TWI516597B - Manufacture of active highly phosphorylated human n-acetylgalactosamine-6-sulfatase and uses thereof - Google Patents

Description

Preparation of active highly phosphorylated human N-acetylgalactosamine-6-sulfatase and use thereof

The present invention relates to the field of cell and molecular biology and medical technology, in particular to the preparation of highly phosphorylated human lysosomal sulfatase enzymes and their management of lysosomal storage associated with lysosomal sulfatase deficiency Use in disease. In particular, the present invention relates to the preparation of active highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and its management of mucopolysaccharidosis IVa (MPS IVa or Moquio) Use of A syndrome) and other lysosomal storage diseases associated with GALNS deficiency.

Lysosomal storage diseases (LSDs) are caused by a lack of specific lysosomal enzymes necessary for cell waste degradation in lysosomes. The lack of such lysosomal enzymes causes undegraded "accumulation" to accumulate in the lysosome, thereby causing lysosomal swelling and dysfunction and ultimately leading to cell and tissue damage. Many lysosomal enzymes have been identified and associated with their associated diseases. Once the missing enzyme has been identified, treatment can be simplified to a single problem of efficient delivery of the replacement enzyme to the affected tissue of the patient.

One way to treat lysosomal storage diseases is intravenous enzyme replacement therapy (ERT) (Kakkis, Expert Opin. Investig. Drugs 11(5): 675-685, 2002). ERT uses vascular structures to transport enzymes from a single administration site to most tissues. Once the enzyme is widely distributed, it must be absorbed into the cells. The basis for uptake into cells is found in the unique characteristics of lysosomal enzymes. Lysosomal enzymes constitute a single class of glycoproteins defined by phosphates at the 6-position of the terminal mannose residue. Mannose-6-phosphate binds to receptors found on the surface of most cells with high affinity and specificity (Munier-Lehmann et al , Biochem. Soc. Trans . 24(1): 133-136, 1996; Marnell et al , J. Cell. Biol . 99(6): 1907-1916, 1984). Each of the polypeptide chains has two mannose-6-phosphate binding sites, the mannose-6-phosphate receptor (MPR) (Tong et al., J. Biol. Chem . 264:7962-7969, 1989). The uptake of the enzyme from the blood into the tissue then mediates intracellular delivery into the lysosome.

Large scale production of lysosomal enzymes involves expression in mammalian cell lines. The goal is to have the recombinase primarily secreted into the surrounding growth medium for collection and downstream processing. In an ideal system for large scale production of lysosomal enzymes, the enzyme will be highly phosphorylated and then directed primarily to the cell surface (i.e., for secretion) rather than primarily to lysosomes. As noted above, this partitioning of phosphorylated lysosomal enzymes is completely opposite to what occurs in normal cells. The preparation of cell lines for the production of lysosomal enzymes is concentrated in maximizing the content of mannose-6-phosphate per molase, but is characterized by a lower specific yield. In vitro attempts to produce lysosomal enzymes containing high levels of mannose-6-phosphate moieties have been successful (Canfield et al., U.S. Patent No. 6,537,785). In vitro enzymes exhibit high levels of mannose-6-phosphate and high levels of unmodified terminal mannose. Competition of lysosomal enzymes between mannose-6-phosphate and mannose receptors necessitates high dose enzymes to achieve efficacy and can result in greater immunogenicity of the lesions to the treated individual.

Sulfate esters constitute a unique class of lysosomal enzymes. Sulfate esters cleave sulfate from a variety of substrates including, for example, steroids, carbohydrates, proteoglycans, and glycolipids. All known eukaryotic sulfatase enzymes contain a cysteine residue at their catalytic site. After sulfatase activity requires this cysteine residue translational modified to C α _- carboxylic acyl glycine (FGly). Cysteine to FGly post-translational enzyme activation immediately after translation, occurs on the unfolded sulfatase in the endoplasmic reticulum before sulfatase targets lysosomes (Dierks et al, Proc. Natl. Acad. Sci. USA 94: 11963-11968, 1997). The methionine producing enzyme catalyzing this reaction is sulfatase modifying factor 1 (SUMF1). Mutations in SUMF1 that cause abnormal FGly formation in lysosomal sulfatase enzymes cause multiple Sulfate Deficiency (MSD) in humans to emphasize the importance of this unique post-translational modification (Diez-Ruiz et al., Annu Rev. Genomics Hum. Genet. 6:355-379, 2005).

Therefore, the therapeutic effectiveness of the lysosomal sulfatase preparation depends on the amount of mannose-6-phosphate in the preparation and the presence of the active enzyme.

Thus, the art is highly efficient in the preparation of therapeutically effective active highly phosphorylated lysosomal sulfatase enzymes for the management of lysosomal storage disorders caused by or associated with such lysosomal sulfatase deficiencies. There is a need for a prolific system.

The present invention relates to the discovery that when a CHO-K1 cell strain derivative (referred to as G71) having a defective endosome acidification is engineered to express recombinant human sulfatase modifying factor 1 (SUMF1), the modified G71 cell is partially borrowed. Highly productive, highly phosphorylated recombinant lysosomal sulfatase enzyme is produced by preventing the loss of material to the lysosomal compartment of the cell strain for preparation. In one embodiment, the invention provides an END3 complementation population cell line that co-expresses recombinant human SUMF1 with recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) to produce a high yield of active hyperphosphate Enzyme. Exemplary cell lines are G71, G71S and derivatives thereof, which retain the desired properties of G71, i.e., are capable of producing high yields of highly phosphorylated recombinant lysosomal sulfatase enzymes. This application of CHO-K1 cell lines, which together express the END3 complementation of recombinant human SUMF1 and recombinant lysosomal sulfatase enzymes, will be particularly useful for the preparation of lysosomal storage diseases to be managed by enzyme replacement therapy (ERT). Active highly phosphorylated lysosomal sulfatase.

In a first aspect, the invention features an active highly phosphorylated recombinant human lysosomal sulfatase enzyme or a biologically active fragment thereof, which is mutated in an amount of END3 complementary CHO cells or derivatives thereof, which is capable of achieving therapeutic use. A novel method of body, variant or derivative. In a broad embodiment, the method comprises the steps of: (a) cultivating CHO-derived END3 complementary population cells or derivatives thereof: (b) preparing for activity heights in CHO-derived END3 complementary population cells or derivatives thereof a first mammalian expression vector that phosphorylates a recombinant human lysosomal sulfatase enzyme or a biologically active fragment, mutant, variant or derivative thereof; (c) is capable of being produced in a CHO-derived END3 complementary population cell or derivative thereof a second mammalian expression vector representing recombinant human sulfatase modifying factor 1 (SUMF1) or a biologically active fragment, mutant, variant or derivative thereof; (d) transfected with CHO derived from the first and second expression vectors END3 complementary population of cells or derivatives thereof; (e) selection and selection of CHO-derived END3 complementary populations that express highly active recombinant human lysosomal sulfatase or biologically active fragments, mutants, variants or derivatives thereof Transfectants of cells or derivatives thereof; and (f) cell culture processing methods for the preparation of highly phosphorylated recombinant human lysosomal sulfatase or biologically active fragments, mutants, variants or derivatives thereof optimization. The recombinant human lysosomal sulfatase enzyme is selected from the group consisting of arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronate-2-sulfatase (IDS), sulfamidase/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-acetylgalactosamine-6 - sulfatase (GALNS).

The method involves the step of transfecting a cDNA encoding all or part of a lysosomal sulfatase enzyme and a cDNA encoding all or part of human SUMF1 into a CHO-derived END3 complementary population cell or derivative thereof. In some embodiments, the first and second expression vectors, each capable of expressing a highly active phosphorylated recombinant human lysosomal sulfatase enzyme and human SUMF1, are simultaneously transfected into a CHO-derived END3 complementary population cell or derivative thereof. . In some embodiments, the first and second expression vectors are sequentially transfected into CHO derived END3 complementary population cells or derivatives thereof. In some embodiments, a cDNA encoding a full length human lysosomal sulfatase enzyme is used, while in other embodiments, a cDNA encoding a biologically active fragment, mutant, variant or derivative thereof is used. In some embodiments, a cDNA encoding full length human SUMF1 is used, while in other embodiments, a cDNA encoding a biologically active fragment, mutant, variant or derivative thereof is used. In some embodiments, human lysosomal sulfatase and human SUMF1 cDNA are simultaneously or sequentially transferred to CHO-derived END3 complementation cells or derivatives thereof using a plurality of expression vectors. In some embodiments, human lysosomal sulfatase and human SUMF1 cDNA are simultaneously transferred into CHO-derived END3 complementary population cells or derivatives thereof using a single expression vector. In a preferred embodiment, the CHO-derived END3 complementary population cell or derivative thereof is a G71 cell line, a G71S cell line, or a G71 or G71S derivative.

In a preferred embodiment, the method comprises producing an active hyperphosphorylated recombinant human lysosomal sulfatase enzyme, such as arylsulfatase A (ARSA), aryl sulphate, from an END3 complementary population CHO cell line or a derivative thereof. Esterase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase ( G6S) or N-acetylgalactosamine-6-sulfatase (GALNS). In a particularly preferred embodiment, the method comprises producing an active hyperphosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) from an END3 complementary population CHO cell line or a derivative thereof. The END3 complementarity cell line is any modified CHO cell line that retains the properties of the END3 complementation cell line, such as defective endosomal acidification. In a preferred embodiment, the CHO-derived END3 complementary population cell or derivative thereof is a G71 cell line, a G71S cell line, or a G71 or G71S derivative.

In a second aspect, the invention provides a mammalian cell line deficient in endosomal acid, characterized in that it is capable of producing an amount of active hyperphosphorylated recombinant human lysozyme capable of achieving therapeutic use of a lysosomal sulfatase enzyme Body sulfatase. In a preferred embodiment, the present invention provides a CHO-K1-derived END3 complementary population cell line designated G71, G71S or a derivative thereof, which is capable of producing a high yield of active highly phosphorylated recombinant human lysosomal sulfatase enzyme, Thereby, large-scale production of such therapeutic lysosomal sulfatase enzymes can be achieved. In a more preferred embodiment, the cell line is at least about 0.5 pg/cell/day, preferably at least about 0.75 pg/cell/day, more preferably at least about 1.0 pg/cell/day and even more preferably at least about 1.25. The picogram/cell/day amount is expressed and secreted recombinant human lysosomal sulfatase.

The END3 complementarity cell line is any modified CHO cell line that retains the properties of the END3 complementation cell line, such as defective endosomal acidification. In one embodiment, the END3 complementary population CHO cell line is derived from G71 or a derivative thereof and comprises (a) a recombinant human sulfatase modifying factor 1 (SUMF1) expression vector and (b) a recombinant human lysosomal sulfatase enzyme A performance vector, wherein the recombinant human lysosomal sulfatase enzyme is selected from the group consisting of arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronic acid-2- Sulfate esterase (IDS), sulfonamide/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-acetylgalactosamine-6-sulfate Enzyme (GALNS). In a preferred embodiment, the END3 complementary population CHO cell line comprises a recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) expression vector. In a more preferred embodiment, the END3 complementary population CHO cell line expresses and secretes recombinant human GALNS. In another preferred embodiment, the END3 complementary population CHO cell line is selected from the group consisting of pure line 4, pure line 5, pure line C6, pure line C2, pure line C5, pure line C7, pure line C10, pure line C11, and pure line C30. In a more preferred embodiment, the END3 complementary population CHO cell line is pure line C2. In another preferred embodiment, the END3 complementary population CHO cell line is suitable for growth in suspension.

In a third aspect, the invention provides a recombinant human lysosomal sulfatase enzyme produced according to the methods of the invention and thereby present in an amount capable of achieving therapeutic use of a lysosomal sulfatase enzyme. The lysosomal sulfatase enzyme can be a full length protein or a fragment, mutant, variant or derivative thereof. In some embodiments, the lysosomal sulfatase enzyme of the invention, or a fragment, mutant, variant or derivative thereof, may be modified as necessary to enhance its stability or pharmacokinetic properties (eg, pegylation, mutation) Induction, fusion, and combination). In a preferred embodiment, the enzyme is a human lysosomal sulfatase enzyme, a human lysosomal sulfatase enzyme having a biologically active fragment of a natural sulfatase enzyme, or a substantial amine group with a human lysosomal sulfatase enzyme. A polypeptide having homology of acid sequence. In some embodiments, the lysosomal sulfatase enzyme is a protein derived or derived from a human or mammalian sequence. In other embodiments, the lysosomal sulfatase enzyme is an enzyme that causes a human disease such as Metachromic Leukodystrophy or MLD (ie, arylsulfatase A (ARSA)). , Maroteaux-Lamy syndrome or MPSVI (also known as arylsulfatase B (ARSB)), Hunter syndrome or MPS II (ie, iduronic acid-2-sulfate) Esterase (IDS), Sanfilippo A syndrome or MPS IIIa (also known as sulfamidase/heparin-N-sulfatase (SGSH)), St. Philippe D syndrome or MPS IIId (also That is, N-acetyl glucosamine-sulfatase (G6S) and Moquiao A syndrome or MPS IVa (i.e., N-acetylgalactosamine-6-sulfatase (GALNS)). In a particularly preferred embodiment, the lysosomal sulfatase enzyme lacks an enzyme that causes Moquia A syndrome or MPS IVa (i.e., N-acetylgalactosamine-6-sulfatase (GALNS)) . In another particularly preferred embodiment, the lysosomal sulfatase enzyme is an enzyme associated with a human disease such as a plurality of sulfatase deficiencies or MSD (ie, N-acetylgalactosamine-6-sulfate) Enzyme (GALNS)).

