcta 840ccatccatgt ccctgcccag catgaagatg gccctgagga tgacccacag ttggtaggca 900tcactgctcg taacattcca cgagggcccc agttggctgc ccagaacttg ggcatcagcc 960tggccaactt gttgctgagc aaaggagcca aaaacatcct ggatgttgca cggcaattga 1020acgatgccca ttaa 103451035DNAHomo sapiens 5atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacgggcagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaagg 420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480gacgagcagc aggagttcag tgtcatcatc ctggcaacag ctggcctgca gcgcatgggc 540tggcacaacc gggttgggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact tgttgctgag caagggagcc aaaaacatcc tggatgttgc acggcaattg 1020aacgatgccc attaa 103561035DNAHomo sapiens 6atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720gaaggaggtt gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020aacgatgccc attaa 103571034DNAHomo sapiens 7atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaatta 1020acgatgccca ttaa 103481035DNAHomo sapiens 8atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggcc 840accatccatg tccctaccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020aacgatgccc attaa 103591260DNAHomo sapiens 9cacaggaaac agctatgacc atgattacgc caagctcgaa attaaccctc actaaaggga 60acaaaagctg gagctccacc gcggtggcgg ccgctctaga actagtggat cccccgggct 120gcaggaattc atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca 180gacggacagt gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat 240tgctatgtcc accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa 300aagcctgttt accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt 360tcactccttg aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg 420caagcgggaa aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct 480agaaaccctg ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct 540gcagagaaag ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct 600tcggaagctg gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca 660gcgcatgggc tggcacaacc gggttgggca gatcctgcac cctgaggaat gcatgtatgc 720tgtgggccag ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct 780ggtgggtgtg ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct 840gaggcacctg gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg 900gcaactgtac ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac 960catgcaggct accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca 1020gttggtaggc atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt 1080gggcatcagc ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc 1140acggcaattg aacgatgccc attaataagc ttatcgatac cgtcgacctc gagggggggc 1200ccggtaccca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa 1260101113DNAHomo sapiens 10cacacagcct actttccaag cggagccatg tctggtaacg gcaatgcggc tgcaacggcg 60gaagaaaaca gcccaaagat gagagtgatt cgcgtgggta cccgcaagag ccagcttgct 120cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct cgtaccctgg cctgcagttt 180gaaatcattg ctatgtccac cacaggggac aagattcttg atactgcact ctctaagatt 240ggagagaaaa gcctgtttac caaggagctt gaacatgccc tggagaagaa tgaagtggac 300ctggttgttc actccttgaa ggacctgccc actgtgcttc ctcctggctt caccatcgga 360gccatctgca agcgggaaaa ccctcatgat gctgttgtct ttcacccaaa atttgttggg 420aagaccctag aaaccctgcc agagaagagt gtggtgggaa ccagctccct gcgaagagca 480gcccagctgc agagaaagtt cccgcatctg gagttcagga gtattcgggg aaacctcaac 540acccggcttc ggaagctgga cgagcagcag gagttcagtg ccatcatcct ggcaacagct 600ggcctgcagc gcatgggctg gcacaaccgg gttgggcaga tcctgcaccc tgaggaatgc 660atgtatgctg tgggccaggg ggccttgggc gtggaagtgc gagccaagga ccaggacatc 720ttggatctgg tgggtgtgct gcacgatccc gagactctgc ttcgctgcat cgctgaaagg 780gccttcctga ggcacctgga aggaggctgc agtgtgccag tagccgtgca tacagctatg 840aaggatgggc aactgtacct gactggagga gtctggagtc tagacggctc agatagcata 900caagagacca tgcaggctac catccatgtc cctgcccagc atgaagatgg ccctgaggat 960gacccacagt tggtaggcat cactgctcgt aacattccac gagggcccca gttggctgcc 1020cagaacttgg gcatcagcct ggccaacttg ttgctgagca aaggagccaa aaacatcctg 1080gatgttgcac ggcaattgaa cgatgcccat taa 1113111380DNAHomo sapiens 11agcaggtcct actatcgcct ccctctagtc tctgcttctt tggatccctg aggagggcag 60aaggaagaaa acagcccaaa gatgagagtg attcgcgtgg gtacccgcaa gagccagctt 120gctcgcatac agacggacag tgtggtggca acattgaaag cctcgtaccc tggcctgcag 180tttgaaatca ttgctatgtc caccacaggg gacaagattc ttgatactgc actctctaag 240attggagaga aaagcctgtt taccaaggag cttgaacatg ccctggagaa gaatgaagtg 300gacctggttg ttcactcctt gaaggacctg cccactgtgc ttcctcctgg cttcaccatc 360ggagccatct gcaagcggga aaaccctcat gatgctgttg tctttcaccc aaaatttgtt 420gggaagaccc tagaaaccct gccagagaag agtgtggtgg gaaccagctc cctgcgaaga 480gcagcccagc tgcagagaaa gttcccgcat ctggagttca ggagtattcg gggaaacctc 540aacacccggc ttcggaagct ggacgagcag caggagttca gtgccatcat cctagcaaca 600gctggcctgc agcgcatggg ctggcacaac cgggttgggc agatcctgca ccctgaggaa 660tgcatgtatg ctgtgggcca gggggccttg ggcgtggaag tgcgagccaa ggaccaggac 720atcttggatc tggtgggtgt gctgcacgat cccgagactc tgcttcgctg catcgctgaa 780agggccttcc tgaggcacct ggaaggaggc tgcagtgtgc cagtagccgt gcatacagct 840atgaaggatg ggcaactgta cctgactgga ggagtctgga gtctagacgg ctcagatagc 900atacaagaga ccatgcaggc taccatccat gtccctgccc agcatgaaga tggccctgag 960gatgacccac agttggtagg catcactgct cgtaacattc cacgagggcc ccagttggct 1020gcccagaact tgggcatcag cctggccaac ttgttgctga gcaaaggagc caaaaccatc 1080ctggatgttg cacggcagct taacgatgcc cattaactgg tttgtggggc acagatgcct 1140gggttgctgc tgtccagtgc ctacatcccg ggcctcagtg ccccattctc actgctatct 1200ggggagtgat taccccggga gactgaactg cagggttcaa gccttccagg gatttgcctc 1260accttggggc cttgatgact gccttgcctc ctcagtatgt gggggcttca tctctttaga 1320gaagtccaag caacagcctt tgaatgtaac caatcctact aataaaccag ttctgaaggt 1380121377DNAHomo sapiens 12cacacagcct actttccaag cggagccatg tctggtaacg gcaatgcggc tgcaacggcg 60gaagaaaaca gcccaaagat gagagtgatt cgcgtgggta cccgcaagag ccagcttgct 120cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct cgtaccctgg cctgcagttt 180gaaatcattg ctatgtccac cacaggggac aagattcttg atactgcact ctctaagatt 240ggagagaaaa gcctgtttac caaggagctt gaacatgccc tggagaagaa tgaagtggac 300ctggttgttc actccttgaa ggacctgccc actgtgcttc ctcctggctt caccatcgga 360gccatctgca agcgggaaaa ccctcatgat gctgttgtct ttcacccaaa atttgttggg 420aagaccctag aaaccctgcc agagaagagt gtggtgggaa ccagctccct gcgaagagca 480gcccagctgc agagaaagtt cccgcatctg gagttcagga gtattcgggg aaacctcaac 540acccggcttc ggaagctgga cgagcagcag gagttcagtg ccatcatcct agcaacagct 600ggcctgcagc gcatgggctg gcacaaccgg gtggggcaga tcctgcaccc tgagaaatgc 660atgtatgctg tgggccaggg ggccttgggc gtggaagtgc gagccaagga ccaggacatc 720ttggatctgg tgggtgtgct gcacgatccc gagactctgc ttcgctgcat cgctgaaagg 780gccttcctga ggcacctgga aggaggctgc agtgtgccag tagccgtgca tacagctatg 840aaggatgggc aactgtacct gactggagga gtctggagtc tagacggctc agatagcata 900caagagacca tgcaggctac catccatgtc cctgcccagc atgaagatgg ccctgaggat 960gacccacagt tggtaggcat cactgctcgt aacattccac gagggcccca gttggctgcc 1020cagaacttgg gcatcagcct ggccaacttg ttgctgagca aaggagccaa aaacatcctg 1080gatgttgcac ggcagcttaa cgatgcccat taactggttt gtggggcaca gatgcctggg 1140ttgctgctgt ccagtgccta catcccgggc ctcagtgccc cattctcact gctatctggg 1200gagtgattac cccgggagac tgaactgcag ggttcaagcc ttccagggat ttgcctcacc 1260ttggggcctt gatgactgcc ttgcctcctc agtatgtggg ggcttcatct ctttagagaa 1320gtccaagcaa cagcctttga atgtaaccaa tcctactaat aaaccagttc tgaaggt 13771310024DNAHomo sapiens 13aatcatgatt gttaattatg ttcatgatta caggcgcggt ggctcacgcc tgtactccca 60gcactttggg aggccgaggt gggcgaatca cctgaggtca ggagttcaag acctgcctga 120ctaacatgga gaaacctcat ctctaccaaa aatacaaaat tagccgggtg tggtggtgcg 180tgcctgtaat cccagctact cggggggctg aggcaggaga attgcttgaa cccgggaggc 240ggaggttgca gtgagctgag atcgtgccat tgcattccag cctgggcaac aagagcgaaa 300ctccgtctca aaaaaaaaaa aaaattatgt tcatgggaaa gcacttttcc taacaagccc 360ttttctcact acatgtaggt ttgtgctccc acttcagtta cttgtcttta ggcatgacct 420ttaatctctc tgaaccagtt tcctcatttt aagaattgaa atgctggctg ggccagtcgt 480cacgcctgta atcccagcac tttgggaggc caaggcgaga tgactgcttg agtccaggag 540ttcgagacta gcctgggcaa catagtgagg ccacctcccc gctgtctcta taaaaaaatc 600tagaaattag tcccacgtgg tgatgtgcgc ctgtagtccc agctgcttgg gaggctgagg 660tggggggatc gctgaagccg ggaggtcaag gctgcagtga cccgtggtca tgccgctgca 720ctctagtctg gggacacagt gagaccccgt atcaaaaaga aaaatgctgc ctatttcaag 780gttgtagcaa agctaagttt gaacagagca aaggaagcgc catagaagct gcactacttg 840ctcatgtcac agctggggaa tggggaggtc gaatggggag gtccactgtc gcaatgttcc 900aattcccgcc cagagggagg gacctcccct tcgagggagg gcgccggaag tgacgcgagg 960ctctgcggag accaggagtc agactgtagg acgacctcgg