Lysosomal sulfatase enzymes may also be derived or derived from human or mammalian sequences. In other embodiments of the invention, in each aspect thereof, the amino acid sequence of the lysosomal sulfatase enzyme is identical to the corresponding portion of the human or mammalian lysosomal sulfatase amino acid sequence. In other embodiments, the polypeptide moiety is a natural lysosomal sulfatase enzyme from a human or a mammal. In other embodiments, the lysosomal sulfatase polypeptide is conjugated to a natural lysosomal sulfate of a human or mammalian enzyme over a length of at least about 25, 50, 100, 150 or 200 amino acids or throughout the length of the polypeptide. The enzyme amino acid sequence is substantially homologous (i.e., the amino acid sequence identity is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the lysosomal sulfatase enzyme is human N-acetylgalactosamine-6-sulfatase (GALNS). The amino acid sequence of human GALNS is set forth in SEQ ID NO: 4, wherein amino acids 27 to 522 correspond to secretory precursor proteins. In some embodiments, the GALNS enzyme comprises or consists of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80 with amino acid 27 to 522 of SEQ ID NO: 4. %, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence; or a sequence identical to amino acid 27 to 522 of SEQ ID NO: 4. The GALNS enzyme preferably retains a catalytic acid amino acid corresponding to Cys (amino acid 79 of SEQ ID NO: 4) at position 53 of the secretory precursor protein, which is capable of being converted to C _α -formin . The GALNS enzyme may also retain other amino acids in the active site cavity, including at least 1, 2, 3, 4, 5, 6, 7, 8, or all charged amino acids: Asp288, Asn289, Asp39, Asp54, His236, Lys140, His142, Lys310 and alpha helix: Arg83. Sukegawa, Human Molecular Genetics , 2000, Vol. 9, No. 9 pp. 1283-1290, which is incorporated herein by reference in its entirety, which describes other mutations which reduce the GALNS activity of the patient and the severity of the various mutations The respective 3D positions within are associated. In other embodiments, the individual to which the lysosomal sulfatase enzyme is administered is a human.

In a preferred embodiment, the lysosomal sulfatase enzyme is a highly phosphorylated recombinant human lysosomal sulfatase enzyme produced by a cell line deficient in endosomal acidification (e.g., a CHO-derived END3 complementary population cell line). The END3 complementarity cell line is any modified CHO cell line that retains the properties of the END3 complementation cell line, such as defective endosomal acidification. In a preferred embodiment, the CHO-derived END3 complementary population cell or derivative thereof is a G71 cell line, a G71S cell line, or a G71 or G71S derivative. Alternatively, the lysosomal sulfatase enzyme can be at a relatively high yield, such as at least about 0.5 pg/cell/day, at least about 0.75 pg/cell/day, at least in a relatively high yield, allowing at least about 0.5 pg/cell/day. Any host cell cultured under conditions of about 1.0 pg/cell/day or at least about 1.25 pg/cell/day of expression and secretion, such as any CHO cell or CHO cell derived strain, is produced.

In a more preferred embodiment, the recombinant human lysosomal sulfatase enzyme has a high level of phosphorylated oligosaccharide (i.e., greater than about 0.25, preferably greater than 0.5, and more preferably greater than about 0.75 diphosphorylated oligosaccharide per protein chain) Mannose chain).

In some embodiments, the invention provides a recombinant human lysosomal sulfatase enzyme, such as GALNS, having a specified high content of phosphorylated oligosaccharides. For example, a lysosomal sulfatase enzyme has from 0.5 to 1.0 diphosphorylated oligomannose chains per monomeric protein chain, or from 0.5 to 0.9 diphosphorylated oligomannose chains per monomeric protein chain, or per a monomeric protein chain of 0.5 to 0.8 diphosphorylated oligomannose chains, or 0.5 to 0.75 diphosphorylated oligomannose chains per monomeric protein chain, or 0.54 to 0.75 diphosphorylation per monomeric protein chain Oligomannose chain. Covering other similar ranges, such as at least 0.4, 0.45, 0.5, 0.55, 0.6 or 0.65 diphosphorylated oligomannose chains per monomeric protein chain, up to 0.7, 0.75, 0.8, 0.85, 0.9 per monomeric protein chain, 0.95, 0.98 or 1.0 diphosphorylated oligomannose chains, or any combination of any of these numbers. In a preferred embodiment, the enzyme is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), such as N-acetosilamine-6-sulfatase of SEQ ID NO: 4.

In some embodiments, the recombinant human lysosomal sulfatase enzyme having an active site cysteine residue to C α _- A high percentage conversion of acyl glycine (FGIy) (i.e., at least about 50%, 55% 60% or 65%, preferably at least about 70%, 75%, 80%, 85%, 90% or 95%). In a preferred embodiment, the enzyme is active recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and the active site cysteine residue is at position 53 (SEQ ID NO: 4) 79) Cys.

In a particular embodiment, the recombinant human lysosomal sulfatase enzyme has a high content of phosphorylated oligosaccharides, such as any range or amount of each of the monomeric protein chains described herein, and a biphosphorylated oligomannose chain; A high percentage of cysteine residues to C _? -methionine (FGly), such as any percentages described herein. In a preferred embodiment, the enzyme is active highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), such as N-acetosilamine-6-sulfate of SEQ ID NO: 4. Esterase.

In any of the foregoing embodiments, at least 99.5%, at least 99.5%, at least as determined by Coomassie Blue staining or by SDS-capillary gel electrophoresis (SDS-CGE) when performing SDS-PAGE under reducing conditions 99%, at least 98.5%, at least 98%, at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% or at least 65% recombinant human lysosomal sulfate The enzyme (e.g., GALNS (SEQ ID NO: 4)) is in the form of a precursor.

Furthermore, lysosomal sulfatase such as GALNS (SEQ ID NO: 4) also appears to be greater than the same amino acid produced in host cells (eg, CHO cells or CHO derived cells) that do not exhibit recombinant human SUMF1. The specific activity of the control lysosomal sulfatase enzyme of the sequence is at least about 30% (eg, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%) Specific activity of 90%, 95%, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times or 50 times).

In any of the foregoing embodiments, the lysosomal sulfatase enzyme such as GALNS (SEQ ID NO: 4) exhibits a specific uptake (Kuptake) to fibroblasts of about 0.1 to 10 nM, Or about 0.1 to 7 nM, or about 0.5 to 5 nM, or about 1 to 5 nM, or about 1 to 3.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM or about 3.5 nM, or any combination of any of these numbers.

In any of the foregoing embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of recombinant human lysosomal sulfatase enzyme (eg, GALNS (SEQ ID NO: 4) binds to a mannose-6-phosphate receptor column.

According to this aspect, there is provided a purified preparation of any of these examples of lysosomal sulfatase enzymes, wherein the method of SMS-PAGE is performed by Somas blue staining or by another method of purity when performing SDS-PAGE under non-reducing conditions (eg SDS-PAGE under reducing or non-reducing conditions followed by staining with Coomassie blue or silver; or layering by HPLC (including C4 reverse phase (RP) or C3RP) or size exclusion chromatography (SEC) The lysosomal sulfatase enzyme component of GALNS (SEQ ID NO: 4) has a purity of at least about 90%, 95%, 97%, 98% or 99% as determined by separation. In some embodiments, Coomassie Blue is performed by Somas blue staining or by another method of detecting the precursor (eg, SDS-PAGE under reducing conditions), such as under SDS-PAGE under reducing conditions. Or silver staining; or chromatographic separation by HPLC (eg C4 reverse phase (RP), C3RP) or size exclusion chromatography (SEC); or combination of electrophoretic separation and chromatographic separation, eg sequential SDS-PAGE and A significant amount of the lysosomal sulfatase enzyme component of the purified preparation is in the form of a secretory precursor as determined by capillary gel electrophoresis (SDS-CGE) (eg, at least 65%, 70%, 75%, 80%, 85) %, 90%, 95%, 97%, 98%, 98.5%, 99% or 99.5% precursor).

In a particular embodiment, the lysosomal sulfatase enzyme component of the purified formulation has a high level of phosphorylated oligosaccharides, such as any range or amount of biphosphorylated oligomannose chains per monomeric protein chain described herein; A high percentage of conversion of the active site cysteine residue to C[ _alpha] -methionine (FGly), such as any percentages described herein. In a more specific embodiment, the purified formulation has a Kuptake as described herein.

In a related aspect, the invention provides a sterile composition comprising any of the lysosomal sulfatase enzymes or purified preparations described herein, as well as sterile pharmaceutically acceptable diluents, carriers, and/or excipients. Such sterilized compositions may be in the form of a lyophilized powder which may be reconstituted in a vial or in a vial by the addition of a sterile diluent.

In a fourth aspect, the invention provides a method of purifying a recombinant human lysosomal sulfatase produced by the method of the invention. In a preferred embodiment, purification is accomplished using a two-column method (dye-ligand chromatography, such as Blue-Sepharose; and cation exchange chromatography, such as SE Hi-Cap). Lysosomal sulfatase enzyme, the method comprises at least five purification steps: (1) filtering the collection, ie expressing the human sulfatase modification factor 1 (SUMF1) and the recombinant human lysosomal sulfatase enzyme END3 complementary group CHO a medium for the cell strain or a derivative thereof; (2) adjusting the pH of the filtered collection to pH 4.5 (to induce contaminating protein precipitation); (3) loading the pH-adjusted filtered collection onto the dye a ligand column (for example, a blue-agarose gel column), washing the column and dissolving the lysosomal sulfatase enzyme from the column; (4) coming from the dye-ligand column The eluent is loaded on a cation exchange column (for example, SE Hi-Cap column), the column is washed and the lysosomal sulfatase is dissolved from the column; and (5) ultrafiltration and diafiltration from cation exchange liquid. The collected filtrate in the step (1) is concentrated 10-20 times by ultrafiltration, as the case may be, and then the pH is adjusted. The lysosomal sulfatase enzyme subjected to ultrafiltration and diafiltration in the step (5) is optionally formulated in the formulation buffer. In a particularly preferred embodiment, the lysosomal enzyme is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

In another preferred embodiment, a three-column method (capture chromatography, such as cation exchange SE Hi-Cap; intermediate chromatography, such as dye-ligand Capto Blue, zinc chelate agarose FF or Capto) is used. Adhere; and polishing chromatography, such as ToyoPearl Butyl 650M, Phenyl Sepharose Hi-Sub or Phenyl Sepharose Low-Sub) to purify lysosomal sulfatase, the method comprising at least five Purification steps: (1) ultrafiltration collection by, for example, Sartocon's Cassettes (30 kDa, Hydrosart), ie, human sulfatase modification factor 1 (SUMF1) and recombinant human lysosomal sulfatase a medium for END3 complementary CHO cell strain or a derivative thereof; (2) adjusting the pH of the filtered collection to pH 4.5 (to induce contaminating protein precipitation); (3) filtering by pH adjustment Loading on a capture column (such as a Fractogel EMD SE Hi-CAP (M) cation exchange column), washing the column and dissolving lysosomal sulfatase from the column; (4) coming from the capture column The eluent is loaded on an intermediate column (eg dye-ligand Capto Blue, zinc chelate agarose FF or Capto Adhere), washing the column and dissolving lysosomal sulfatase from the column; and (5) loading the eluate on a purified column (eg, ToyoPearl Butyl 650M, Phenyl Sepharose Hi- On a Sub or Phenyl Sepharose Low-Sub), the column was washed and the lysosomal sulfatase was dissolved from the column. The dissolved lysosomal sulfatase enzyme from step (5) is formulated in a formulation buffer. Optionally, ultrafiltration of the dissolved lysosomal sulfatase from step (5) is then formulated in a formulation buffer. Optionally, the lysosomal sulfatase enzyme from the column in step (4) is exposed to an environment of pH 3.5 to achieve low pH virus inactivation and subsequently loaded onto the purified column in step (5). In a particularly preferred embodiment, the lysosomal sulfatase enzyme is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

In another preferred embodiment, the lysosomal sulfatase enzyme system uses a different three-column method (capture or immobilization) designed to reduce proteolytic digestion (ie, clipping) of lysosomal sulfatase enzymes. Metal affinity chromatography (IMAC), such as dye-ligand Capto Blue, zinc chelate agarose gel FF or Capto Adhere; intermediate chromatography, such as Fractogel EMD SE Hi-Cap cation exchange; and refined chromatography, such as ToyoPearl Purification of butyl 650M, phenyl sepharose gel Hi-Sub or phenyl sepharose Low-Sub, the method comprises at least six purification steps: (1) filtering the collection, ie expressing human sulfatase modification Mammalian cell line of factor 1 (SUMF1) and recombinant human lysosomal sulfatase, such as END3 complementary group CHO cell strain or a derivative thereof, filtered collection by, for example, Sartocon calyx (30 kDa, Hydrosart Ultrafiltration/diafiltration to produce a concentrated filtration collection, such as 20-fold concentration, and charcoal filtration of the concentrated filtration collection; (2) loading the charcoal-filtered concentrated collection onto a capture or IMAC column (eg, dye-matching) Position Capto Blue, zinc chelate agar On the gel FF or Capto Adhere), the trap column is washed under the condition that the lysosomal sulfatase enzyme is retained on the trapping column, and the lysosomal sulfatase enzyme is dissolved from the trapping column; (3) as the case may be, Filter the trapping column to remove the virus with a filter (such as Mustang Q filter); (4) adjust the pH of the trap column or the filtered eluate to an acidic pH, such as pH 4.5 ± 0.1, then Filtering the trapping column to an acidic pH or a filtered eluate; (5) filtering the trapping column to an acidic pH or a filtered eluent to the intermediate column (eg Fractogel) On the EMD SE Hi-CAP cation exchange column, the intermediate column is washed under the condition that the lysosomal sulfatase remains on the intermediate column, and the lysosomal sulfatase is dissolved from the intermediate column; (6) Adjusting the pH of the intermediate column's eluent to a low pH (eg, pH 3.5 ± 0.1) to render the virus inactive; and (7) loading the low pH virus inactivated eluate of the intermediate cation exchange column onto the refining column ( For example, hydrophobic interaction chromatography (HIC) columns, such as ToyoPearl Butyl 650M, phenyl agar On the gel Hi-Sub or phenyl agarose gel Low-Sub, the column is washed and purified by leaving the lysosomal sulfatase on the purified column, and the lysosomal sulfuric acid is dissolved from the refined column. Esterase. In a preferred embodiment, step (3) is included in the purification process. In another preferred embodiment, step (3) is omitted in the purification process. Optionally, (8) the buffered lysosomal sulfatase enzyme of step (7) is buffer exchanged into a formulation, including, for example, but not limited to, a formulation as described herein, such as 20 mM NaOAc/HOAc, 50 mM NaH ₂ PO ₄ , 30 mM arginine HCl, 2% (w/v) sorbitol (pH 5.4), and adjust the concentration of the dissolved lysosomal sulfatase in the formulation to an appropriate concentration, for example 3 mg/mL; (9) removal of any residual virus and DNA in the formulation of purified lysosomal sulfatase by filtration through a virus filter and DNA filter; and (10) nonionic interfacial activity A dose such as polysorbate 20 (PS20 or Tween-20) is added to the formulation of the purified lysosomal sulfatase enzyme. The final formulation of the purified lysosomal sulfatase enzyme (bulk drug) is stored at 2-8 ° C or frozen. In a particularly preferred embodiment, steps (8) through (10) are included in the purification process. In a particularly preferred embodiment, the lysosomal sulfatase enzyme is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

In some embodiments, the collection is collected in step (1) at a pH of about 6.5. In some embodiments, the charcoal filter in step (1) is a Zeta Plus R55 activated carbon filter. In some embodiments, the capture column in step (2) is a Zn-IMAC column. In some embodiments, the Zn-IMAC column is a Zn chelated agarose gel FF column. In some embodiments, the filter in step (3) is a Mustang Q filter. In some embodiments, the acidic pH of the eluent or filtered eluate from step (2) or (3) is adjusted to about 4.5 ± 0.1 in step (4). In some embodiments, the intermediate column in step (5) is a cation exchange column. In some embodiments, the cation exchange column is a Fractogel EMD SE Hi-Cap column. In some embodiments, the low pH of the eluate from the intermediate column of step (6) is adjusted to about 3.5 ± 0.1 in step (6). In some embodiments, the refined column in step (7) is a hydrophobic interaction chromatography (HIC) column. In some embodiments, the HIC column is a ToyoPearl Butyl 650M column.

In some embodiments, the formulation comprises 20 mM NaOAc/HOAc, 50 mM NaH ₂ PO ₄ , 30 mM arginine HCl, 2% (w/v) sorbitol (pH 5.4). In some embodiments, the nonionic surfactant is polysorbate 20 (PS20). In some embodiments, the concentration of lysosomal sulfatase enzyme in the formulation is adjusted to about 3 mg/mL. In some embodiments, the virus filter is a DV20 filter and the DNA filter is a Mustang Q filter. In some embodiments, the nonionic surfactant added to the formulation is Polysorbate 20 (PS20) with a final concentration of 0.01% (w/v).

In a fifth aspect, the present invention provides a purified preparation of active highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active mutant, variant or derivative thereof. Suitable for the treatment of lysosomes caused by GALNS deficiency (eg, type Ia mucopolysaccharidosis (MPS IVa) or Moquio A syndrome) or associated with GALNS enzyme deficiency (eg multiple sulfatase deficiency (MSD)) Individuals who accumulate disease. In a preferred embodiment, the purified preparation of active highly phosphorylated recombinant human GALNS has a GALNS enzyme component having (a) at least about 90%, 95%, 97%, 98% or 99% purity, as in Determined by Coomassie blue staining or silver staining on SDS-PAGE under non-reducing conditions; (b) Cystein acid residue at position 53 to C _a -methionine (FGly) (SEQ ID NO) : position 4 of 79) at least about 80%, 85%, 90% or 95% conversion; (c) N-linked glycosylation of the indoleamine residue at positions 178 and 397, with position 178 Some of the oligomannose chains attached to the asparagine residue are diphosphorylated; (d) 0.5 to 1.0, or 0.5 to 0.9, or 0.5 to 0.8, or 0.5 to 0.75, or 0.54 to each monomeric protein chain 0.75 diphosphorylated oligomannose chains (eg, at least 0.4, 0.45, 0.5, 0.55, 0.6 or 0.65 and up to 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.98 or 1.0 diphosphoric acid per monomeric protein chain) Oligomannose chain, or any combination of any of these numbers; and (e) at least 65% or at least 70% (eg, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%) , 98.5%, 99% or 99.5%) of the GALNS enzyme system is in the form of a precursor , Or by Coomassie Blue staining SDS- determined by capillary gel electrophoresis (SDS-CGE) When SDS-PAGE as performed under reducing conditions. In addition, the GALNS enzyme may also (f) exhibit a specific activity greater than that of the control GALNS enzyme having the same amino acid sequence produced in a host cell (eg, CHO cells or CHO-derived cells) that does not exhibit recombinant human SUMF1, at least about 30. % (eg 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 2 times, 2.5 times, 3 times) Specific activity of 4x, 5x, 10x, 15x, 20x, 30x, 40x or 50x). Optionally, the GALNS enzyme exhibits a specific uptake (Kuptake) to fibroblasts of from about 0.1 to 10 nM, or from about 0.1 to 7 nM, or from about 0.5 to 5 nM, or from about 1 to 5 nM, or from about 1 to 3.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM or about 3.5 nM, or any combination of any of these numbers.

The purified active hyperphosphorylated recombinant human GALNS consists of SDS-PAGE under reducing conditions or as determined by SDS-CGE consisting of a major band of about 55-60 kDa (ie, at least about 75% of the visible protein). Or at least 80%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%. Human GALNS) and sub-bands of about 39 kDa and about 19 kDa (ie, less than about 25%, less than about 20%, preferably less than about 15%, more preferably less than about 10%, and even better) Less than about 5%, less than about 3%, less than about 2%, less than about 1.5%, less than about 1%, or less than about 0.5% mature or processed human GALNS). In a particularly preferred embodiment, the purified active hyperphosphorylated recombinant human GALNS consists essentially of SDS-PAGE under reducing conditions or as determined by SDS-CGE consisting essentially of: a single of about 55-60 kDa Belt (also known as precursor human GALNS). In one embodiment, the purified active highly phosphorylated recombinant human GALNS is suitable for treating MPS IVa or Moquiao A syndrome. In one embodiment, the purified active highly phosphorylated recombinant human GALNS is suitable for the treatment of MSD.

In a sixth aspect, the invention provides a method of treating a disease caused, in whole or in part, by a lysosomal sulfatase deficiency or associated with a lysosomal sulfatase deficiency. The method comprises administering a therapeutic recombinant human lysosomal sulfatase enzyme produced by the method of the invention, wherein the lysosomal sulfatase enzyme binds to the MPR receptor and is transported across the cell membrane, into the cell and passed to the lysin in the cell body.

In one embodiment, the method comprises treating an individual suffering from a lysosomal sulfatase deficiency comprising administering to a subject in need thereof a therapeutically effective amount of the lysosomal sulfatase, wherein the lysosomal sulfate The enzyme is a recombinant human lysosomal sulfatase enzyme or a biologically active fragment, mutant, variant or derivative thereof produced by CHO derived END3 complementary population cells or derivatives thereof. In some embodiments, the method comprises administering a therapeutic recombinant human lysosomal sulfatase enzyme or a biologically active fragment, mutant, variant thereof, alone or in combination with a pharmaceutically acceptable carrier, diluent or excipient. Body or derivative. Preferred embodiments include optimizing the dosage to best address the lysosomal sulfatase deficiency in response to the needs of the individual to be treated, preferably a mammal and the best human.

Such therapeutic lysosomal sulfatase enzymes are particularly useful, for example, in the treatment of patients suffering from lysosomal storage diseases caused by lysosomal sulfatase deficiency, such as dystrophic leukodystrophy or MLD, type VI adhesion. Polysaccharide disease (MPS VI) or Marathi's syndrome, type II mucopolysaccharidosis (MPS II) or Hunter syndrome, type IIIa mucopolysaccharidosis (MPS IIIa) or St. Philippe A syndrome, type IIId Patients with polysaccharide disease (MPS IIId) or St. Philippe D syndrome and type IVa mucopolysaccharidosis (MPS IVa) or Moquiao A syndrome. In a particularly preferred embodiment, the lysosomal storage disease is MPS IVa or Moquiao A syndrome and the lysosomal sulfatase enzyme is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS). In other embodiments, the invention also provides a pharmaceutical composition comprising a lysosomal sulfatase enzyme that causes a deficiency in a lysosomal storage disease and a pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, the method comprises treating an individual afflicted with a lysosomal storage disease associated with one or more lysosomal sulfatase deficiencies, the treatment comprising administering to the individual in need thereof a therapeutically effective amount of a lysosome A sulfatase enzyme, wherein the lysosomal sulfatase enzyme is a recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) produced by CHO-derived END3 complementary population cells or a derivative thereof, or a biologically active fragment thereof , mutant, variant or derivative. In some embodiments, the methods comprise administering a therapeutic recombinant human GALNS enzyme, or a biologically active fragment, mutant, variant or derivative thereof, alone or in combination with a pharmaceutically acceptable carrier, diluent or excipient. . In a particularly preferred embodiment, the lysosomal storage disease is a plurality of sulfatase deficiency (MSD).

In a particularly preferred embodiment, the CHO-derived END3 complementary population cell or derivative thereof is a G71 cell line, a G71S cell line, or a G71 or G71S derivative thereof.

In another embodiment, the invention provides a method of enzyme replacement therapy by administering to a subject in need of the enzyme replacement therapy a therapeutically effective amount of a lysosomal sulfatase enzyme, wherein the patient's cells have A lysosomal sulfatase lysosome containing insufficient amount to prevent or reduce damage to cells, whereby the amount of lysosomal sulfatase enzyme sufficient to prevent or reduce damage to cells enters the lysosome. The cells may be separated from the blood within or outside the CNS or by capillary walls, the endothelial cells of which are tightly sealed by tight engagement to prevent diffusion of the active agent.

In a specific embodiment, the present invention provides a composition comprising a biologically active recombinant human lysosomal sulfatase enzyme and a pharmaceutical composition, wherein the active recombinant human lysosomal sulfatase is reduced in a target lysosome, Lack or non-existence, and is cast into individuals. Preferred active human lysosomal sulfatase enzymes include, but are not limited to, arylsulfatase A, arylsulfatase B, iduronic acid-2-sulfatase, sulfonamide/acetyl heparin -N-sulfatase, N-acetylglucosamine-6-sulfatase and N-acetylgalactosamine-6-sulfatase. In a preferred embodiment, N-acetylgalactosamine-6-sulfatase is an active recombinant human lysosomal sulfatase enzyme.

In a preferred embodiment, the invention provides a therapeutically effective amount of any of the recombinant human N-acetylgalactosamine-6 described herein by administering to an individual suffering from MPS IVa or Moquio A syndrome or MSD. - A method of treating the individual with a sulfatase enzyme (GALNS), a purified preparation and/or a sterile composition.

In a more preferred embodiment, the invention provides a therapeutically effective amount of recombinant human N-acetylgalactosamine produced by END3 complementary population cells by administering to a subject suffering from MPS IVa or Moquio A syndrome or MSD 6-sulfatase (GALNS) to treat the subject, wherein the recombinant human GALNS of the active site cysteine residue to C α _- a higher acyl glycine (FGIy) the degree of conversion (also That is, at least about 50%, preferably at least about 70%, more preferably at least about 90%, even more preferably at least about 95% conversion) and the degree of phosphorylation of the recombinant human GALNS is higher (ie, each protein chain is greater than about 0.25, preferably greater than 0.5 and more preferably greater than about 0.75 diphosphorylated oligomannose chains).

In a particularly preferred embodiment, the invention provides a method for administering a therapeutically effective amount of purified active highly phosphorylated recombinant human GALNS having a GALNS enzyme component to an individual suffering from MPS IVa or Moquio A syndrome or MSD. Formulations for treating the individual, the GALNS enzyme component having: (a) at least about 90%, 95%, 97%, 98% or 99% purity, such as by SDS-PAGE under non-reducing conditions Determined by Coomassie blue staining or silver staining; (b) the cysteine residue at position 53 is at least about 80% to C _? -methionine (FGly) (position 79 of SEQ ID NO: 4), 85%, 90% or 95% conversion; (c) 0.5 to 1.0, or 0.5 to 0.9, or 0.5 to 0.8, or 0.5 to 0.75, or 0.54 to 0.75 diphosphorylated oligomannose per monomeric protein chain a sugar chain (eg, at least 0.4, 0.45, 0.5, 0.55, 0.6 or 0.65 and at most 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.98 or 1.0 diphosphorylated oligomannose chains per monomeric protein chain, or any Any combination of these numbers); and (d) at least 65% or at least 70% (eg, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 98.5%, 99%, or 99.5) %) of the GALNS enzyme system is a precursor Form, or by Coomassie Blue staining SDS- determined by capillary gel electrophoresis (SDS-CGE) When SDS-PAGE as performed under reducing conditions. In addition, the GALNS enzyme may also (e) exhibit at least about 30 specific activities greater than the control GALNS enzyme having the same amino acid sequence produced in a host cell (eg, CHO cells or CHO-derived cells) that does not exhibit recombinant human SUMF1. % (eg 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 2 times, 2.5 times, 3 times) Specific activity of 4x, 5x, 10x, 15x, 20x, 30x, 40x or 50x). Optionally, the GALNS enzyme exhibits a specific uptake (Kuptake) to fibroblasts of from about 0.1 to 10 nM, or from about 0.1 to 7 nM, or from about 0.5 to 5 nM, or from about 1 to 5 nM, or from about 1 to 3.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM or about 3.5 nM, or any combination of any of these numbers.