gtcccacgtg tccccggtac 1020tcgccggccg gagcctccgg cttcccgggg ccgggggacc ttagcggcac ccacacacag 1080cctactttcc aagcggagcc atgtctggta acggcaatgc ggctgcaacg gcggtgagtg 1140ctgagccggt gaccagcaca ctttgggctt ctggacgagc cgtgcagcga ttggccccag 1200gttgccatcc tcagtcgtct attggtcaga acggctatct tttttttttt tttttttttt 1260tttttggtcc gagtagcttt taaagggcca gtagctcggt tgccctccgg aaggaatggg 1320gaaatcagag agcggtgata ctgggttaag agtggaagga ttgtttggaa cggaactccg 1380gtccctgcgg gcatctgggt gggattccca tcaggcctgg gatgcacggc tctagattta 1440gtgacccaga ccaagaacgt tcgtctacac agacggggtc ctttcattcg aggctgggct 1500gaggcggatg cagatacggc ccctttggga agacacgttc cacttttgat tcataggaga 1560gagtatcagc caagcctccg aactgcacac aaacgtctta gaagtgcgcc ttctttttgt 1620gttatagtgg tctcccagcc acagccaacg ctccaagtcc ccagctgtga cacacctact 1680gaattactac cgtgggtggg aggccgccgt gggcctttcc attacgagcc tgcttgccga 1740gccctgggct tgtgcacaga caaactgcag agctggtgga ggccactgcc aggccgagat 1800aagaaagaga tggggagctg ctaatctccc cctgtccagc ctgttggtga gggctgggat 1860ctttgctctt gcagtcattc cagagccctg gactaggagt aggaagatct gaattgtggc 1920cccaactctc tttcggttat tagctctgtg accctaggca agtcacctca tcccttgatg 1980ccacccgttg cttctgtaac atggtcccaa aggtgcctgt cttgtccacc tgataggatt 2040tttgagacga caacaatatg caaaagcaat agcttcaaca tagaagtgct cagtgtttta 2100ttttttaatg aaacggtttg acttggatat gctgtgcaca ttcaatgaac ttaaggaatt 2160gtttgaacct agtagttctg ggaccttaga gtcctttctg tgggctccct gtggcccaga 2220tttttggtgg ccacgtttaa tatcaagcct agcctaattt gcaaagggtc tcccagggtt 2280aatttattgg agtgatcaca tggagtagac cagagtctga gggcagaaag ctgtcacctg 2340cttcggcaat agaggcccca gatgtctggg tgcaaaagaa ctccatagca ccccgaccaa 2400catggtgaaa ccccgtctct actaaaaata taaaaattag gccgagcaca gtggctcatg 2460cctgtaatcc tagcactttg ggaggccgag gcaggtggat tgcctgagct caggagttcg 2520agaccagcct agggaacaca gtgaaacccc gtttctacta aaaatacaaa aaattagccg 2580acgtggtggc atgcgcctgc agtcccagct acttgggagg ctaagacagg agaatcgctt 2640gaacctggga ggtggaggtt gcactgagcc gagaccgcgc cattgcactc cagcctgggt 2700gacagagcgc aactccccct caaaaaaaga aaaaaatata tatatatata tatatatata 2760tacacacata ttttagctgg gcatggtggt gtgcgtctgt agtagtccca gctacttggg 2820aggctgagtc aggagaatcg cttgaacctg gaaggcagtg gttgtagtta gctgagaaca 2880tgccactgca ctccagcctg ggcaacagag ggagactctg tctcaaaaaa aaaaaaaaaa 2940aggaactaca taggatgaac atcccagatc agggaatgtt gactgtcgac agtatcagta 3000tctacagtgg ctactgtctg atgtagaaag aaatgggatc aggctaggcg tggtggctca 3060cgcctgtaat cccagctctt tgggaggctg gggcaggagg atcacaagtt cgagaccagc 3120ctggccaaca cagtgaaacc ccgtctctac taaaaatgtg aaaattagct gggcatggtg 3180gaacatgctg tagttccagc ttgaacccag gggtggaggt tgtagtgagc ctagatcacg 3240ccactgcact ccagcctgag caaaacagtg agactctgtc taaaaaaaaa aaaaaaaaaa 3300agagaaatgg gacctccgtc ttagactgaa gaattcagtt ctacgtgctt agcagtgaat 3360acttttgtcc aaggtactct ggcaggagga agaggcgtgt cctcttgagt tcttgacttg 3420ggctctggcc tgttaatatt tccatgttgg tgaaaccaga ggcagcactc taggtgaacg 3480aactttaggc agcgcagcct cctagtctta tggaacatct gaggcagaag aaacctgagt 3540ccaacctttt cattttatag atgaacaaac agatcctgat gggacagtgt acccaaggtc 3600acccagccaa gaggctgagc aggactgtac gtcagatccg tttacctcag tccttaatgc 3660atgcagtcca gccagattaa gggaccctta atactgtcag ctttccccac tgtgggatct 3720tcatcctctt gacttctttt gtagccagac atctgggcct cttgctggag aaggtggcag 3780cttgctgctc ttagactcta gtctactcca tgtggcatct ggatggcact gaaattttct 3840caagtgcctt gtctgttgta gataatgaat ctatcctcca gtgactcagc acaggttccc 3900cagtgtggtc ctggctgccc tgcccctgcc agctgcaggc cccacccttc ctgtggccag 3960gctgatgggc cttatctctt tacccacctg gctgtgcaca gcactcccac tgacaactgc 4020cttggtcaag gtgggcttca gggctcagtg tcctggttac tgcagcggca gcaacagcag 4080gtcctactat cgcctccctc tagtctctgc ttctctggat ccctgaggag ggcagaaggt 4140actgaggaag gttaaaggga ccagccttgg agtatttccc cactctgaga ctcagctggc 4200cacaggccag gttctgaatt tcctttcttc caagccagtg attctggttc ttggacaagg 4260tgttgaggaa cactagaaac agaggggact gtgacctggg gactttttct gcaggaagaa 4320aacagcccaa agatgagagt gattcgcgtg ggtacccgca agagccaggt gggtgcagga 4380gccggggtgg aggaggtttg tcagaacagt tatgatgctc acagcatcac aaattggggg 4440actcagaggg ttagttccta gtatgaagga gatggggtgg ctgggcgtta agttccccgg 4500gaaatggcag attacattct atggcaagat catccctagg ctgggaaaat tgttggagtg 4560cagagggctc ccaagcccct tctcatgccc agatggaaat tccagtccct tcaggatctg 4620cctaacctgt gacagtctaa agagtctgag ccgtggctgg gaagggcagg actaatccaa 4680atctctaccc gcagcttgct cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct 4740cgtaccctgg cctgcagttt gaaatcagtg agttttctgg aaaggagtgg aagctaatgg 4800gaagcccagt accccgagag gagagaacac aacatttctg gctttgccta tagctaaagc 4860ccgtcccgct gccccgagat tccttctggg ctgctcccag ttctgaaggt gctttcctct 4920gaatacctcc agctctgact acctggatta gcctggcatt taacatcttg agctttgggt 4980ctttttatga gtgtttctgg tcttcctgct cgattgtata tactcagagg gcaggaacca 5040gggattatgt gcctctgtcc ccatcatgaa tcgtagcaca gtgctaggct cagtaaatgc 5100tgatcaataa tgagcacctg attgattgac tctctcctca gttgctatgt ccaccacagg 5160ggacaagatt cttgatactg cactctctaa ggtaacaaca tcttcctccc cagttcttgt 5220ccccactctt ctttccttcc ctgaagggat tcactcaggc tctttctgtc cggcagattg 5280gagagaaaag cctgtttacc aaggagcttg aacatgccct ggagaagaat gagtaagtaa 5340agataggaga gtgtggtgcc ctcccagtct cttgctggga ccctagtatg ctaggtctct 5400tgctgggacc cggggtgtca gataggctgc tgggcttaaa ccctcagaga ggctgaaggc 5460agctcatagg tgggtttttt caggcttcag aaaaggagag tgtctggttc tgagccatct 5520ggctgcctgg actgcaagaa tggctggggg agggagggta ggagggagag taggagggag 5580agtgagagga gagcagtttt catgctcctg agatcttgag aaggtgtgct tcctgaactg 5640ccctaggctc caccactgaa gtagaggcag gggtgggtgg agaaggggtg aaggctggct 5700gctcataccc tttctctttg cccccctctc ccatctctat agagtggacc tggttgttca 5760ctccttgaag gacctgccca ctgtgcttcc tcctggcttc accatcggag ccatctgcaa 5820gtaagagtct tgcaagtaag gggcttgggc aggggtaggc atcatgtgaa cctttgcctt 5880tccctttggg gcctgaccct ctgcttcagg gttatctcct ctgccctgag gagtgttgac 5940tggtggcaga aaactcaaga aataccagtg agttggcaat cgagagagaa tagaggtgat 6000ctgaacttaa atctcttccc tcattctgtg cccttccctc ctcccccagg cgggaaaacc 6060ctcatgatgc tgttgtcttt cacccaaaat ttgttgggaa gaccctagaa accctgccag 6120agaagaggta agtggggcct ggataggcag cttggtggga tgtgcccaga agatgcaggg 6180atgggaggag gaggaaagga acagtgactg cctagtgtta aaatctcatt gtaacttctc 6240tctgggcagt gtggtgggaa ccagctccct gcgaagagca gcccagctgc agagaaagtt 6300cccgcatctg gagttcagga gtattgtatc cttttagaag agtgacggat ccttttggaa 6360gagtgacgga gacagcagcc aaggaaaaag acaaggtcta gagggctctg ggagtccgga 6420gagtggaagg ggcttccagc aagcagcccg tggggtcagt ggcctgtctg tctttccatg 6480cactcatccg tccactcatt tacagtctaa tgttttctta gccccagaca agtgttcaga 6540gtgcaaggca ttggggataa tggtgagcaa gataaacatt cccctgcata tgtagagttt 6600acgtcttact tagggataat gcagttatac tgaactgaat agtgactact tctggaggga 6660tagggagtac ttcctttttt tttttttttt tttctgagac ggagtctcgc tctgttgccc 6720aggttggagt gcagtggcgc aatctaggct cactgcaact tctgcctcct gagttcaagc 6780aatcttcctg cctcagcctc ctaagtagtt gggattacag gtgccaccac acctggctaa 6840tttttgtatt tttagtagag actgggtttc accatgttag tcaggctggt ctcaaactcc 6900tgacctcagg tgatccacca gcctcggcct cccaaagggc tgggattaca ggcttgagcc 6960ccgcacccgg tcagtacttc catttttata tgctactata ttgtcttgac ttttacaatg 7020aatatgtagt acatttcata aaactaaatt taaaaatagt atgtgctaag tgctccaata 7080agtgaagttg ggaattttct ggaaacttct agttggaaca tctaaacaca gaagtctggg 7140gtgtcaggga aggtttctca gaggtcttgt aaccttggca agttatttag cctccctatg 7200tcattttcct tatctgtaaa gtggggataa taatactacc ttcctcacag ggttgttgtg 7260aagatgaaat gagctgacat atggaaagta cttttagagc agtgtctggc atgtagtaag 7320tatgatgtaa ctgttagctg ttaacattaa gctgagagct ggaagatgac tgaaagtcag 7380ccagctagag agggaaagac agactcaggc agagggaacc gcacgaggcc ccagattgcc 7440cgacactgtg gtccttagca actctccaca gcggggaaac ctcaacaccc ggcttcggaa 7500gatggacgag cagcaggagt tcagtgccat catcctggca acagctggcc tgcagcgcat 7560gggctggcac aaccgggttg ggcaggtagg gcctgcccct atcctctccc cagctcatct 7620gcatctcctt tctgccttac agtcatcccc aatttaggat ttttagactt tatgattgtg 7680tgaaagcgat atacgttcag tagaaactgt acttagtacc catacagcca ttctgttttt 7740tactttcagt acagtattca ttacatgaga tattcacttt