The purified active hyperphosphorylated recombinant human GALNS consists of SDS-PAGE under reducing conditions or as determined by SDS-CGE consisting of a major band of about 55-60 kDa (ie, at least about 75% of the visible protein). Or at least about 80%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99% or At least about 99.5% of the precursor human GALNS) and sub-bands of about 39 kDa and about 19 kDa (ie, less than about 25%, or less than about 20%, preferably less than about 15%, more preferably less than about 20% of the visible protein). 10%, and even more preferably less than about 5%, less than about 3%, less than about 2%, less than about 1.5%, less than about 1%, or less than about 0.5% of mature or processed human GALNS). In SDS-PAGE under reducing conditions or as determined by SDS-CGE, in a more particularly preferred embodiment, the purified active highly phosphorylated recombinant human GALNS consists essentially of a single band of about 55-60 kDa ( That is, the composition of the precursor human GALNS).

In some embodiments, the individual is suffering from MPS IVa or Moquiao A syndrome. In some embodiments, the individual suffers from MSD.

The corresponding use of the highly hyperphosphorylated lysosomal sulfatase enzyme of the present invention, preferably produced by the method of the present invention, in the preparation of a medicament for the treatment of the aforementioned lysosomal storage diseases is also encompassed.

In a seventh aspect, the invention provides a pharmaceutical composition comprising as described above for use in the treatment of a disease caused, in whole or in part, by a lysosomal sulfatase deficiency or associated with a lysosomal sulfatase deficiency Active highly phosphorylated recombinant human lysosomal sulfatase enzyme, and one or more pharmaceutically acceptable carriers, diluents or excipients. In a preferred embodiment, the pharmaceutical composition comprises the highly hyperphosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) produced by the method of the invention, or a biologically active fragment thereof, a mutant thereof, A variant or derivative, and one or more pharmaceutically acceptable carriers, diluents or excipients. Such pharmaceutical compositions may be suitable for administration by a number of routes, such as intrathecal, parenteral, topical, intranasal, inhalation or oral administration. In a preferred embodiment, the pharmaceutical composition is suitable for parenteral administration. Included within the scope of this aspect is an embodiment characterized by a nucleic acid sequence encoding a full-length lysosomal sulfatase enzyme or a fragment, mutant, variant or derivative thereof, which can be administered in vivo by a lysosomal enzyme deficiency In the cell.

In a more preferred embodiment, the pharmaceutical composition comprises an active highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) produced by the method of the invention or a biologically active fragment, mutant, variant thereof The body or derivative, and one or more pharmaceutically acceptable carriers, diluents or excipients are included in a formulation comprising one or more buffering agents and one or more stabilizers. In certain embodiments, the composition comprises a phosphate buffering agent effective to reduce the amount of dephosphorylation of the GALNS enzyme; and a stabilizing amount of one or more stabilizers selected from the group consisting of: an amine acid salt, An amino acid buffer, a surfactant, and a polyol; wherein the pH of the formulation is between about 5.0 and 5.8.

In some embodiments, the GALNS enzyme comprises an amino acid sequence that is at least 95% identical to the amino acids 27 to 522 of SEQ ID NO: 4, and has: (i) at least about 95% purity, as in non-reducing conditions determined by Coomassie blue staining when carried out under SDS-PAGE, (ii) the position of the 53 cysteine residues in the C α _- at least about 80% of the conversion of p-acyl glycine (FGly), and ( Iii) 0.5 to 0.8 biphosphorylated oligomannose chains per monomeric protein chain, wherein at least 70% of the GALNS enzyme system is in the form of a precursor, such as by SDS-PAGE under reducing conditions Determined by mas blue staining. In some embodiments, the purity of the GALNS enzyme is at least 95% as determined by RP-HPLC. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% of the GALNS enzyme is in the form of a precursor, It was determined by Coomassie blue staining when performing SDS-PAGE under reducing conditions. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% of the GALNS enzyme is in the form of a precursor, As determined by SDS-capillary gel electrophoresis. In some embodiments, the GALNS enzyme has at least about 90% conversion of the cysteine residue at position 53 to C[ _alpha] -methionine (FGly). In some embodiments, 50% to 80% GALNS enzyme binds to a mannose-6-phosphate receptor column. In some embodiments, the GALNS enzyme exhibits a specific uptake (Kuptake) to fibroblasts of between about 1 and 5 nM. In some embodiments, the GALNS enzyme exhibits a Kuptake to fibroblasts of from about 1 to 3.5 nM.

The concentration of GALNS or a biologically active fragment, mutant, variant or derivative thereof in the formulation is from about 0.1 to 10 mg/mL, preferably from about 0.5 to 5 mg/mL, and more preferably from about 0.5 to 1.5 mg/mL.

In certain embodiments, the formulation comprises a phosphate buffer that is effective to reduce the amount of dephosphorylation of the GALNS enzyme. In a related embodiment, the phosphate buffer is NaH ₂ PO ₄ or its equivalent. In another embodiment, the formulation additionally comprises a second buffer. In one embodiment, the second buffer is an acetate buffer. In another embodiment, the acetate buffer is NaOAc/HOAc or an equivalent thereof. Exemplary buffers are described in more detail in [Embodiment].

The concentration of NaOAc/HOAc or its equivalent in the intended formulation is from about 5 to 100 mM, preferably from about 5 to 50 mM, and more preferably from about 10 to 30 mM. In a related embodiment, the concentration of NaH ₂ PO ₄ or its equivalent in the formulation is from about 5 to 100 mM, preferably from about 25 to 100 mM, and more preferably from about 25 to 75 mM. In certain embodiments, the pH of the formulation is about pH 4.5-6.5, preferably about pH 5.0-6.0, and more preferably about pH 5.0-5.8.

In another embodiment, the formulation comprises one or more stabilizers selected from the group consisting of: an amine acid salt, an amino acid buffer, a surfactant, and a polyol. In one embodiment, the stabilizer is arginine or a histidine or buffer, optionally a arginine hydrochloride. In a related embodiment, the stabilizer is a polysorbate, optionally a polysorbate 20. In another embodiment, the stabilizer is a ternary or trihydric sugar alcohol, optionally sorbitol. Exemplary stabilizers are described in more detail in [Embodiment].

In certain embodiments, the stabilizer is selected from the group consisting of arginine hydrochloride or its equivalent, Tween-20 (polysorbate 20) or its equivalent, and sorbitol or its equivalent. In some embodiments, the concentration of the arginine hydrochloride or its equivalent in the formulation is from about 5 to 200 mM, preferably from about 10 to 100 mM, and more preferably from about 10 to 50 mM. In another embodiment, the concentration of Tween-20 or its equivalent in the formulation is from about 0.001 to 1.0% (w/v), preferably from about 0.005 to 0.2% (w/v), and more preferably about 0.005. To 0.015% (w/v). In a related embodiment, the concentration of sorbitol or its equivalent in the formulation is from about 0.1 to 10% (w/v), preferably from about 0.5 to 5% (w/v), and more preferably about 1.0. Up to 3.0% (w/v). In one embodiment, the formulation comprises a arginine or buffer, a polysorbate, and a polyol.

The invention further provides a method of preventing dephosphorylation of a recombinant human GALNS enzyme comprising mixing a GALNS enzyme with a phosphate buffer such that the final concentration of phosphate buffer is between about 25 mM and 75 mM. In an exemplary embodiment, for example, when stored at room temperature (eg, 25 ° C) for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 After the month of testing, the amount of dephosphorylation was reduced compared to the formulation of the same enzyme in 1 mM phosphate buffer.

In a particularly preferred embodiment, the pharmaceutical composition comprises an active highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) produced by the method of the invention, or a biologically active fragment thereof, a mutant thereof, a variant or derivative, and one or more pharmaceutically acceptable carriers, diluents or excipients comprising NaOAc/HOAc and NaH ₂ PO ₄ as a buffer, and arginine hydrochloride, Tween-20 (Polysorbate 20) and sorbitol as stabilizer formulations. The concentration of GALNS in the formulation was about 1.0 +/- 0.5 mg/mL. The concentration of NaOAc/HOAc in the formulation was about 20 +/- 10 mM and the concentration of NaH ₂ PO _{4 in} the formulation was about 50 +/- 25 mM. The pH of the formulation is pH 5.4 +/- 0.4. The concentration of arginine hydrochloride in the formulation was about 30 +/- 20 mM. The concentration of Tween-20 in the formulation is about 0.01 +/- 0.005% (w/v). The concentration of sorbitol in the formulation was about 2.0 +/- 1.0% (w/v).

In another aspect, the invention provides a method for detecting lysosomal sulfatase activity comprising (a) cultivating a patient suffering from lysosomal sulfatase deficiency in a condition that promotes maintenance of chondrocyte differentiation (for example, chondrocytes of patients suffering from Moquio syndrome; (b) contacting the chondrocytes with lysosomal sulfatase which degrades keratan sulfate; and (c) detecting keratin in cells The content of the prime, wherein the decrease in the keratan sulfate content in the cells contacted with the lysosomal sulfatase is indicative of lysosomal sulfatase activity compared to cells not contacted with the lysosomal sulfatase. In some embodiments, the lysosomal sulfatase enzyme is N-acetylgalactosamine-6-sulfatase (GALNS). In some embodiments, the cultivation is carried out in a medium comprising insulin growth factor 1 (IGF1), transforming growth factor beta (TGF-beta), transferrin, insulin, and ascorbic acid. In some embodiments, keratan sulfate is detected by confocal microscopy or by binding to an anti- keratan sulfate antibody. The method can be carried out using any lysosomal sulfatase enzyme, including naturally occurring or recombinant human enzymes or fragments or variants thereof, including at least 80%, 85% of the human enzyme or its mature form comprising or without a signal sequence precursor. A variant of the 90%, 95% or 100% identical amino acid sequence.

In another aspect, the invention provides a cell-based assay for measuring the activity of a recombinant human lysosomal enzyme to degrade a natural receptor. The method comprises (a) culturing an isolated human cell lacking a lysosomal enzyme under conditions in which the natural receptor of the lysosomal enzyme is accumulated; (b) contacting the cell with the lysosomal enzyme; (c) dissolving the (d) adding an enzyme to the cell lysate, the enzyme (i) is specific for the natural receptor, and (ii) cleavage of the small oligosaccharide from the natural receptor; (e) Measuring the small oligosaccharides; (f) isolating the labeled small oligosaccharides as appropriate; (g) detecting the labeled small oligosaccharides; and (h) by comparing (i) The amount of labeled small oligosaccharides of the cells contacted by the lysosomal enzyme is (ii) the amount of labeled small oligosaccharides from cells not contacted with the lysosomal enzyme to determine the natural receptor of the lysosomal enzyme degradation. The activity, wherein (h) (i) reduces the activity of the lysosomal enzyme to degrade the natural receptor compared to (h) (ii). In one embodiment, the small oligosaccharide is a monosaccharide, a disaccharide or a trisaccharide. In a related embodiment, the small oligosaccharide is a disaccharide. In some embodiments, the lysosomal enzyme is selected from the group consisting of arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide/heparin- N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-acetylgalactosamine-6-sulfatase (GALNS). In some embodiments, the lysosomal enzyme is alpha-L-iduronidase (IDU). In some embodiments, the lysosomal enzyme is an acid alpha-glucosidase (GAA). In some embodiments, the lysosomal enzyme is beta-glucoronidase (GUSB). In some embodiments, the lysosomal enzyme is beta-galactosidase (GLB1).

Suitable human cells that can be used in cell-based assays include any lysosomal enzyme deficiency to be tested, thus accumulating any human cells that naturally interfere with the lysosomal enzyme. For example, cells that exhibit complete (100%) or partial deficiency (eg, 30%, 50%, 70%, 80%, 90%, 95%, or 95% reduction in activity) can be used. A cell expressing a mutant enzyme having reduced activity, or a cell derived from a patient suffering from a lysosomal storage disease such as mucopolysaccharidosis may be used. Cells that are recombinantly altered to knock out or reduce lysosomal enzyme activity, for example, by introducing mutations into the coding gene or its promoter or other regulatory regions, can be used. Cells that have been treated to reduce lysosomal enzymatic activity (e.g., treated with antisense or RNAi to reduce enzyme performance) can be used.

Suitable enzymes which cleave (digest) small oligosaccharides from carbohydrates and which are "specific" to the natural receptor of the lysosomal enzyme (i.e., primarily digesting such natural receptors) can be selected by the average skilled person. For example, for detecting the activity of GALNS or GLB1 (an enzyme that degrades keratan sulfate), the enzyme of step (d) may be Keratinase II or any enzyme that acts primarily on keratan sulfate. As another example, for detecting IDU, ARSB, IDS, or GUSB (an enzyme that degrades dermatan sulfate), the enzyme of step (d) may be Chondroitinase ABC or mainly dermatan sulfate. Any enzyme that works. As another example, for detecting IDU, IDS, SGHS, G6S or GUSB (an enzyme that degrades heparan sulfate), the enzyme of step (d) may be Heparanase I or B. Heparinase II or both. As another example, for detecting GAA (an enzyme that degrades glycogen), the enzyme of step (d) can be an alpha-amylase or any enzyme that acts primarily on hepatic glucose.

This cell-based method is extremely sensitive in detecting lysosomal enzyme activity. In some embodiments, when the concentration of the lysosomal enzyme is as low as about 10 nM, or about 5 nM, or about 1 nM, or about 0.75 nM, or about 0.5 nM, or about 0.25 nM, or about 0.1 nM, or Lysosomal enzyme activity can be detected at about 0.05 nM, or about 0.01 nM, or about 0.005 nM, or about 1 pM, or about 0.5 pM.

Other features and advantages of the present invention will become apparent from the following description. However, it is to be understood that the preferred embodiments of the present invention are given by way of example only, and in accordance with the Modifications will become apparent to those skilled in the art.

The present invention relates to the discovery of a method for the preparation of recombinant lysosomal sulfatase enzymes on a large scale with highly highly phosphorylated lysosomal sulfatase enzyme products which are highly effective against lysosomes and therefore therapeutically effective. Consistent demand.

The therapeutic effectiveness of the lysosomal enzyme preparation depends on the amount of mannose-6-phosphate in the preparation. Phosphate esters are added to the target glycoprotein by post-translational modification in the endoplasmic reticulum and early high-kilth (Golgi). The folded lysosomal enzyme displays a unique tertiary determinant recognized by the oligosaccharide modifying enzyme. The determinant consists of a set of isolated amino acids separated from each other and found on most lysosomal enzymes, even in the absence of primary sequence homology. The modification enzyme UDP-GlcNAc phosphotransferase binds to a protein determinant and adds GlcNAc-1-phosphate to the 6-position of the terminal mannose residue adjacent to the binding site on the oligosaccharide: second enzyme phosphodiester α-GlcNAc The enzyme then cleaves the GlcNAc-phosphate linkage to produce a mannose-6-phosphate terminal oligosaccharide (Canfield et al., U.S. Patent No. 6,537,785). The purpose of mannose-6-phosphate modification is to shift the lysosomal enzyme autocrine pathway to the intracellular lysosomal pathway. The mannose-6-phosphate-bearing enzyme is bound by MPR in the trans-high-base and transported to the lysosome rather than the cell surface.

In addition to the presence of the mannose-6-phosphate label on the lysosomal enzyme oligosaccharide, the lysosomal transport of the enzyme depends on the acidification of the transport endosomes that occur at the end of the trans-high-base stack. The quenching of the acidic environment in these endosomes by diffusion-diffused basic molecules causes the vesicle contents (including lysosomal enzymes) to be expelled into the extracellular environment (Braulke et al., Eur. J. Cell Biol . 43 ( 3): 316-321, 1987). Acidification requires specific vacuolar ATPase embedded in the endosomal membrane (Nishi et al, Nat. Rev. Mol. Cell Biol. 3(2): 94-103, 2002). The failure of this ATPase is expected to increase the secretion of lysosomal enzymes at the expense of lysosomal transport. It is expected that a cell line for preparation carrying a vacuolar ATPase deficiency will prevent the non-productive transfer of phosphorylated recombinase to the intracellular lysosomal compartment.

In 1984, a Chinese hamster ovary (CHO) cell mutant with specific defects in endosome acidification was generated and characterized (Park et al, Somat. Cell Mol. Genet. 17(2): 137-150, 1991). CHO-K1 cells are induced by chemical mutations and are selected for survival at elevated temperatures in the presence of toxins. These toxins require endosome acidification to fully manifest their lethality (Marnell et al, J. Cell. Biol. 99(6): 1907-1916, 1984). In previous studies, a mixture of two toxins with different mechanisms of action was chosen to avoid selection of toxin-specific resistance. The principle is that although the probability of causing an accidental mutation that is resistant to a particular toxin is small, the probability of two simultaneous accidental mutations specific for two completely different toxins is absent. Selection is made at elevated temperatures to allow for temperature sensitive mutations. This genetic screening yielded two mutants, one of which is called G.7.1 (G71), which is resistant to toxins at elevated temperatures. The damage in G71 is not due to the uptake or mechanism of action of the two toxins, but is caused by the inability of the pure line to acidify the endosomes at elevated temperatures. This is not obvious at the permissible temperature (34 ° C), but to a lesser extent. G71 cells were also found to be iron auxotrophic at elevated temperatures despite the normal uptake of transferrin from the culture medium (Timchak et al, J. Biol. Chem. 261 (30): 14154-14159, 1986). Since iron is only released from transferrin at low pH, despite the normal uptake of transferrin, there is still an iron auxotrophy indicating endosome acidification. Another study demonstrates that acidification defects are predominantly manifested in endosomes rather than lysosomes (Stone et al, J. Biol. Chem . 262(20): 9883-9886, 1987). The data on G71 is consistent with the conclusion that mutations lead to the destabilization of vacuolar ATPase responsible for endosomal acidification. Destabilization is most pronounced at high temperatures (39.5 ° C), but is partially manifested even at lower temperatures (34 ° C). Studies on the transport of two endogenous lysosomal enzymes cathepsin D and alpha-glucosidase in G71 cells (Park et al, Somat. Cell Mol. Genet . 17(2): 137- 150, 1991) showed that both enzymes were quantitatively secreted at high temperatures, and the glycosylation of the enzyme was not affected. The secretion of phosphorylated acid alpha-glucosidase is significantly enhanced at unlicensed temperatures.

The therapeutic effectiveness of the lysosomal sulfatase preparation depends not only on the amount of mannose-6-phosphate but also on the presence of the active enzyme in the formulation. All known sulfatase enzymes contain a cysteine residue at their catalytic site; this cysteine residue is post-translationally modified to C _? -methionine (FGly) to activate the enzyme. This cysteine to FGly post-translational enzyme activation catalyzed by sulfatase modification factor 1 (SUMF1) immediately after translation, before sulfatase targeting lysosomes, unfolded sulfatase in the endoplasmic reticulum Occurs (Dierks et al, Proc. Natl. Acad. Sci. USA 94: 11963-11968, 1997). Mutations in SUMF1 that cause abnormal FGly formation in lysosomal sulfatase enzymes cause multiple sulfatase deficiencies (MSD) in humans to emphasize the importance of this unique post-translational modification (Diez-Ruiz et al., Annu. Rev. Genomics) Hum. Genet . 6:355-379, 2005).

Therefore, G71 cells, also known as endosome acidification-deficient mutant CHO cells, express the ability of recombinant human sulfatase-modifying enzyme (SUMF1) and human lysosomal sulfatase enzymes for large-scale production and are suitable for the management of such lysozymes. The activity of the lysosomal storage disorder that causes or is associated with such lysosomal sulfatase deficiency is highly phosphorylated by recombinant human lysosomal sulfatase enzymes.

I. Definition

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. The following bibliography provides a general definition of many of the terms used in the present invention to those skilled in the art: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988). THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

The disclosures, patent applications, patents, and other references cited herein are hereby incorporated by reference in their entirety in their entirety in their entirety herein

It is to be understood that the singular forms ""

As used herein, the following terms have their meaning unless otherwise specified.

"Dual gene variant" refers to any of two or more polymorphic forms of a gene occupying the same genetic locus. Dual gene variants are usually produced via mutations and can result in phenotypic polymorphism within the population. The gene mutation can be silent (ie, the encoded polypeptide is unchanged) or can encode a polypeptide with altered amino acid sequence. "Dual gene variant" also refers to cDNA derived from the mRNA transcript of a genetically identical variant, and the protein encoded thereby.

"Amplification" refers to any means used to replicate a polynucleotide sequence and thereby expand to a greater number of polynucleotide molecules, such as by reverse transcription, polymerase chain reaction, and ligase chain reaction.

If the polynucleotide of the first sequence is specifically hybridized to the polynucleotide of the second sequence, the first sequence is an "antisense sequence" relative to the second sequence.

"cDNA" refers to DNA that is complementary or identical to mRNA in either single or double stranded form.

A conventional notation is used herein to describe a polynucleotide sequence: the left-hand end of the single-stranded polynucleotide sequence is the 5' end; the left-hand direction of the double-stranded polynucleotide sequence is referred to as the 5' direction. The direction in which 5' to 3' nucleotides are added to the nascent RNA transcript is called the transcriptional direction. A DNA strand with the same sequence as mRNA is called a "coding strand"; a sequence on the same DNA strand as the mRNA transcribed from the DNA and located 5' to the 5' end of the RNA transcript is called "upstream sequence"; in sequence and RNA A sequence on the same DNA strand that is 3' to the 3' end of the encoded RNA transcript is referred to as a "downstream sequence."

"Complementary" refers to the topological compatibility of the interaction surfaces of two polynucleotides or the matching of such surfaces. Thus, the two molecules can be described as complementary and, in addition, the contact surface features are complementary to one another. The first polynucleotide is complementary to the second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, a polynucleotide of sequence 5'-TATAC-3' is complementary to a polynucleotide of sequence 5'-GTATA-3'. A nucleotide sequence is "substantially complementary" to a reference nucleotide sequence if the sequence complementary to the target nucleotide sequence is substantially identical to the reference nucleotide sequence.

"Conservative substitution" refers to the replacement of an amino acid in a polypeptide with a functionally similar amino acid. The following six groups each contain an amino acid that is conservatively substituted for each other:

1) alanine (A), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) aspartame (N), glutamic acid (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), proline (V);

6) Amphetamine (F), tyrosine (Y), tryptophan (W).

The term "fragment" when used in reference to a polypeptide refers to a polypeptide that is shorter than the full length polypeptide by truncation at the N-terminus or C-terminus or both ends of the protein and/or by deleting the internal portion or region of the protein. Fragments of the polypeptide can be produced by methods known in the art.

The term "mutant" when used in reference to a polypeptide refers to a polypeptide in which one or more of the amino acids have been substituted with a different amino acid. The amino acid substitution can be a conservative substitution as defined above or can be a non-conservative substitution. Mutant polypeptides can be produced by methods known in the art.

The term "derivative" when used in reference to a polypeptide refers to a polypeptide that is chemically modified by techniques such as, but not limited to, the following: ubiquitination, labeling (eg, with radionuclides or various enzymes), such as polyethylation The covalent polymer linkage of the diolation (i.e., derivatized with polyethylene glycol) and insertion or substitution by chemical synthesis of an amino acid such as ornithine which is not normally found in human proteins. Derivatized polypeptides can be produced by methods known in the art.

The term "derivative" when used in reference to a cell line refers to a cell line that is a progeny of the parent cell line; for example, the term includes cells or parents that have been subcultured or subcultured from the parent cell and retain the desired properties. Progeny of the cell line that have been mutated and selected for retention of the desired property, and progeny cell lines have been transformed into progeny containing different expression vectors or different exogenously added nucleic acids.

"Detection" means determining the presence, absence or amount of an analyte in a sample, and may include quantifying the amount of analyte in or on each cell in the sample.

"Detectable part" or "marker" means a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, suitable labels include ³² P, ³⁵ S, fluorescent dyes, electron-dense reagents, enzymes (eg, commonly used in ELISA), biotin-streptavadin, digoxigenin, The hapten and protein of the antiserum or monoclonal antibody, or the nucleic acid molecule whose sequence is complementary to the target, can be utilized. The detectable portion often produces a measurable signal, such as a radioactive signal, a chromogenic signal, or a fluorescent signal, that can be used to quantify the amount of bound detectable moiety in the sample. The detectable moiety can be incorporated into the primer or probe in a covalent manner or via an ion, van der Waals or hydrogen bond, or linked to a primer or probe, such as a radioactive nucleotide, or can be resistant to Biotin-labeled nucleotides recognized by streptavidin. The detectable portion can be detected directly or indirectly. Indirect detection may involve combining a second directly or indirectly detectable portion with a detectable portion. For example, the detectable moiety can be a ligand that binds to a partner, such as biotin, which is a binding partner of streptavidin; or a nucleotide sequence, which is specific for it A binding partner for the complementary sequence of the hybrid. The binding partner itself can be directly detectable, for example, the antibody itself can be labeled with a fluorescent molecule. The binding partner may also be indirectly detectable. For example, a nucleic acid having a complementary nucleotide sequence may be part of a branched DNA molecule, which in turn may be detected by hybridization with other labeled nucleic acid molecules. . (See, for example, Fahrlander et al, Bio/Technology 6: 1165, 1988). Quantification of the signal is achieved by, for example, scintillation counting, densitometry, or flow cytometry.

"Diagnosis" means the identification of the presence or nature of a pathological condition. Diagnostic methods differ in their specificity and selectivity. Although a particular diagnostic method may not provide a definitive diagnosis of the condition, it is sufficient if the method provides a positive indication that aids in the diagnosis.

The term "effective amount" means a dose sufficient to produce the desired result or sufficient diagnostic achievement for an individual's health, condition, and disease. The desired result may include a subjective or objective improvement in the dose recipient. By "therapeutically effective amount" is meant an amount of an agent that produces a predetermined beneficial effect on health.

"Coding" means that a particular nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) acts as a biological process for the synthesis of a defined nucleotide sequence (ie rRNA, tRNA and mRNA) or to determine an amine group. The intrinsic properties of the other polymers of the acid sequence and the template of the macromolecules and the resulting biological properties. Thus, if the transcription and translation of mRNA produced by a gene produces a protein in a cell or other biological system, the gene encodes a protein. The coding strand of a gene or cDNA (whose nucleotide sequence is identical to the mRNA sequence and usually provided in the sequence listing) and the non-coding strand used as a transcriptional template can be referred to as a protein or other product encoding the gene or cDNA. Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate forms of each other and which encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA can include introns.

"Equivalent dose" means a dose containing the same amount of active agent.

"Expression control sequence" refers to a nucleotide sequence in a polynucleotide that modulates the expression (transcription and/or translation) of a nucleotide sequence to which it is operably linked. "Operably linked" refers to a functional relationship between two parts, some of which (eg, the ability to regulate transcription) contribute to another (eg, transcription of a sequence). Expression control sequences can include, for example, but are not limited to, promoters (eg, inducible or constitutive promoters), enhancers, transcription terminators, initiation codons (ie, ATGs), intron splicing signals, and termination codes. The sequence of the child.

By "expression vector" is meant a vector comprising a recombinant polynucleotide comprising a display control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises a cis-acting element sufficient for expression; the other elements for expression can be supplied by a host cell or an in vitro expression system. The expression vector includes all expression vectors known in the art, such as a vesicle, a plastid (e.g., a naked body or a plastid contained in a liposome), and a virus having a recombinant polynucleotide.

"Highly phosphorylated", "highly phosphorylated" and "highly phosphorylated oligosaccharide" means at least 50% of lysosomal sulfatase enzymes via phosphorylated oligosaccharides and cation-independent mannose-6-phosphates. Body-bound lysosomal sulfatase enzyme preparation. The combination is further characterized by sensitivity to competition with mannose-6-phosphate. Highly phosphorylated lysosomal sulfatase enzymes may also refer to lysosomal sulfatase enzymes having at least 0.25, preferably at least 0.5 and more preferably at least 0.75 diphosphorylated oligomannose chains per protein chain. Alternatively, highly phosphorylated lysosomal sulfatase (GALNS) may refer to a Kuptake (concentration of enzyme/ligand that produces half the maximum uptake value) in fibroblasts of about 0.1 to 10 nM, or about 0.1 to 7 nM, or about 0.5 to 5 nM, or about 1 to 5 nM, or about 1 to 3.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM or about 3.5 nM, Or an enzyme of any combination of any of these numbers.