attgtaaaac aggcttggtg 7800tcagatgatt ttgtccaact ataataggct aatcttaagt gttctgagca catgtaaggt 7860aggctaggtg tattaaatgc attttcagct tgttttcaac ttaacaatgg gtttatcagg 7920atgtaaccct attgtaagtc aaggaccatc tgtcttcact tcttgaccac cccacctcta 7980acaccgtagg ctgggaagat tgtgaatcag aggccagact ctaggctttc atggagaaaa 8040tttacaaaaa aaaaaaaaag aggccagact cacacttagg cctacccagg ctttctagat 8100gatagggaac tcccatctca ctgccaggtg cttttagaca cccccgtgtc cacccttttg 8160actccctgtt ccgcctccac agatcctgca ccctgaggaa tgcatgtatg ctgtgggcca 8220ggtacacttg accagggaag ccacatggtg acatatgcct tccctttgtt ctcaaccaag 8280aagcttgtct cacaaccttc tgcatctgct tccccagaat agcattctca gggaggggca 8340gaccttggga tgctaccggt ccaaaaggcg ctggggagca agtagataga ggtggtccca 8400tgctttgcgc cattggttgg ggaaagatca ggcctgatgt cctaggatgt ttttccatca 8460gggggccttg ggcgtggaag tgcgagccaa ggaccaggac atcttggatc tggtgggtgt 8520gctgcacgat cccgagactc tgcttcgctg catcgctgaa agggccttcc tgaggcacct 8580ggtagggcct gtgctccacc tgtggagggc tggggacttg gagagctggg aaaggtggca 8640gggaagattt cttacatgaa tgctctgtat acagtgctaa ctcattcttg ttgaatgttg 8700tgtatggata ggaccaggtc tgggcccaca gttgcctttt cagtgatgtc ctcaggtctg 8760tggtcacagg gtggtgttaa gagcccttgc agctcacaag aacttcttgt tacaggaagg 8820aggctgcagt gtgccagtag ccgtgcatac agctatgaag gatgggcaag taagtggggg 8880gaaatgggcg ggaagccagg gaaaggagga ctgtggcatt tcttcctgtg catcccaggt 8940ttctaggtag tcccctctca gactgtgctg aggcaactgt tttcttcccc agctgtacct 9000gactggagga gtctggagtc tagacggctc agatagcata caagagacca tgcaggctac 9060catccatgtc cctgcccagg taccaaagct ggagggcgag ggggtaataa acaagagtgc 9120atataatctc ttgttctcac caaatcccac ctccttccct catacagcat gaagatggcc 9180ctgaggatga cccacagttg gtaggcatca ctgctcgtaa cattccacga gggccccagt 9240tggctgccca gaacttgggc atcagcctgg ccaacttgtt gctgagcaaa ggagccaaaa 9300acatcctgga tgttgcacgg cagcttaacg atgcccatta actggtttgt ggggcacaga 9360tgcctgggtt gctgctgtcc agtgcctaca tcccgggcct cagtgcccca ttctcactgc 9420tatctgggga gtgattaccc cgggagactg aactgcaggg ttcaagcctt ccagggattt 9480gcctcacctt ggggccttga tgactgcctt gcctcctcag tatgtggggg cttcatctct 9540ttagagaagt ccaagcaaca gcctttgaat gtaaccaatc ctactaataa accagttctg 9600aaggtgttgt gtgtgcgcgt gtggagttgg cgggaagata ggaacaaaca caaagccctt 9660tcatccttac ctcagaggct gggacttttg cccagagttc tcctggtacg tcctttctgc 9720ttctgcctca atagttttca tttcacacag aataaattgt ctcccaggaa caccaagaaa 9780cagagccaca atcttaaatt cctatggttt gccccttcag ttaacagtag agcctgttta 9840tattgcatgg cccctcccac ccctattatc aggaaagtat agaaagtcac taattctaca 9900actctcttgc aaaatgaaaa caaatgctcc atttaaaaaa aaaacaatcc tttaataaaa 9960ttagtccatc taaaactccc caatgcctaa ggttctagtc gtggaagggt tagctgcaga 10020attc 1002414361PRTHomo sapiens 14Met Ser Gly Asn Gly Asn Ala Ala Ala Thr Ala Glu Glu Asn Ser Pro1 5 10 15Lys Met Arg Val Ile Arg Val Gly Thr Arg Lys Ser Gln Leu Ala Arg 20 25 30Ile Gln Thr Asp Ser Val Val Ala Thr Leu Lys Ala Ser Tyr Pro Gly 35 40 45Leu Gln Phe Glu Ile Ile Ala Met Ser Thr Thr Gly Asp Lys Ile Leu 50 55 60Asp Thr Ala Leu Ser Lys Ile Gly Glu Lys Ser Leu Phe Thr Lys Glu65 70 75 80Leu Glu His Ala Leu Glu Lys Asn Glu Val Asp Leu Val Val His Ser 85 90 95Leu Lys Asp Leu Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala 100 105 110Ile Cys Lys Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys 115 120 125Phe Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val Val Gly 130 135 140Thr Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg Lys Phe Pro His145 150 155 160Leu Glu Phe Arg Ser Ile Arg Gly Asn Leu Asn Thr Arg Leu Arg Lys 165 170 175Met Asp Glu Gln Gln Glu Phe Ser Ala Ile Ile Leu Ala Thr Ala Gly 180 185 190Leu Gln Arg Met Gly Trp His Asn Arg Val Gly Gln Ile Leu His Pro 195 200 205Glu Glu Cys Met Tyr Ala Val Gly Gln Gly Ala Leu Gly Val Glu Val 210 215 220Arg Ala Lys Asp Gln Asp Ile Leu Asp Leu Val Gly Val Leu His Asp225 230 235 240Pro Glu Thr Leu Leu Arg Cys Ile Ala Glu Arg Ala Phe Leu Arg His 245 250 255Leu Glu Gly Gly Cys Ser Val Pro Val Ala Val His Thr Ala Met Lys 260 265 270Asp Gly Gln Leu Tyr Leu Thr Gly Gly Val Trp Ser Leu Asp Gly Ser 275 280 285Asp Ser Ile Gln Glu Thr Met Gln Ala Thr Ile His Val Pro Ala Gln 290 295 300His Glu Asp Gly Pro Glu Asp Asp Pro Gln Leu Val Gly Ile Thr Ala305 310 315 320Arg Asn Ile Pro Arg Gly Pro Gln Leu Ala Ala Gln Asn Leu Gly Ile 325 330 335Ser Leu Ala Asn Leu Leu Leu Ser Lys Gly Ala Lys Asn Ile Leu Asp 340 345 350Val Ala Arg Gln Leu Asn Asp Ala His 355 36015344PRTHomo sapiens 15Met Arg Val Ile Arg Val Gly Thr Arg Lys Ser Gln Leu Ala Arg Ile1 5 10 15Gln Thr Asp Ser Val Val Ala Thr Leu Lys Ala Ser Tyr Pro Gly Leu 20 25 30Gln Phe Glu Ile Ile Ala Met Ser Thr Thr Gly Asp Lys Ile Leu Asp 35 40 45Thr Ala Leu Ser Lys Ile Gly Glu Lys Ser Leu Phe Thr Lys Glu Leu 50 55 60Glu His Ala Leu Glu Lys Asn Glu Val Asp Leu Val Val His Ser Leu65 70 75 80Lys Asp Leu Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala Ile 85 90 95Cys Lys Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys Phe 100 105 110Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val Val Gly Thr 115 120 125Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg Lys Phe Pro His Leu 130 135 140Glu Phe Arg Ser Ile Arg Gly Asn Leu Asn Thr Arg Leu Arg Lys Met145 150 155 160Asp Glu Gln Gln Glu Phe Ser Ala Ile Ile Leu Ala Thr Ala Gly Leu 165 170 175Gln Arg Met Gly Trp His Asn Arg Val Gly Gln Ile Leu His Pro Glu 180 185 190Glu Cys Met Tyr Ala Val Gly Gln Gly Ala Leu Gly Val Glu Val Arg 195 200 205Ala Lys Asp Gln Asp Ile Leu Asp Leu Val Gly Val Leu His Asp Pro 210 215 220Glu Thr Leu Leu Arg Cys Ile Ala Glu Arg Ala Phe Leu Arg His Leu225 230 235 240Glu Gly Gly Cys Ser Val Pro Val Ala Val His Thr Ala Met Lys Asp 245 250 255Gly Gln Leu Tyr Leu Thr Gly Gly Val Trp Ser Leu Asp Gly Ser Asp 260 265 270Ser Ile Gln Glu Thr Met Gln Ala Thr Ile His Val Pro Ala Gln His 275 280 285Glu Asp Gly Pro Glu Asp Asp Pro Gln Leu Val Gly Ile Thr Ala Arg 290 295 300Asn Ile Pro Arg Gly Pro Gln Leu Ala Ala Gln Asn Leu Gly Ile Ser305 310 315 320Leu Ala Asn Leu Leu Leu Ser Lys Gly Ala Lys Asn Ile Leu Asp Val 325 330 335Ala Arg Gln Leu Asn Asp Ala His 340162022DNAHomo sapiens 16ccggtaccgg ctcctcctgg gctccctcta gcgccttccc cccggcccga ctgcctggtc 60agcgccaagt gacttacgcc cccgaccctg agcccggacc gctaggcgag gaggatcaga 120tctccgctcg agaatctgaa ggtgccctgg tcctggagga gttccgtccc agccctgcgg 180tctcccggta ctgctcgccc cggccctctg gagcttcagg aggcggccgt cagggtcggg 240gagtatttgg gtccggggtc tcagggaagg gcggcgcctg ggtctgcggt atcggaaaga 300gcctgctgga gccaagtagc cctccctctc ttgggacaga cccctcggtc ccatgtccat 360gggggcaccg cggtccctcc tcctggccct ggctgctggc ctggccgttg cccgtccgcc 420caacatcgtg ctgatctttg ccgacgacct cggctatggg gacctgggct gctatgggca 480ccccagctct accactccca acctggacca gctggcggcg ggagggctgc ggttcacaga 540cttctacgtg cctgtgtctc tgtgcacacc ctctagggcc gccctcctga ccggccggct 600cccggttcgg atgggcatgt accctggcgt cctggtgccc agctcccggg ggggcctgcc 660cctggaggag gtgaccgtgg ccgaagtcct ggctgcccga ggctacctca caggaatggc 720cggcaagtgg caccttgggg tggggcctga gggggccttc ctgccccccc atcagggctt 780ccatcgattt ctaggcatcc cgtactccca cgaccagggc ccctgccaga acctgacctg 840cttcccgccg gccactcctt gcgacggtgg ctgtgaccag ggcctggtcc ccatcccact 900gttggccaac ctgtccgtgg aggcgcagcc cccctggctg cccggactag aggcccgcta 960catggctttc gcccatgacc tcatggccga cgcccagcgc caggatcgcc ccttcttcct 1020gtactatgcc tctcaccaca cccactaccc tcagttcagt gggcagagct ttgcagagcg 1080ttcaggccgc gggccatttg gggactccct gatggagctg gatgcagctg tggggaccct 1140gatgacagcc ataggggacc tggggctgct tgaagagacg ctggtcatct tcactgcaga 1200caatggacct gagaccatgc gtatgtcccg aggcggctgc tccggtctct tgcggtgtgg 1260aaagggaacg acctacgagg gcggtgtccg agagcctgcc ttggccttct ggccaggtca 1320tatcgctccc ggcgtgaccc acgagctggc cagctccctg gacctgctgc ctaccctggc 1380agccctggct ggggccccac tgcccaatgt caccttggat ggctttgacc tcagccccct 1440gctgctgggc acaggcaaga gccctcggca gtctctcttc ttctacccgt cctacccaga 1500cgaggtccgt ggggtttttg ctgtgcggac tggaaagtac aaggctcact tcttcaccca 1560gggctctgcc cacagtgata ccactgcaga ccctgcctgc cacgcctcca gctctctgac 1620tgctcatgag cccccgctgc