As used herein, "diphosphorylated oligomannose chain" refers to a mannose-containing oligosaccharide chain that is N-linked to an aspartic acid residue in a lysosomal sulfatase enzyme and comprises two mannose-6 - Phosphate residue. Typically, the bisphosphorylated oligomannose chain has seven mannose residues, namely mannose diphosphate 7 (BPM7), which are linked to two G1cNAc residues, which in turn are lysosomal sulfates. The aspartic acid residue in the enzyme is linked.

"Active", "activated" and "highly activated" means at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90% or 95% protein The active site cysteine residue has been translated and modified into a lysosomal sulfatase enzyme preparation of Cα-methionine (FGly). Alternatively, "activity", "activation" and "high degree of activation" mean that the specific activity is greater than that of a host cell (eg, CHO cells or CHO-derived cells) that does not exhibit recombinant human SUMF1. The specific activity of the control lysosomal sulfatase enzyme is at least about 30% (eg, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90) A lysosomal sulfatase enzyme preparation of %, 95%, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times or 50 times). Suitable control preparations for lysosomal sulfatase enzymes preferably have the same amino acid sequence as the highly active preparation, and the same promoter is used by the same gene in the same host cell (with the exception that the host cell does not exhibit recombinant human SUMF1) Or regulatory sequence performance, produced under the same or similar culture conditions (including continued same culture period), and optionally purified to the same or similar extent as the highly active preparation.

"Active hyperphosphorylation" means that at least 50%, preferably at least 70%, more preferably at least 90% and even more preferably at least 95% of the protein active site cysteine residues have been translated and modified into C _? -A A lysosomal sulfatase enzyme preparation having at least 0.25, preferably at least 0.5, and more preferably at least 0.75 diphosphorylated oligomannose chains per glycoprotein (FGly).

The term "biologically active" means a polypeptide (ie, an enzyme) fragment that retains at least a substantial amount (eg, at least about 50%, preferably at least about 70%, and more preferably at least about 90%) of the full length polypeptide, one or more biological activities, A mutant, variant or derivative thereof. When used with respect to lysosomal sulfatase, the biologically active fragment, mutant, variant or derivative retains at least a substantial amount of sulfatase activity (i.e., the sulfate is cleaved from its target substrate). When used with sulfatase modification factor 1 (SUMF1), its biologically active fragments, mutants, variants or derivatives retain at least a substantial amount of the activity to produce methotrexate (ie, lysosomal sulfatase) The active site cysteine residue is modified to C _α -methionine (FGly).

The term "purity" or "pure" when used in reference to a polypeptide refers to the amount of the polypeptide being analyzed as compared to any contaminant that can be detected using a particular method. For the recombinant lysosomal sulfatase enzyme of the present invention, "purity" can be separated by SDS-PAGE electrophoresis under reduced or non-reducing conditions, followed by staining with Coomassie blue or silver, or by HPLC chromatography separation (eg, C4 reverse phase (RP), C3 RP) or by any other chromatographic separation (eg, size exclusion (SEC) and the like). Using any of these methods, the purified recombinant lysosomal sulfatase enzyme of the invention has a purity of at least about 80% or at least about 85%, preferably at least about 90%, more preferably at least about 95%, and even More preferably at least about 97%, 98% or 99%.

The term "precursor" or "precursor form" refers to a form of recombinant lysosomal sulfatase secreted by mammalian cells, ie lacking a signal sequence, but lacking certain modifications normally present in lysosomes, such as proteins. Internal cracking. The terms "mature", "mature form", "processed" or "processed form" refer to the form of a recombinant lysosomal sulfatase enzyme normally present in lysosomes. For the recombinant lysosomal sulfatase enzyme of the present invention, the relative abundance of "precursor" or "precursor form" and "mature", "mature form", "processed" or "processed form" can be The sulfatase preparation is subjected to SDS-PAGE electrophoresis under reducing conditions, followed by staining with Coomassie blue or silver, or by HPLC chromatography (eg, C4 reverse phase (RP), C3RP) or by any other chromatography. Separation (eg, size exclusion (SEC) and the like), or a combination of electrophoretic separation and chromatographic separation (eg, SDS-PAGE followed by capillary gel electrophoresis (SDS-CGE)) is determined. Using such methods, the purified recombinant lysosomal sulfatase enzyme of the invention is at least about 65%, 70% or 75%, preferably at least about 80% or 85%, more preferably at least about 90%, and even more preferably. At least about 95%, 97%, 98%, 98.5%, 99% or 99.5% of the "precursor" or "precursor form". Alternatively, using such methods, the purified recombinant lysosomal sulfatase enzyme of the invention is less than about 35%, 30% or 25%, preferably less than about 20% or 15%, more preferably less than about 10%, and even More preferably less than about 5%, 3%, 2%, 1.5%, 1% or 0.5% of "mature", "mature form", "processed" or "processed form". In some embodiments, only the "precursor" or "precursor form" is detected, that is, the sulfatase preparation is subjected to SDS-PAGE under reducing conditions or by SDS-CGE, and is basically determined by a single detectable. The tape composition; or consists essentially of a single peak when analyzed by HPLC.

In the case of two or more polynucleotide or polypeptide sequences, the term "consistent" or "consistency" refers to two or more sequences or sub-sequences when compared and aligned for maximum correspondence. The same nucleotide or amino acid residues of the same sequence or with a specified percentage are measured using one of the following sequence comparison algorithms or by visual inspection.

"Linker" refers to a molecule that joins two other molecules in a covalent manner or via ionic, van der Waals, or hydrogen bonding, for example, hybridizing to a complementary sequence at the 5' end and hybridizing to another complementary sequence at the 3' end. Thereby, the nucleic acid molecules of the two non-complementary sequences are joined.

"Low-level phosphorylation" or "hypophosphorylation" refers to a lysosomal sulfatase enzyme preparation in which the uptake to fibroblasts has a maximum concentration of more than 10 nM or is bound to mannose-6-phosphate. The fraction of lysosomal sulfatase enzyme in the body column is less than about 25%.

"Natural presence" when applied to an object means that the object is visible in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by humans in the laboratory is naturally occurring.

"Pharmaceutical composition" means a composition suitable for pharmaceutical use in a target animal, including humans and mammals. The pharmaceutical compositions comprise a pharmacologically effective amount of a therapeutic lysosomal sulfatase enzyme and also comprise one or more pharmaceutically acceptable carriers, diluents or excipients. A pharmaceutical composition encompasses a composition comprising an active ingredient and an inert ingredient comprising a carrier, diluent or excipient, and a combination, combination or aggregation of any two or more components or decomposition by one or more Any product produced directly or indirectly by other types of reactions or interactions of one or more components. Accordingly, the pharmaceutical compositions of the present invention encompass any composition prepared by incorporating a lysosomal sulfatase enzyme of the invention with one or more pharmaceutically acceptable carriers, diluents or excipients.

"Pharmaceutically acceptable carrier, diluent or excipient" means any standard pharmaceutical carrier, diluent, buffer, and excipient such as, but not limited to, phosphate buffered saline solution, 5% A solution of dextrose, and an emulsion (such as an oil/water or water/oil emulsion), and various types of humectants and/or adjuvants. Suitable pharmaceutical carriers, diluents or excipients and formulations are described in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers, diluents or excipients will depend on the intended mode of administration of the active agent. Typical modes of administration include, for example, but not limited to, enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal) injections; or topical, transdermal, or transmucosal administration.

A "pharmaceutically acceptable salt" is a salt which can be formulated into a lysosomal sulfatase enzyme for medical use, and includes, for example, a metal salt (sodium, potassium, magnesium, calcium, etc.) and a salt of ammonia or an organic amine.

"Polynucleotide" means a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") as well as nucleic acid analogs. A nucleic acid analog comprises a nucleotide comprising a non-naturally occurring base, a linkage to other nucleotides (in addition to a naturally occurring phosphodiester bond) or a linkage via a bond other than a phosphodiester bond. A nucleic acid analog of a base. Thus, nucleotide analogs include, for example, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, phosphonic acids. Methyl ester, p-palmitine methyl phosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA) and analogs thereof. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to a larger polynucleotide. The term "oligonucleotide" generally refers to a shorter polynucleotide that generally does not exceed about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences (i.e., A, U, G, C), wherein "U" replaces "T".

"Polypeptide" means a polymer composed of amino acid residues, their associated naturally occurring structural variants, and synthetic non-naturally occurring analogs linked via peptide bonds, their associated naturally occurring structural variants and synthetic non-natural An analogue of existence. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term "protein" generally refers to a larger polypeptide. The term "peptide" generally refers to a shorter polypeptide. A conventional notation is used herein to depict a polypeptide sequence: the left-hand end of the polypeptide sequence is the amino terminus; the right-hand end of the polypeptide sequence is the carboxy terminus.

"Introduction" refers to a polynucleotide that is capable of specifically hybridizing to a given polynucleotide template and providing a starting point for the synthesis of a complementary polynucleotide. This synthesis occurs when the polynucleotide primer is placed under conditions that induce synthesis (i.e., in the presence of nucleotides, complementary polynucleotide templates, and reagents for polymerization such as DNA polymerase). The primer is usually a single strand, but it can be double stranded. Primers are usually deoxyribonucleic acids, but a variety of synthetic and naturally occurring primers are also suitable for many applications. The primer is complementary to the template (the primer is designed to hybridize to the template to serve as the starting site for the synthesis), but does not need to reflect the exact sequence of the template. In this case, the specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, for example, chromogenic, radioactive or fluorescent moieties and used as detectable moieties.

"Probe" when used in reference to a polynucleotide refers to a polynucleotide that is capable of specifically hybridizing to a specified sequence of another polynucleotide. The probe specifically hybridizes to the target complementary polynucleotide, but does not need to reflect the exact complement of the template. In this case, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. The probe can be labeled, for example, with a color-producing, radioactive or fluorescent moiety and used as a detectable moiety.

"Prophylactic" treatment is a treatment administered to an individual who does not exhibit signs of disease or exhibits only early signs for the purpose of reducing the risk of developing a lesion. The compounds of the present invention can be administered as a prophylactic treatment to reduce the likelihood of developing a lesion or, if manifested, to minimize the severity of the lesion.

"Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. The amplified or assembled recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a host cell. A host cell comprising a recombinant polynucleotide is referred to as a "recombinant host cell." The gene is then expressed in a recombinant host cell to produce, for example, a "recombinant polypeptide." Recombinant polynucleotides can also function as non-coding (eg, promoter, origin of replication, ribosome binding site, etc.).

"Specific hybridization with", "specific hybridization" or "selective hybridization with" refers to a nucleic acid molecule when a specific nucleotide sequence is present in a complex mixture (eg, total cell) DNA or RNA. It binds preferentially to the sequence, duplexing or hybridization under stringent conditions.

The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target subsequence and, to a lesser extent, to other sequences or not to other sequences at all. In the case of nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization" and "stringent hybridization wash conditions" are dependent on the sequence and differ under different environmental parameters. A broad guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Chapter 2, Part I, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York in. In general, highly stringent hybridization and washing conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) of the particular sequence at the determined ionic strength and pH. Tm is the temperature at which the 50% target sequence hybridizes to a perfectly matched probe (under determining ionic strength and pH). Extremely stringent conditions are chosen to be equal to the Tm of a particular probe.

An example of stringent hybridization conditions for hybridizing complementary nucleic acids having more than 100 complementary residues on a filter in a Southern or Northern dot method is: 50% formalin containing 1 mg heparin at 42 °C , cross-breeding overnight. An example of one of the highly stringent washing conditions is: 0.15 M NaCl at 72 ° C for about 15 minutes. An example of stringent washing conditions is: washing with 0.2 x SSC at 65 °C for 15 minutes (for a description of SSC buffer, see Sambrook et al.). Typically, a low stringency wash is performed prior to high stringency wash to remove background probe signals. An exemplary medium stringency wash, such as a double helix of more than 100 nucleotides, is: 1 x SSC at 45 °C for 15 minutes. For example, an exemplary low stringency wash of a double helix of more than 100 nucleotides is: 4-6 x SSC at 40 °C for 15 minutes. In general, in a particular hybridization assay, the signal to noise ratio is 2 times (or more than 2 times) the signal to noise ratio observed for an unrelated probe indicating that specific hybridization is detected.

The "individual" of diagnosis or treatment is a human or non-human animal, including mammals or primates.

In the case of two nucleic acids or polypeptides, the phrase "substantially homologous" or "substantially identical" generally means that when compared and aligned for maximum correspondence, two or more sequences or subsequences have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residue identity, as used by one of the following sequence comparison algorithms or by Visually inspect the measurements. Preferably, the substantial identity is within a sequence region of at least about 50 residues in length, more preferably within a region of at least about 100 residues, and optimally, the sequence is at least about 150 residues. The scope is substantially consistent. In a preferred embodiment, the sequences are substantially identical over the entire length of either or both of the biopolymers.

For sequence comparisons, typically one sequence serves as a reference sequence against which the test sequence is compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

For example, by Smith and Waterman,Adv.Appl.Math2:482,1981 Local homology algorithm, by Needleman and Wunsch,J.Mol.Biol. 48:443, 1970 homology alignment algorithm, by Pearson and Lipman,Proc. Natl. Acad. Sci. USA 85:2444, 1988, similarity search method, computerized tool by this algorithm (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI in GAP) , BESTFTT, FASTA, and TFASTA) or by visual inspection to make an optimal alignment of the sequences for comparison.