tctatgacct gtccaaggac cctggtgaga actacaacct 1680gctggggggt gtggccgggg ccaccccaga ggtgctgcaa gccctgaaac agcttcagct 1740gctcaaggcc cagttagacg cagctgtgac cttcggcccc agccaggtgg cccggggcga 1800ggaccccgcc ctgcagatct gctgtcatcc tggctgcacc ccccgcccag cttgctgcca 1860ttgcccagat ccccatgcct gagggcccct cggctggcct gggcatgtga tggctcctca 1920ctgggagcct gtgggggagg ctcaggtgtc tggagggggt ttgtgcctga taacgtaata 1980acaccagtgg agacttgcac atctgaaaaa aaaaaaaaaa aa 2022171524DNAHomo sapiens 17atgggggcac cgcggtccct cctcctggcc ctggctgctg gcctggccgt tgcacgtccg 60cccaacatcg tgctgatctt tgccgacgac ctcggctatg gggacctggg ctgctatggg 120caccccagct ctaccactcc caacctggac cagctggcgg cgggagggct gcggttcaca 180gacttctacg tgcctgtgtc tctgtgcaca ccctctaggg ccgccctcct gaccggccgg 240ctcccggttc ggatgggcat gtaccctggc gtcctggtgc ccagctcccg ggggggcctg 300cccctggagg aggtgaccgt ggccgaagtc ctggctgccc gaggctacct cacaggaatg 360gccggcaagt ggcaccttgg ggtggggcct gagggggcct tcctgccccc ccatcagggc 420ttccatcgat ttctaggcat cccgtactcc cacgaccagg gcccctgcca gaacctgacc 480tgcttcccgc cggccactcc ttgcgacggt ggctgtgacc agggcctggt ccccatccca 540ctgttggcca acctgtccgt ggaggcgcag cccccctggc tgcccggact agaggcccgc 600tacatggctt tcgcccatga cctcatggcc gacgcccagc gccaggatcg ccccttcttc 660ctgtactatg cctctcacca cacccactac cctcagttca gtgggcagag ctttgcagag 720cgttcaggcc gcgggccatt tggggactcc ctgatggagc tggatgcagc tgtggggacc 780ctgatgacag ccatagggga cctggggctg cttgaagaga cgctggtcat cttcactgca 840gacaatggac ctgagaccat gcgtatgtcc cgaggcggct gctccggtct cttgcggtgt 900ggaaagggaa cgacctacga gggcggtgtc cgagagcctg ccttggcctt ctggccaggt 960catatcgctc ccggcgtgac ccacgagctg gccagctccc tggacctgct gcctaccctg 1020gcagccctgg ctggggcccc actgcccaat gtcaccttgg atggctttga cctcagcccc 1080ctgctgctgg gcacaggcaa gagccctcgg cagtctctct tcttctaccc gtcctaccca 1140gacgaggtcc gtggggtttt tgctgtgcgg actggaaagt acaaggctca cttcttcacc 1200cagggctctg cccacagtga taccactgca gaccctgcct gccacgcctc cagctctctg 1260actgctcatg agcccccgct gctctatgac ctgtccaagg accctggtga gaactacaac 1320ctgctggggg gtgtggccgg ggccacccca gaggtgctgc aagccctgaa acagcttcag 1380ctgctcaagg cccagttaga cgcagctgtg accttcggcc ccagccaggt ggcccggggc 1440gaggaccccg ccctgcagat ctgctgtcat cctggctgca ccccccgccc agcttgctgc 1500cattgcccag atccccatgc ctga 152418507PRTHomo sapiens 18Met Gly Ala Pro Arg Ser Leu Leu Leu Ala Leu Ala Ala Gly Leu Ala1 5 10 15Val Ala Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly 20 25 30Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn 35 40 45Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val 50 55 60Pro Val Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg65 70 75 80Leu Pro Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser 85 90 95Arg Gly Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala 100 105 110Ala Arg Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val 115 120 125Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe 130 135 140Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr145 150 155 160Cys Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu 165 170 175Val Pro Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro 180 185 190Trp Leu Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu 195 200 205Met Ala Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala 210 215 220Ser His His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu225 230 235 240Arg Ser Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala 245 250 255Ala Val Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu 260 265 270Glu Thr Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg 275 280 285Met Ser Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr 290 295 300Thr Tyr Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly305 310 315 320His Ile Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu 325 330 335Leu Pro Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr 340 345 350Leu Asp Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser 355 360 365Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg 370 375 380Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr385 390 395 400Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala 405 410 415Ser Ser Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser 420 425 430Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala 435 440 445Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala 450 455 460Gln Leu Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly465 470 475 480Glu Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg 485 490 495Pro Ala Cys Cys His Cys Pro Asp Pro His Ala 500 50519489PRTHomo sapiensMOD_RES(51)..(51)C-alpha Formylglycine 19Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly Tyr Gly1 5 10 15Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn Leu Asp 20 25 30Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val Pro Val 35 40 45Ser Leu Xaa Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro 50 55 60Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly65 70 75 80Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala Ala Arg 85 90 95Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val Gly Pro 100 105 110Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe Leu Gly 115 120 125Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr Cys Phe 130 135 140Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu Val Pro145 150 155 160Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro Trp Leu 165 170 175Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu Met Ala 180 185 190Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala Ser His 195 200 205His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu Arg Ser 210 215 220Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala Ala Val225 230 235 240Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu Glu Thr 245 250 255Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg Met Ser 260 265 270Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr Thr Tyr 275 280 285Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly His Ile 290 295 300Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu Leu Pro305 310 315 320Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr Leu Asp 325 330 335Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser Pro Arg 340 345 350Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg Gly Val 355 360 365Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr Gln Gly 370 375 380Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala Ser Ser385 390 395 400Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser Lys Asp 405 410 415Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala Thr Pro 420 425 430Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala Gln Leu 435 440 445Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly Glu Asp 450 455 460Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg Pro Ala465 470 475 480Cys Cys His Cys Pro Asp Pro His Ala 48520489PRTHomo sapiens 20Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly Tyr Gly1 5 10 15Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn Leu Asp 20 25 30Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val Pro Val 35 40 45Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro 50 55 60Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly65 70 75 80Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala Ala Arg 85 90 95Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val Gly Pro 100 105 110Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe Leu Gly 115 120 125Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr Cys Phe 130 135 140Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu Val Pro145 150 155 160Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro Trp Leu 165 170 175Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu Met Ala 180 185 190Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala Ser His 195 200 205His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu Arg Ser 210 215 220Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala Ala Val225 230 235 240Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu Glu Thr 245 250 255Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg Met Ser 260 265 270Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr Thr Tyr 275 280 285Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly His Ile 290 295 300Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu Leu Pro305 310 315 320Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr Leu Asp 325 330 335Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser Pro Arg 340 345 350Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg Gly Val 355 360 365Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr Gln Gly 370 375 380Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala Ser Ser385 390 395 400Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser Lys Asp 405 410 415Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala Thr Pro 420 425 430Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala Gln Leu 435 440 445Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly Glu Asp 450 455 460Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg Pro Ala465 470 475 480Cys Cys His Cys Pro Asp Pro His Ala 485211011PRTHomo sapiens 21Met Gly Ala Tyr Ala Arg Ala Ser Gly Val Cys Ala Arg Gly Cys Leu1 5 10 15Asp Ser Ala Gly Pro Trp Thr Met Ser Arg Ala Leu Arg Pro Pro Leu 20 25 30Pro Pro Leu Cys Phe Phe Leu Leu Leu Leu Ala Ala Ala Gly Ala Arg 35 40 45Ala Gly Gly Tyr Glu Thr Cys Pro Thr Val Gln Pro Asn Met Leu Asn 50 55 60Val His Leu Leu Pro His Thr His Asp Asp Val Gly Trp Leu Lys Thr65 70 75 80Val Asp Gln Tyr Phe Tyr Gly Ile Lys Asn Asp Ile Gln His Ala Gly 85 90 95Val Gln Tyr Ile Leu Asp Ser Val Ile Ser Ala Leu Leu Ala Asp Pro 100 105 110Thr Arg Arg Phe Ile Tyr Val Glu Ile Ala Phe Phe Ser Arg Trp Trp 115 120 125His Gln Gln Thr Asn Ala Thr Gln Glu Val Val Arg Asp Leu Val Arg 130 135 140Gln Gly Arg Leu Glu Phe Ala Asn Gly Gly Trp Val Met Asn Asp Glu145 150 155 160Ala Ala Thr His Tyr Gly Ala Ile Val Asp Gln Met Thr Leu Gly Leu 165 170 175Arg Phe Leu Glu Asp Thr Phe Gly Asn Asp Gly Arg Pro Arg Val Ala 180 185 190Trp His Ile Asp Pro Phe Gly His Ser Arg Glu Gln Ala Ser Leu Phe 195 200 205Ala Gln Met Gly Phe Asp Gly Phe Phe Phe Gly Arg Leu Asp Tyr Gln 210 215 220Asp Lys Trp Val Arg Met Gln Lys Leu Glu Met Glu Gln Val Trp Arg225 230 235 240Ala Ser Thr Ser Leu Lys Pro Pro Thr Ala Asp Leu Phe Thr Gly Val 245 250 255Leu Pro Asn Gly Tyr Asn Pro Pro Arg Asn Leu Cys Trp Asp Val Leu 260 265 270Cys Val Asp Gln Pro Leu Val Glu Asp Pro Arg Ser Pro Glu Tyr Asn 275 280 285Ala Lys Glu Leu Val Asp Tyr Phe Leu Asn Val Ala Thr Ala Gln Gly 290 295 300Arg Tyr Tyr Arg Thr Asn His Thr Val Met Thr Met Gly Ser Asp Phe305 310 315 320Gln Tyr Glu Asn Ala Asn Met Trp Phe Lys Asn Leu Asp Lys Leu Ile 325 330 335Arg Leu Val Asn Ala Gln Gln Ala Lys Gly Ser Ser Val His Val Leu 340 345 350Tyr Ser Thr Pro Ala Cys Tyr Leu Trp Glu Leu Asn Lys Ala Asn Leu 355 360 365Thr Trp Ser Val Lys His Asp Asp Phe Phe Pro Tyr Ala Asp Gly Pro 370 375 380His Gln Phe Trp Thr Gly Tyr Phe Ser Ser Arg Pro Ala Leu Lys Arg385 390 395 400Tyr Glu Arg Leu Ser Tyr Asn Phe Leu Gln Val Cys Asn Gln Leu Glu 405 410 415Ala Leu Val Gly Leu Ala Ala Asn Val Gly Pro Tyr Gly Ser Gly Asp 420 425 430Ser Ala Pro Leu Asn Glu Ala Met Ala Val Leu Gln His His Asp Ala 435 440 445Val Ser Gly Thr Ser Arg Gln His Val Ala Asn Asp Tyr Ala Arg Gln 450 455 460Leu Ala Ala Gly Trp Gly Pro Cys Glu Val Leu Leu Ser Asn Ala Leu465 470 475 480Ala Arg Leu Arg Gly Phe Lys Asp His Phe Thr Phe Cys Gln Gln Leu 485 490 495Asn Ile Ser Ile Cys Pro Leu Ser Gln Thr Ala Ala Arg Phe Gln Val 500 505 510Ile Val Tyr Asn Pro Leu Gly Arg Lys Val Asn Trp Met Val Arg Leu 515 520 525Pro Val Ser Glu Gly Val Phe Val Val Lys Asp Pro Asn Gly Arg Thr 530 535 540Val Pro Ser Asp Val Val Ile Phe Pro Ser Ser Asp Ser Gln Ala His545 550 555 560Pro Pro Glu Leu Leu Phe Ser Ala Ser Leu Pro Ala Leu Gly Phe Ser 565 570 575Thr Tyr Ser Val Ala Gln Val Pro Arg Trp Lys Pro Gln Ala Arg Ala 580 585 590Pro Gln Pro Ile Pro Arg Arg Ser Trp Ser Pro Ala Leu Thr Ile Glu 595 600 605Asn Glu His Ile Arg Ala Thr Phe Asp Pro Asp Thr Gly Leu Leu Met 610 615 620Glu Ile Met Asn Met Asn Gln Gln Leu Leu Leu Pro Val Arg Gln Thr625 630 635 640Phe Phe Trp Tyr Asn Ala Ser Ile Gly Asp Asn Glu Ser Asp Gln Ala