An example of a suitable algorithm is PILEUP. PILEUP uses progressive pairwise alignments to generate multiple sequence alignments of a set of related sequences to show relationship and percent sequence identity. It also plots a tree or tree that shows the clustering relationship used to generate the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng and Doolittle, J. Mol. Evol. 35:351-360,1987. The method used is similar to that described by Higgins and Sharp, CABIOS 5: 151-153, 1989. The program can align up to 300 sequences, each of which can have a maximum length of 5,000 nucleotides or an amino acid. The multiple alignment program begins with a pair of identical sequences and produces a cluster of two aligned sequences. This cluster is then aligned with the cluster of the next most relevant sequence or aligned sequence. The two sequence clusters are aligned by simply extending the alignment pairs of the two individual sequences. The final comparison is achieved by a series of progressive comparisons. The program is executed by specifying the amino acid or nucleotide coordinates of a particular sequence and its region for sequence comparison and by specifying program parameters. For example, the following parameters can be used to compare a reference sequence to other test sequences to determine a percent sequence identity: preset gap weight (3.00), preset gap length weight (0.10), and weighted end gap. Another algorithm suitable for generating multiple alignments of sequences is Clustal W (Thompson et al, Nucleic Acids Research 22: 4673-4680, 1994).

Another example of an algorithm suitable for determining sequence identity and percent sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215: 403-410, 1990. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying a high-scoring sequence pair (HSP) by identifying a short word of length W in the query sequence that matches or satisfies a certain positive threshold when compared to a word of the same length in the database sequence. T. T is referred to as the proximity word score threshold (Altschul et al, J. Mol. Biol. 215: 403-410, 1990). These initial neighborhood word matches serve as seeds for the initial search to find longer HSPs containing them. Word matching then extends in both directions along the sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (a reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For the amino acid sequence, a scoring matrix is used to calculate the cumulative score. The extension of the word match in each direction is stopped in the following cases: the cumulative comparison scores from the maximum amount X that it achieves; due to the accumulation of one or more negative score residues, the cumulative score changes Be 0 or below; or reach the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses word length (W) 11, expected value (E) 10, M = 5, N = -4, and a comparison of the two strands as a preset value. For amino acid sequences, the BLASTP program uses word length (W) 3, expected value (E) 10, and BLOSUM62 scoring matrix as presets (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 , 1989).

In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between the two sequences (see, eg, Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001 in comparison to the test nucleic acid and the reference nucleic acid.

As described below, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid can be immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. Thus, for example, when two peptides differ only in conservative substitutions, the polypeptide will generally be substantially identical to the second polypeptide. As indicated herein, another indicator that the two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

"Substantially pure" or "separated" means that the target substance is the main substance present (ie, in molar amount, greater than any other individual macromolecular substance in the composition), and the substantially purified part is the target. A composition comprising at least about 50% (in moles) of all macromolecular species present. In general, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition are related purified materials. If the composition consists essentially of a single macromolecular material, the target material is purified to substantial homogeneity (contaminants in the composition cannot be detected by conventional detection methods). For the purposes of this definition, solvent species, small molecules (<500 Daltons), stabilizers (eg, BSA), and elemental ionic species are not considered macromolecular materials. In some embodiments, the lysosomal sulfatase enzymes of the invention are substantially pure or isolated. In some embodiments, the lysosomal sulfatase enzymes of the invention are substantially pure or isolated relative to the macromolecular starting materials used in their synthesis. In some embodiments, a pharmaceutical composition of the invention comprises a substantially purified or isolated therapeutic lysosomal sulfatase enzyme blended with one or more pharmaceutically acceptable carriers, diluents or excipients.

"Therapeutic" treatment is the treatment administered to an individual exhibiting their signs or symptoms for the purpose of attenuating or eliminating the signs or symptoms of the lesion. Signs or symptoms can be biochemical, cellular, tissue, functional, subjective or objective signs or symptoms. The lysosomal sulfatase enzyme of the present invention can be administered as a therapeutic therapeutic agent or for achieving a diagnosis.

"Therapeutic index" means a dose range (amount and/or timing) above the minimum therapeutic amount and below an unacceptable toxic amount.

"Treatment" means prophylactic or therapeutic or diagnostic treatment.

The term "unit dosage form" as used herein refers to a physical individual unit suitable for use as a single dose in human and animal subjects, each unit containing a predetermined amount of a lysosomal sulfatase enzyme of the invention, the predetermined amount being The amount of one or more pharmaceutically acceptable carriers, diluents or excipients combined is sufficient to produce the desired effect. The specifications of the novel unit dosage forms of the invention depend on the particular lysosomal sulfatase enzyme used and the effect to be achieved and the pharmacodynamics associated with each lysosomal sulfatase enzyme in the host.

II. Production of lysosomal sulfatase

In one aspect, the invention features a novel method of producing such enzymes capable of achieving therapeutic use of an active highly phosphorylated lysosomal sulfatase enzyme. In general, the method is characterized by using a cDNA encoding human sulfatase modification factor 1 (SUMF1) or a biologically active fragment, mutant, variant or derivative thereof and encoding a full-length lysosomal sulfatase enzyme or biological activity thereof The cDNA transformation of fragments, mutants, variants or derivatives is suitable for cell lines. Those skilled in the art can prepare expression constructs other than the expression constructs expressly described herein to achieve optimal production of such lysosomal sulfatase enzymes in suitable transfected cell lines. In addition, those skilled in the art can readily design biologically active fragments, variants, mutants or derivatives encoding naturally occurring SUMF1 or a lysosomal sulfatase enzyme having the same or similar biological activity as a naturally occurring full length enzyme. Fragment of cDNA.

Host cell

The host cell for producing a recombinant lysosomal sulfatase enzyme is a cell strain lacking endosome acidification, characterized in that it is capable of producing such lysosomes capable of achieving therapeutic use of such lysosomal sulfatase enzymes. Sulfate esterase. The present invention provides a CHO-K1-derived END3 complementary population cell line called G71. The present invention also provides a G71 cell line suitable for growth in serum-free suspension culture, referred to as G71S. The present invention also provides derivatives of G71 and G71S cell lines which have been further sub-selected or contain different expression plastids.

Cells containing and expressing DNA or RNA encoding a recombinant protein are referred to herein as genetically modified cells. A mammalian cell containing and expressing DNA or RNA encoding a recombinant protein is referred to as a genetically modified mammalian cell. Introduction of DNA or RNA into cells by known transfection methods such as, but not limited to, electroporation, microinjection, microprojectile bombardment, calcium phosphate precipitation, modified calcium phosphate precipitation, cationic lipids Treatment, photoporation, fusion methods, receptor-mediated transfer, or polybrene precipitation are achieved. Alternatively, DNA or RNA can be introduced by infection with a viral vector. Methods for producing cells (including mammalian cells) that express DNA or RNA encoding recombinant proteins are described in the following applications: Richard F Selden, Douglas A. Treco, and Michael W. Heartlein (November 4, 1994) U.S. Patent Application Serial No. 08/334,797, entitled "In Vivo Protein Production and Delivery System for Gene Therapy"; Richard F Selden, Douglas A. Treco, and Michael W. Heartlein (Application on November 4, 1994) U.S. Patent Application Serial No. 08/334,455, entitled "In Vivo Production and Delivery of Erythropoietin or Insulinotropin for Gene Therapy"; and Douglas A. Treco, Michael W. Heartlein, and Richard F Selden (April 20, 1994) U.S. Patent Application Serial No. 08/231,439, entitled "Targeted Introduction of DNA Into Primary or Secondary Cells and Their Use for Gene Therapy". The teachings of each of these applications are expressly incorporated herein by reference in their entirety.

In a preferred embodiment, the host cell for producing a recombinant lysosomal sulfatase enzyme is a cell line lacking for endosomal acidification, characterized in that it is capable of producing a therapeutic use capable of achieving such lysosomal sulfatase enzymes. The amount of these lysosomal sulfatase enzymes. In a preferred embodiment, the present invention provides a CHO-K1-derived END3 complementary population cell line designated G71, and a G71 cell line designated G71S suitable for growth in serum-free suspension culture. The cell line expresses human sulfatase-modifying factor 1 (SUMF1) and recombinant lysosomal sulfatase, and is therefore capable of producing high-yield active highly phosphorylated lysosomal sulfatase enzymes as specified in the definition. This enables a large-scale production of therapeutic lysosomal sulfatase enzymes. In a preferred embodiment, the G71 or G71S cell line or derivative thereof is at least about 0.5 pg/cell/day, preferably at least about 0.75 pg/cell/day, more preferably at least about 1.0 pg/cell/day. And even more preferably at least about 1.25 picograms per cell per day of expression and secretion of recombinant lysosomal sulfatase.

Vector and nucleic acid construct

A nucleic acid construct for expressing a recombinant protein (human sulfatase modifying factor 1 (SUMF1) or lysosomal sulfatase or both) may be a nucleic acid construct for extrachromosomal (free) expression in transfected mammalian cells A nucleic acid construct that is integrated into the genome of the recipient cell via homologous recombination, either randomly or at a pre-selected targeting site. In addition to the recombinant protein coding sequence, the extrachromosomal construct also contains sequences sufficient to express the protein in the cell and, where appropriate, the replication of the construct. It typically includes a promoter, a recombinant protein encoding DNA, and a polyadenylation site. The DNA encoding the recombinant protein is positioned in the construct in such a way that its expression is under the control of the promoter. Optionally, the construct may contain additional components, such as one or more of the following: a splicing site, an enhancer sequence, a selectable marker gene under the control of an appropriate promoter, an amplifiable marker gene under the control of an appropriate promoter, and A matrix attachment region (MAR) or other element known in the art that enhances the performance of the region into which it is inserted.

In those embodiments in which the DNA construct is integrated into the genome of the cell, it is desired to include only the recombinant protein encoding nucleic acid sequence. Optionally, it may include a promoter and enhancer sequence, a polyadenylation site, a splicing site, a nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an amplifiable marker, a matrix attachment region (MAR), or the like. Other elements known to enhance the performance of the region into which they are inserted, and/or DNA homologous to genomic DNA in the recipient cell, such that integration of DNA targets selected sites in the genome (to target DNA) Or DNA sequence).

Cell culture method

Mammalian cells containing DNA or RNA encoding the recombinant protein are cultured under conditions suitable for cell growth and DNA or RNA expression. The cells expressing the recombinant protein can be identified using known methods and methods described herein, and the recombinant protein can be isolated and purified using known methods and methods described herein, in the presence or absence of expanded recombinant protein production. . Genetically modified mammalian cells indicative of the presence of a phenotype of DNA or RNA encoding a recombinant protein can be displayed, for example, via screening (such as PCR screening, screening by Southern blot analysis, or screening for expression of recombinant proteins). To identify. Selection of cells containing the combined recombinant protein-encoding DNA can be achieved by including a selectable marker in the DNA construct, followed by culturing the selectable marker gene under conditions suitable for the survival of only those cells expressing the selectable marker gene. Transfected or infected cells. Further amplification of the introduced DNA construct can be achieved by culturing genetically modified mammalian cells under appropriate conditions (eg, a drug concentration that is viable in cells containing only multiple copies of the amplifiable marker gene) A genetically modified mammalian cell containing an amplifiable marker gene is cultured in the presence thereof.

As described herein, genetically modified mammalian cells expressing recombinant proteins can be identified by detecting performance products. For example, mammalian cells that exhibit active highly phosphorylated lysosomal sulfatase enzymes can be identified by sandwich enzyme immunoassays. The antibody can be directed to the active agent moiety.

Variant of lysosomal sulfatase

In certain embodiments, active highly phosphorylated lysosomal sulfatase mutants or variants can be prepared and will be suitable for a variety of applications in which active hyperphosphorylated lysosomal sulfatase enzymes can be used. The amino acid sequence mutant or variant of the polypeptide may be a substituted, inserted or deleted mutant or variant. Deletion mutants or variants lack one or more residues of the native protein that are not required for functional or immunogenic activity. One common type of deletion mutant or variant is a deletion mutant or variant lacking a secretion signal sequence or a signal sequence that directs binding of a protein to a particular portion of a cell. Insertion mutants or variants typically involve the addition of a substance at a non-terminal end of the polypeptide. This may include insertion of an immunoreactive epitope or only a single residue. End additives, also known as fusion proteins, are discussed below.

The variant may be substantially homologous or substantially identical to the unmodified lysosomal sulfatase enzyme as described above. Preferred variants are variants which are active highly phosphorylated lysosomal sulfatase polypeptide variants which retain at least some of the biological activity (e.g., sulfatase activity) of the lysosomal sulfatase enzyme. Other preferred variants include human N-acetylgalactosamine-6-sulfatase polypeptide variants that retain at least some of the sulfatase activity of human N-acetylgalactosamine-6-sulfatase.

A substituted mutant or variant typically exchanges one amino acid of the wild type polypeptide to another amino acid at one or more sites within the protein and can be designed to be free of loss of other functions or properties. One or more properties of the polypeptide are modulated, such as, but not limited to, stability against proteolytic cleavage. Such substitutions are preferably conservative, i.e., an amino acid is replaced by an amino acid having a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamic acid or histidine; aspartate To glutamic acid; cysteine to serine; glutamic acid to aspartame; glutamic acid to aspartic acid; glycine to valine; histidine to aspartame or Branic acid; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or iso-white Aminic acid; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan Or phenylalanine; and valine to isoleucine or leucine.

One aspect of the invention encompasses the production of a glycosylation site mutant or variant in which the O-linked or N-linked glycosylation site of the lysosomal sulfatase has been mutated. Such mutants or variants will produce important information about the biological activity, physical structure and binding potential of the active highly phosphorylated lysosomal sulfatase enzyme. In particular aspects, it is contemplated that other mutants or variants of the active highly phosphorylated lysosomal sulfatase polypeptide will be produced which retain biological activity but are increased or decreased in binding activity. Likewise, mutations in the active or catalytic regions are specifically contemplated to produce protein mutants or variants that are altered in binding activity. In these examples, the sequence of the active highly phosphorylated lysosomal sulfatase enzyme is compared to the sequences of other related enzymes and the selected residues are specifically mutated.

The numbering of the amino acids of the mature protein is numbered from the putative amine terminus of the amino acid No. 1, and exemplary exemplary mutations include, for example, substitution of all or some of the potentially glycosylated aspartames, including recombinant human N- Positions 178 and 397 of acetaminosamine-6-sulfatase (GALNS) (see Figure 5 ).