645 650 655Ser Gly Ala Tyr Ile Phe Arg Pro Asn Gln Gln Lys Pro Leu Pro Val 660 665 670Ser Arg Trp Ala Gln Ile His Leu Val Lys Thr Pro Leu Val Gln Glu 675 680 685Val His Gln Asn Phe Ser Ala Trp Cys Ser Gln Val Val Arg Leu Tyr 690 695 700Pro Gly Gln Arg His Leu Glu Leu Glu Trp Ser Val Gly Pro Ile Pro705 710 715 720Val Gly Asp Thr Trp Gly Lys Glu Val Ile Ser Arg Phe Asp Thr Pro 725 730 735Leu Glu Thr Lys Gly Arg Phe Tyr Thr Asp Ser Asn Gly Arg Glu Ile 740 745 750Leu Glu Arg Arg Arg Asp Tyr Arg Pro Thr Trp Lys Leu Asn Gln Thr 755 760 765Glu Pro Val Ala Gly Asn Tyr Tyr Pro Val Asn Thr Arg Ile Tyr Ile 770 775 780Thr Asp Gly Asn Met Gln Leu Thr Val Leu Thr Asp Arg Ser Gln Gly785 790 795 800Gly Ser Ser Leu Arg Asp Gly Ser Leu Glu Leu Met Val His Arg Arg 805 810 815Leu Leu Lys Asp Asp Gly Arg Gly Val Ser Glu Pro Leu Met Glu Asn 820 825 830Gly Ser Gly Ala Trp Val Arg Gly Arg His Leu Val Leu Leu Asp Thr 835 840 845Ala Gln Ala Ala Ala Ala Gly His Arg Leu Leu Ala Glu Gln Glu Val 850 855 860Leu Ala Pro Gln Val Val Leu Ala Pro Gly Gly Gly Ala Ala Tyr Asn865 870 875 880Leu Gly Ala Pro Pro Arg Thr Gln Phe Ser Gly Leu Arg Arg Asp Leu 885 890 895Pro Pro Ser Val His Leu Leu Thr Leu Ala Ser Trp Gly Pro Glu Met 900 905 910Val Leu Leu Arg Leu Glu His Gln Phe Ala Val Gly Glu Asp Ser Gly 915 920 925Arg Asn Leu Ser Ala Pro Val Thr Leu Asn Leu Arg Asp Leu Phe Ser 930 935 940Thr Phe Thr Ile Thr Arg Leu Gln Glu Thr Thr Leu Val Ala Asn Gln945 950 955 960Leu Arg Glu Ala Ala Ser Arg Leu Lys Trp Thr Thr Asn Thr Gly Pro 965 970 975Thr Pro His Gln Thr Pro Tyr Gln Leu Asp Pro Ala Asn Ile Thr Leu 980 985 990Glu Pro Met Glu Ile Arg Thr Phe Leu Ala Ser Val Gln Trp Lys Glu 995 1000 1005Val Asp Gly 1010228079DNAArtificial SequenceDescription of Artificial Sequence Synthetic expression plasmid pLamanExp1 22agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840tgcagtgact ctcttaaggt agccttgcag aagttggtcg tgaggcactg ggcaggtaag 900tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag 960aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct 1020ctccacaggt gtccactccc agttcaatta cagctcttaa ggctagagta cttaatacga 1080ctcactatag gctagcctcg agaattcgcc gccatgggcg cctacgcgcg ggcttcgggg 1140gtctgcgctc gaggctgcct ggactcagca ggcccctgga ccatgtcccg cgccctgcgg 1200ccaccgctcc cgcctctctg ctttttcctt ttgttgctgg cggctgccgg tgctcgggcc 1260gggggatacg agacatgccc cacagtgcag ccgaacatgc tgaacgtgca cctgctgcct 1320cacacacatg atgacgtggg ctggctcaaa accgtggacc agtactttta tggaatcaag 1380aatgacatcc agcacgccgg tgtgcagtac atcctggact cggtcatctc tgccttgctg 1440gcagatccca cccgtcgctt catttacgtg gagattgcct tcttctcccg ttggtggcac 1500cagcagacaa atgccacaca ggaagtcgtg cgagaccttg tgcgccaggg gcgcctggag 1560ttcgccaatg gtggctgggt gatgaacgat gaggcagcca cccactacgg tgccatcgtg 1620gaccagatga cacttgggct gcgctttctg gaggacacat ttggcaatga tgggcgaccc 1680cgtgtggcct ggcacattga ccccttcggc cactctcggg agcaggcctc gctgtttgcg 1740cagatgggct tcgacggctt cttctttggg cgccttgatt atcaagataa gtgggtacgg 1800atgcagaagc tggagatgga gcaggtgtgg cgggccagca ccagcctgaa gcccccgacc 1860gcggacctct tcactggtgt gcttcccaat ggttacaacc cgccaaggaa tctgtgctgg 1920gatgtgctgt gtgtcgatca gccgctggtg gaggaccctc gcagccccga gtacaacgcc 1980aaggagctgg tcgattactt cctaaatgtg gccactgccc agggccggta ttaccgcacc 2040aaccacactg tgatgaccat gggctcggac ttccaatatg agaatgccaa catgtggttc 2100aagaaccttg acaagctcat ccggctggta aatgcgcagc aggcaaaagg aagcagtgtc 2160catgttctct actccacccc cgcttgttac ctctgggagc tgaacaaggc caacctcacc 2220tggtcagtga aacatgacga cttcttccct tacgcggatg gcccccacca gttctggacc 2280ggttactttt ccagtcggcc ggccctcaaa cgctacgagc gcctcagcta caacttcctg 2340caggtgtgca accagctgga ggcgctggtg ggcctggcgg ccaacgtggg accctatggc 2400tccggagaca gtgcacccct caatgaggcg atggctgtgc tccagcatca cgacgccgtc 2460agcggcacct cccgccagca cgtggccaac gactacgcgc gccagcttgc ggcaggctgg 2520gggccttgcg aggttcttct gagcaacgcg ctggcgcggc tcagaggctt caaagatcac 2580ttcacctttt gccaacagct aaacatcagc atctgcccgc tcagccagac ggcggcgcgc 2640ttccaggtca tcgtttataa tcccctgggg cggaaggtga attggatggt acggctgccg 2700gtcagcgaag gcgttttcgt tgtgaaggac cccaatggca ggacagtgcc cagcgatgtg 2760gtaatatttc ccagctcaga cagccaggcg caccctccgg agctgctgtt ctcagcctca 2820ctgcccgccc tgggcttcag cacctattca gtagcccagg tgcctcgctg gaagccccag 2880gcccgcgcac cacagcccat ccccagaaga tcctggtccc ctgctttaac catcgaaaat 2940gagcacatcc gggcaacgtt tgatcctgac acagggctgt tgatggagat tatgaacatg 3000aatcagcaac tcctgctgcc tgttcgccag accttcttct ggtacaacgc cagtataggt 3060gacaacgaaa gtgaccaggc ctcaggtgcc tacatcttca gacccaacca acagaaaccg 3120ctgcctgtga gccgctgggc tcagatccac ctggtgaaga cacccttggt gcaggaggtg 3180caccagaact tctcagcttg gtgttcccag gtggttcgcc tgtacccagg acagcggcac 3240ctggagctag agtggtcggt ggggccgata cctgtgggcg acacctgggg gaaggaggtc 3300atcagccgtt ttgacacacc gctggagaca aagggacgct tctacacaga cagcaatggc 3360cgggagatcc tggagaggag gcgggattat cgacccacct ggaaactgaa ccagacggag 3420cccgtggcag gaaactacta tccagtcaac acccggattt acatcacgga tggaaacatg 3480cagctgactg tgctgactga ccgctcccag gggggcagca gcctgagaga tggctcgctg 3540gagctcatgg tgcaccgaag gctgctgaag gacgatggac gcggagtatc ggagccacta 3600atggagaacg ggtcgggggc gtgggtgcga gggcgccacc tggtgctgct ggacacagcc 3660caggctgcag ccgccggaca ccggctcctg gcggagcagg aggtcctggc ccctcaggtg 3720gtgctggccc cgggtggcgg cgccgcctac aatctcgggg ctcctccgcg cacgcagttc 3780tcagggctgc gcagggacct gccgccctcg gtgcacctgc tcacgctggc cagctggggc 3840cccgaaatgg tgctgctgcg cttggagcac cagtttgccg taggagagga ttccggacgt 3900aacctgagcg cccccgttac cttgaacttg agggacctgt tctccacctt caccatcacc 3960cgcctgcagg agaccacgct ggtggccaac cagctccgcg aggcagcctc caggctcaag 4020tggacaacaa acacaggccc cacaccccac caaactccgt accagctgga cccggccaac 4080atcacgctgg aacccatgga aatccgcact ttcctggcct cagttcaatg gaaggaggtg 4140gatggttagg tctgctggga tgggccctct agagtcgacc cgggcggccg cttcccttta 4200gtgagggtta atgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac 4260aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 4320tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 4380tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 4440taaaatccga taaggatcga tccgggctgg cgtaatagcg aagaggcccg caccgatcgc 4500ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa 4560gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 4620ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 4680ctctaaatcg ggggctccct ttagggttcc gatttagagc tttacggcac ctcgaccgca 4740aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 4800gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 4860cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct 4920attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 4980gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca 5040tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 5100gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 5160tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 5220ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 5280gcttttttgg aggcctaggc ttttgcaaaa agctcccggg atggttcgac cattgaactg 5340catcgtcgcc gtgtcccaaa atatggggat tggcaagaac ggagacctac cctggcctcc 5400gctcaggaac gagttcaagt acttccaaag aatgaccaca acctcttcag tggaaggtaa 5460acagaatctg gtgattatgg gtaggaaaac ctggttctcc attcctgaga agaatcgacc 5520tttaaaggac agaattaata tagttctcag tagagaactc aaagaaccac cacgaggagc 5580tcattttctt gccaaaagtt tggatgatgc cttaagactt attgaacaac cggaattggc 5640aagtaaagta gacatggttt ggatagtcgg aggcagttct gtttaccagg aagccatgaa 5700tcaaccaggc caccttagac tctttgtgac aaggatcatg caggaatttg aaagtgacac 5760gtttttccca gaaattgatt tggggaaata taaacttctc ccagaatacc caggcgtcct 5820ctctgaggtc caggaggaaa aaggcatcaa gtataagttt gaagtctacg agaagaaaga 5880ctaattcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc cgcaataaaa 5940tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gcgataagga 6000tccgcgtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 6060gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 6120acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 6180cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 6240taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 6300tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 6360aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 6420ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6480aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 6540acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 6600ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6660gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6720atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 6780acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 6840tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 6900ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6960aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 7020aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 7080ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 7140atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 7200aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 7260accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 7320tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 7380tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7440tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7500cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7560caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7620cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7680cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7740gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7800acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7860atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7920cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7980gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 8040tcctggcctt ttgctggcct tttgctcaca tggctcgac 8079233761DNAHomo sapiens 23ggctactctc ggcttcctgg caacgccgag cgaaagctat gactgcggcc gcgggttcgg 60cgggccgcgc cgcggtgccc ttgctgctgt gtgcgctgct ggcgcccggc ggcgcgtacg 120tgctcgacga ctccgacggg ctgggccggg agttcgacgg catcggcgcg gtcagcggcg 180gcggggcaac ctcccgactt ctagtaaatt acccagagcc ctatcgttct cagatattgg 240attatctctt taagccgaat tttggtgcct ctttgcatat tttaaaagtg gaaataggtg 300gtgatgggca gacaacagac ggcactgagc cctcccacat gcattatgca ctagatgaga 360attatttccg aggatacgag tggtggttga tgaaagaagc taagaagagg aatcccaata 420ttacactcat tgggttgcca tggtcattcc ctggatggct gggaaaaggt ttcgactggc 480cttatgtcaa tcttcagctg actgcctatt atgtcgtgac ctggattgtg ggcgccaagc 540gttaccatga tttggacatt gattatattg gaatttggaa tgagaggtca tataatgcca 600attatattaa gatattaaga aaaatgctga attatcaagg tctccagcga gtgaaaatca 660tagcaagtga taatctctgg gagtccatct ctgcatccat gctccttgat gccgaactct 720tcaaggtggt tgatgttata ggggctcatt atcctggaac ccattcagca aaagatgcaa 780agttgactgg gaagaagctt tggtcttctg aagactttag cactttaaat agtgacatgg 840gtgcaggctg ctggggtcgc attttaaatc agaattatat caatggctat atgacttcca 900caatcgcatg gaatttagtg gctagttact atgaacagtt gccttatggg agatgcgggt 960tgatgacggc ccaagagcca tggagtgggc actacgtggt agaatctcct gtctgggtat 1020cagctcatac cactcagttt actcaacctg gctggtatta cctgaagaca gttggccatt 1080tagagaaagg aggaagctac gtagctctga ctgatggctt agggaacctc accatcatca 1140ttgaaaccat gagtcataaa cattctaagt gcatacggcc atttcttcct tatttcaatg 1200tgtcacaaca atttgccacc tttgttctta agggatcttt tagtgaaata ccagagctac 1260aggtatggta taccaaactt ggaaaaacat ccgaaagatt tctttttaag cagctggatt 1320ctctatggct ccttgacagc gatggcagtt tcacactgag cctgcatgaa gatgagctgt 1380tcacactcac cactctcacc actggtcgca aaggcagcta cccgcttcct ccaaaatccc 1440agcccttccc aagtacctat aaggatgatt tcaatgttga ttacccattt tttagtgaag 1500ctccaaactt tgctgatcaa actggtgtat ttgaatattt tacaaatatt gaagaccctg 1560gcgagcatca cttcacgcta cgccaagttc tcaaccagag acccattacg tgggctgccg 1620atgcatccaa cacaatcagt attataggag actacaactg gaccaatctg actataaagt 1680gtgatgttta catagagacc cctgacacag gaggtgtgtt cattgcagga agagtaaata 1740aaggtggtat tttgattaga agtgccagag gaattttctt ctggattttt gcaaatggat 1800cttacagggt tacaggtgat ttagctggat ggattatata tgctttagga cgtgttgaag 1860ttacagcaaa aaaatggtat acactcacgt taactattaa gggtcatttc gcctctggca 1920tgctgaatga caagtctctg tggacagaca tccctgtgaa ttttccaaag aatggctggg 1980ctgcaattgg aactcactcc tttgaatttg cacagtttga caactttctt gtggaagcca 2040cacgctaata cttaacaggg catcatagaa tactctggat tttcttccct tctttttggt 2100tttggttcag agccaattct tgtttcattg gaacagtata tgaggctttt gagactaaaa 2160ataatgaaga gtaaaagggg agagaaattt atttttaatt taccctgtgg aagattttat 2220tagaattaat tccaagggga aaactggtga atctttaaca ttacctggtg tgttccctaa 2280cattcaaact gtgcattggc cataccctta ggagtggttt gagtagtaca gacctcgaag 2340ccttgctgct aacactgagg tagctctctt catcttattt gcaagcggtc ctgtagatgg 2400cagtaacttg atcatcactg agatgtattt atgcatgctg accgtgtgtc caagtgagcc 2460agtgtcttca tcacaagatg atgctgccat aatagaaagc tgaagaacac tagaagtagc 2520tttttgaaaa ccacttcaac ctgttatgct ttatgctcta aaaagtattt ttttattttc 2580ctttttaaga tgatactttt gaaatgcagg atatgatgag tgggatgatt ttaaaaacgc 2640ctctttaata aactacctct aacactattt ctgcggtaat agatattagc agattaattg 2700ggttatttgc attatttaat ttttttgatt ccaagttttg gtcttgtaac cactataact 2760ctctgtgaac gtttttccag gtggctggaa gaaggaagaa aacctgatat agccaatgct 2820gttgtagtcg tttcctcagc ctcatctcac tgtgctgtgg tctgtcctca catgtgcact 2880ggtaacagac tcacacagct gatgaatgct tttctctcct tatgtgtgga aggaggggag 2940cacttagaca tttgctaact cccagaattg gatcatctcc taagatgtac ttacttttta 3000aagtccaaat atgtttatat ttaaatatac gtgagcatgt tcatcatgtt gtatgattta 3060tactaagcat taatgtggct ctatgtagca aatcagttat tcatgtaggt aaagtaaatc 3120tagaattatt tataagaatt actcattgaa ctaattctac tatttaggaa tttataagag 3180tctaacatag gcttagctac agtgaagttt tgcattgctt ttgaagacaa gaaaagtgct 3240agaataaata agattacaga gaaaattttt tgttaaaacc aagtgatttc cagctgatgt 3300atctaatatt ttttaaaaca aacattatag aggtgtaatt tatttacaat aaaatgttcc 3360tactttaaat atacaattca gtgagttttg ataaattgat atacccatgt aaccaacact 3420ccagtcaagc ttcagaatat ttccatcacc ccagaaggtt ctcttgtata cctgctcagt 3480cagttccttt cactcccaat tgttggcagc cattgatagg aattctatca ctataggtta 3540gttttctttg ttccagaaca tcatgaaagc ggcgtcatgt actgtgtatt cttatgaatg 3600gtttctttcc atcagcataa tgatttgaga ttggtccatg ttgtgtgatt cagtggtttg 3660ttccttctta tttctgaaga gttttccatt gtatgaatat accacaattt gtttcctccc 3720caccagtttc tgatactaca attaaaactg tctacattta c 376124669PRTHomo sapiens 24Met Thr Ala Ala Ala Gly Ser Ala Gly Arg Ala Ala Val Pro Leu Leu1 5 10 15Leu Cys Ala Leu Leu Ala Pro Gly Gly Ala Tyr Val Leu Asp Asp Ser 20 25 30Asp Gly Leu Gly Arg Glu Phe Asp Gly Ile Gly Ala Val Ser Gly Gly 35 40 45Gly Ala Thr Ser Arg Leu Leu Val Asn Tyr Pro Glu Pro Tyr Arg Ser 50 55 60Gln Ile Leu Asp Tyr Leu Phe Lys Pro Asn Phe Gly Ala Ser Leu His65 70 75 80Ile Leu Lys Val Glu Ile Gly Gly Asp Gly Gln Thr Thr Asp Gly Thr 85 90 95Glu Pro Ser His Met His Tyr Ala Leu Asp Glu Asn Tyr Phe Arg Gly 100 105 110Tyr Glu Trp Trp Leu Met Lys Glu Ala Lys Lys Arg Asn Pro Asn Ile 115 120 125Thr Leu Ile Gly Leu Pro Trp Ser Phe Pro Gly Trp Leu Gly

Lys Gly 130 135 140Phe Asp Trp Pro Tyr Val Asn Leu Gln Leu Thr Ala Tyr Tyr Val Val145 150 155 160Thr Trp Ile Val Gly Ala Lys Arg Tyr His Asp Leu Asp Ile Asp Tyr 165 170 175Ile Gly Ile Trp Asn Glu Arg Ser Tyr Asn Ala Asn Tyr Ile Lys Ile 180 185 190Leu Arg Lys Met Leu Asn Tyr Gln Gly Leu Gln Arg Val Lys Ile Ile 195 200 205Ala Ser Asp Asn Leu Trp Glu Ser Ile Ser Ala Ser Met Leu Leu Asp 210 215 220Ala Glu Leu Phe Lys Val Val Asp Val Ile Gly Ala His Tyr Pro Gly225 230 235 240Thr His Ser Ala Lys Asp Ala Lys Leu Thr Gly Lys Lys Leu Trp Ser 245 250 255Ser Glu Asp Phe Ser Thr Leu Asn Ser Asp Met Gly Ala Gly Cys Trp 260 265 270Gly Arg Ile Leu Asn Gln Asn Tyr Ile Asn Gly Tyr Met Thr Ser Thr 275 280 285Ile Ala Trp Asn Leu Val Ala Ser Tyr Tyr Glu Gln Leu Pro Tyr Gly 290 295 300Arg Cys Gly Leu Met Thr Ala Gln Glu Pro Trp Ser Gly His Tyr Val305 310 315 320Val Glu Ser Pro Val Trp Val Ser Ala His Thr Thr Gln Phe Thr Gln 325 330 335Pro Gly Trp Tyr Tyr Leu Lys Thr Val Gly His Leu Glu Lys Gly Gly 340 345 350Ser Tyr Val Ala Leu Thr Asp Gly Leu Gly Asn Leu Thr Ile Ile Ile 355 360 365Glu Thr Met Ser His Lys His Ser Lys Cys Ile Arg Pro Phe Leu Pro 370 375 380Tyr Phe Asn Val Ser Gln Gln Phe Ala Thr Phe Val Leu Lys Gly Ser385 390 395 400Phe Ser Glu Ile Pro Glu Leu Gln Val Trp Tyr Thr Lys Leu Gly Lys 405 410 415Thr Ser Glu Arg Phe Leu Phe Lys Gln Leu Asp Ser Leu Trp Leu Leu 420 425 430Asp Ser Asp Gly Ser Phe Thr Leu Ser Leu His Glu Asp Glu Leu Phe 435 440 445Thr Leu Thr Thr Leu Thr Thr Gly Arg Lys Gly Ser Tyr Pro Leu Pro 450 455 460Pro Lys Ser Gln Pro Phe Pro Ser Thr Tyr Lys Asp Asp Phe Asn Val465 470 475 480Asp Tyr Pro Phe Phe Ser Glu Ala Pro Asn Phe Ala Asp Gln Thr Gly 485 490 495Val Phe Glu Tyr Phe Thr Asn Ile Glu Asp Pro Gly Glu His His Phe 500 505 510Thr Leu Arg Gln Val Leu Asn Gln Arg Pro Ile Thr Trp Ala Ala Asp 515 520 525Ala Ser Asn Thr Ile Ser Ile Ile Gly Asp Tyr Asn Trp Thr Asn Leu 530 535 540Thr Ile Lys Cys Asp Val Tyr Ile Glu Thr Pro Asp Thr Gly Gly Val545 550 555 560Phe Ile Ala Gly Arg Val Asn Lys Gly Gly Ile Leu Ile Arg Ser Ala 565 570 575Arg Gly Ile Phe Phe Trp Ile Phe Ala Asn Gly Ser Tyr Arg Val Thr 580 585 590Gly Asp Leu Ala Gly Trp Ile Ile Tyr Ala Leu Gly Arg Val Glu Val 595 600 605Thr Ala Lys Lys Trp Tyr Thr Leu Thr Leu Thr Ile Lys Gly His Phe 610 615 620Ala Ser Gly Met Leu Asn Asp Lys Ser Leu Trp Thr Asp Ile Pro Val625 630 635 640Asn Phe Pro Lys Asn Gly Trp Ala Ala Ile Gly Thr His Ser Phe Glu 645 650 655Phe Ala Gln Phe Asp Asn Phe Leu Val Glu Ala Thr Arg 660 6652511PRTArtificial SequenceDescription of Artificial Sequence Synthetic 11 residue basic peptide from HIV TAT protein 25Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 102611PRTArtificial SequenceDescription of Artificial Sequence Synthetic TAT peptide 26Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala1 5 10

Claims

1-28. (canceled)

29. A composition comprising 50-300 mg/ml Aryl sulfatase A for the manufacture of a medicament for subcutaneous injection into a mammal.

30. A method of treating a mammal for metachromatic leukodystrophy comprising a subcutaneous injection of a composition according to claim 29.

31. A composition comprising 50-300 mg/ml porphobilinogen deaminase for the manufacture of a medicament for subcutaneous injection into a mammal.

32. A method of treating a mammal for porphobilinogen deaminase acute intermittent porphyria comprising a subcutaneous injection of a composition according to claim 31.

33. A composition comprising 50-300 mg/ml alpha-mannosidase for the manufacture of a medicament for subcutaneous injection into a mammal.

34. A method of treating a mammal for the lysosomal storage disorder alpha-mannosidosis comprising a subcutaneous injection of a composition according to claim 33.

35. A composition comprising 50-300 mg/ml galactosylcerebrosidase for the manufacture of a medicament for subcutaneous injection into a mammal.

36. A method of treating a mammal for Krabbe disease comprising a subcutaneous injection of a composition according to claim 35.

37. A method of concentrating a composition comprising Aryl sulfatase A, functionally equivalent parts and analogues hereof, said method comprising: (i) centrifugation and/or filtration of a composition comprising Aryl sulfatase A and/or a functionally equivalent part or analogue hereof, to obtain a resulting solution; and (ii) concentrating said resulting solution.

38. A method of concentrating a composition comprising porphobilinogen deaminase, functionally equivalent parts and analogues hereof, said method comprising: (i) centrifugation and/or filtration of a composition comprising prophobilinogen deaminase and/or a functionally equivalent part or analogue hereof, to obtain a resulting solution; and (ii) concentrating said resulting solution.

39. A method of concentrating a composition comprising alfa mannosidase, functionally equivalent parts and analogues hereof, said method comprising: (i) centrifugation and/or filtration of a composition comprising alfa mannosidase and/or a functionally equivalent part or analogue hereof, to obtain a resulting solution; and (ii) concentrating said resulting solution.

40. A method of concentrating a composition comprising galactosylcerebrosidase, functionally equivalent parts and analogues hereof, said method comprising: (i) centrifugation and/or filtration of a composition comprising galactosylcerebrosidase and/or a functionally equivalent part or analogue hereof, to obtain a resulting solution; and (ii) concentrating said resulting solution.