The binding of the substrate can be altered by mutations at/near the active site of the lysosomal sulfatase enzyme. Given that such mutations are exemplary, those skilled in the art will recognize that other mutations can be made to the enzyme sequence to provide additional structural and functional information about the protein and its activity.

In order to construct mutants or variants such as those described above, those skilled in the art can employ well-known standard techniques. In particular, N-terminal deletions, C-terminal deletions, internal deletions, and random and point mutation induction are contemplated.

The N-terminal and C-terminal deletions are deletion mutant-inducing forms that utilize, for example, a single restriction site in the vicinity of the end of the C-terminus or N-terminal region. The DNA is cleaved at this site and the cleavage ends are degraded by nucleases such as BAL31, exonuclease III, DNase I and S1 nuclease. Rejoining both ends produces a series of DNAs with different sizes of deletions around the restriction sites. Appropriate biological functions, such as enzymatic activity, of a protein expressed by such a mutant can be assayed using standard techniques in the art and techniques described in this specification. A similar technique can be employed for internal deletion mutants by using two appropriately placed restriction sites, thereby allowing for the creation of precisely defined deletions and rejoining the ends as described above.

Partially digested mutants are also covered. In such cases, those skilled in the art will employ "frequent cutters" which cut DNA at a variety of locations depending on the length of the reaction. Thus, by varying the reaction conditions, it will be possible to generate a series of mutants of different sizes which can then be screened for activity.

Random insertional mutagenesis can also be performed by cleavage of the DNA sequence with, for example, DNase I and insertion of a stretch of nucleotides encoding 3, 6, 9, 12 (etc.) amino acids and ligating the ends. Once such a mutation is produced, the mutant can be screened for various activities exhibited by the wild-type protein.

Point mutation induction can also be employed to specifically identify which amino acid residues are important in the particular activity associated with lysosomal sulfatase biological activity. Thus, those skilled in the art will be able to generate single base changes in DNA strands to produce altered codons and missense mutations.

The amino acid of a particular protein can be altered to produce an equivalent, or even a modified second generation molecule. Such alterations encompass the substitution of a predetermined amino acid of a protein without significant loss of the ability to bind to the interaction of a structure such as an antigen binding region of an antibody or a binding site on a receptor molecule or receptor. Because the ability and nature of protein interactions define the biological functional activity of a protein, certain amino acid substitutions can be made in the protein sequence and its underlying DNA coding sequence, and proteins with similar properties are still obtained. Thus, as discussed below, various changes can be made in the DNA sequence of a gene without significant loss of biological utility or activity.

In making these changes, the hydropathic index of the amino acid can be considered. It is recognized that the relatively hydrophilic nature of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (eg, enzymes, receptors, receptors, DNA, antibodies, antigens, and the like) . Each amino acid has been assigned a hydrophilicity index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, J. Mol. Biol. , 157(1): 105-132, 1982, incorporated herein by reference). In general, the amino acid can be substituted with other amino acids having a similar hydrophilicity index or score and still produce a protein with similar biological activity, i.e., a protein that still achieves biologically functional equivalents.

In addition, a similar amino acid-based substitution can be efficiently performed based on hydrophilicity. U.S. Patent No. 4,554,101, the disclosure of which is incorporated herein by reference in its entirety the entire entire entire entire entire entire entire entire entire entire entire portion Likewise, the amino acid can be substituted with another amino acid having a similar hydrophilicity value and still obtain a bioequivalent and immunologically equivalent protein.

Exemplary amino acid substitutions useful in this aspect of the invention include, but are not limited to, the exchange of arginine and lysine; glutamic acid and aspartic acid; serine and threonine; Acid and aspartame; and valine, leucine and isoleic acid. Those skilled in the art will be familiar with other such substitutions that require retention of some or all of the biological activity while altering the secondary structure of the protein.

Another type of variant contemplated for use in the preparation of the polypeptides of the invention is the use of peptidomimetics. The mimetic is a peptide-containing molecule that mimics the elements of the secondary structure of the protein. See, for example, Johnson et al., "Peptide Turn Mimetics", BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Chapman and Hall, New York (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of the protein is primarily present in a manner that facilitates molecular interactions, such as the interaction of the antibody with the molecules of the antigen, to direct the amino acid side chains. Peptide mimetics are expected to allow molecular interactions similar to natural molecules. These principles can be used in conjunction with the above principles to engineer second generation molecules having many of the natural properties of lysosomal sulfatase enzymes but with altered characteristics and even improved.

Modified glycosylation of lysosomal sulfatase

An active highly phosphorylated lysosomal sulfatase variant having a glycosylation pattern modified relative to the parent polypeptide, such as deletion of one or more carbohydrate moieties and/or addition of one or more non-existing natural polypeptides, may also be produced. The glycosylation site in the middle.

Glycosylation is typically an N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartate residue. The tripeptide sequence aspartame-X-serine and aspartame-X-threonine (X is any amino acid other than proline) is a carbohydrate moiety and an aspartic acid side chain Enzyme-linked recognition sequence. The presence of any of these tripeptide sequences in the polypeptide will result in a potential glycosylation site. Thus, an N-linked glycosylation site can be added to the polypeptide by altering the amino acid sequence such that it contains one or more of these tripeptide sequences. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxylamino acid, most commonly serine or threonine, but 5-hydroxyindole can also be used. Amine acid or 5-hydroxy lysine. The O-linked glycosylation site can be added by insertion or substitution of one or more serine or threonine residues in the sequence of the original polypeptide.

Domain conversion

The various parts of the lysosomal sulfatase protein have substantial sequence homology. Mutations in the lysosomal sulfatase polypeptide that alter its function can be identified. These studies may be important for at least two reasons. First, it provides the reasonable expectation that other homologs, dual gene variants, and mutants of this gene may be present in related species, such as rats, rabbits, monkeys, gibbons, chimpanzees, baboons, baboons, Among cows, pigs, horses, sheep and cats. Certain activities or domains can be identified after isolation of such homologs, variants, and mutants, and with other assays. Second, this will provide a starting point for further mutation analysis of the molecules described above. One way to take advantage of this information is in "domain conversion."

Domain switching involves the use of different but related polypeptides to produce recombinant molecules. For example, by comparing the sequence of a lysosomal sulfatase enzyme (eg, N-acetylgalactosamine-6-sulfatase) with a sequence of a similar lysosomal sulfatase enzyme from another source, and Comparing with mutants of such polypeptides and dual gene variants, predictions can be made regarding functionally important regions of such molecules. It is then possible to switch the relevant domains of these molecules in an attempt to determine the critical extent of these regions for enzyme function and effect in lysosomal storage disorders. Such molecules may have other value because such "chimeras", although different from natural molecules, may provide the same or even enhanced function.

Further analysis of the effects of mutations and their predictions on secondary structure will deepen this understanding based on the numerous lysosomal sulfatase enzymes currently identified. Mutants that are expected to switch between domains between lysosomal sulfatase enzymes will provide useful information about the structural/functional relationship of such molecules to the polypeptide with which they interact.

Fusion protein

In addition to the above mutations, the invention additionally encompasses the production of a particular class of insertional variants known as fusion proteins. This molecule typically has all or a substantial portion of the native molecule that is linked at the N-terminus or C-terminus to all or a portion of the second polypeptide. For example, fusions typically employ a leader sequence from other species to allow for recombinant expression of the protein in a heterologous host. Another suitable fusion involves the addition of an immunologically active domain such as an antibody epitope to facilitate purification of the fusion protein. Including a cleavage site at or near the fusion junction will facilitate removal of the foreign polypeptide after purification. Other suitable fusions include a functional domain (such as an active site from an enzyme), a glycosylation domain, a cell targeting signal, or a junction of a transmembrane region.

A variety of commercially available fusion protein expression systems are available for use in the present invention. Particularly suitable systems include, but are not limited to, glutathione S-transferase (GST) system (Pharmacia, Piscataway, NJ), maltose binding protein system (NEB, Beverley, MA), FLAG system (IBI, New Haven, CT), 6 x His system (Qiagen, Chatsworth, CA). Such systems are capable of producing recombinant polypeptides that carry only a small amount of other amino acids that are unlikely to affect the antigenic ability of the recombinant polypeptide. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be weakly antigenic and do not adversely affect the folding of the polypeptide into its native configuration. Another N-terminal fusion that is expected to be applicable is the fusion of the Met-Lys dipeptide at the N-terminal region of the protein or peptide. Such fusions can advantageously increase protein expression or activity.

A particularly useful fusion construct can be a fusion construct in which an active highly phosphorylated lysosomal sulfatase polypeptide or a fragment thereof is fused to a hapten to enhance the immunogenicity of a lysosomal sulfatase fusion construct. This can be applied to the production of antibodies against active highly phosphorylated lysosomal sulfatase enzymes to enable detection of proteins. In other embodiments, fusion constructs that target a lysosomal sulfatase-related composition to a particular site or cell can be prepared.

Other fusion constructs include heterologous peptides having the desired properties, such as targeting lysosomal sulfatase enzymes to specific organs, tissues or cell types. In a preferred embodiment, a fusion construct comprising a peptide targeting a bone, such as 6 aspartate residues (6 x Asp or 6D) fused to a lysosomal sulfatase enzyme, can target the enzyme to the bone a specific part of it.

Other fusion constructs comprising a heterologous polypeptide having the desired properties (eg, an Ig constant region that extends serum half-life or an antibody or fragment thereof for targeting) are also contemplated. Other fusion systems produce polypeptide hybrids in which the fusion partner needs to be excised from the desired polypeptide. In one embodiment, the fusion partner is linked to a recombinant active highly phosphorylated lysosomal sulfatase polypeptide by a peptide sequence comprising a protease-specific recognition sequence. An example of a suitable sequence is the sequence recognized by Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA).

derivative

As noted above, a derivative refers to a polypeptide that is chemically modified by techniques such as, but not limited to, ubiquitination, labeling (eg, with radionuclides or various enzymes), such as pegylation (using polyethylene glycol) The covalent polymer linkage of the alcohol derivative) and the insertion or substitution by chemical synthesis of an amino acid such as ornithine. Derivatives of lysosomal sulfatase enzymes are also suitable as therapeutic agents and can be produced by the methods of the invention.

Polyethylene glycol (PEG) can be linked to a lysosomal sulfatase enzyme produced by the methods of the invention to provide a longer in vivo half-life. The PEG group can have any suitable molecular weight and can be straight or branched. The average molecular weight of the PEG will preferably range from about 2 kilodaltons ("kDa") to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, optimally from about 5 kDa to about 10 kDa. The PEG group will typically be via a reactive group (eg, an aldehyde group, an amine group, a thiol group, or an ester group) on the PEG moiety and a reactive group (eg, an aldehyde group, an amine group, or an ester group) on the protein moiety. Deuterated or reductive alkylation to attach to the lysosomal sulfatase enzyme of the invention. The addition of the PEG moiety to the relevant polypeptide can be carried out using techniques well known in the art. See, for example, International Publication No. WO 96/11953 and U.S. Patent No. 4,179,337.

The attachment of the lysosomal sulfatase polypeptide to PEG typically occurs in the aqueous phase and can be readily monitored by reverse phase analytical HPLC. The pegylated peptide can be readily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis, and laser desorption mass spectrometry.

mark

In some embodiments, the therapeutic lysosomal sulfatase enzyme is labeled to facilitate its detection. A "marker" or "detectable portion" is a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, labels suitable for use in the present invention include, but are not limited to, radioactive labels (eg, ³² P), fluorophores (eg, luciferin), electronic densities, enzymes (eg, commonly used in ELISA), biotin , digoxin, or hapten and protein, such haptens and proteins can be detected, for example, by incorporating a radioactive label into a hapten or peptide, or for detecting specificity with a hapten or peptide An antibody to a sexual reaction.

Examples of labels suitable for use in the present invention include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), Radiolabel (eg ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), enzymes (eg horseradish peroxidase, alkaline phosphatase) and commonly used in ELISA Other enzymes) and colorimetric labels (such as colloidal gold, tinted glass or plastic beads (eg, polystyrene, polypropylene, latex, etc.)).

The label can be coupled directly or indirectly to the desired component of the lysosomal sulfatase enzyme according to methods well known in the art. Preferably, in one embodiment, an isocyanate reagent is used in conjunction with an active agent of the invention to covalently bind a label to a lysosomal sulfatase enzyme. In one aspect of the invention, the bifunctional isocyanate reagent of the present invention can be used to bind a label to a lysosomal sulfatase enzyme to form a labeled lysosomal sulfatase conjugate that is not linked to an active agent. The labeled lysosomal sulfatase conjugate can be used as an intermediate in the synthesis of the labeled conjugates of the invention or can be used to detect lysosomal sulfatase conjugates. As indicated above, a variety of labels can be used, wherein the choice of label depends on the sensitivity desired, the ease of binding to the desired components of the lysosomal sulfatase enzyme, stability requirements, available instrumentation and disposal regulations. Non-radioactive labels are often linked by indirect means. In general, a ligand molecule (eg, biotin) is covalently bound to a lysosomal sulfatase enzyme. The ligand then binds to another molecule, such as streptavidin, which is inherently detectable or covalently associated with a signaling system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Combine.

The lysosomal sulfatase enzyme of the invention may also bind directly to a signal generating compound, for example to an enzyme or a fluorophore. Suitable enzymes for labeling include, but are not limited to, hydrolases, in particular phosphatases, esterases and glycosidases; or oxidases, in particular peroxidases. Fluorescent compounds (i.e., fluorophores) suitable for labeling include, but are not limited to, luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Other examples suitable for fluorophores include, but are not limited to, eosin, TRITC-amine, quinine, luciferin W, acridine yellow, lissamine Rhodamine), B sulfonyl chloride erythroscein, bismuth (paraben, bipyridyl), Texas red, nicotinamide adenine dinucleotide, flavin gland Flavin adenine dinucleotide and the like. Chemiluminescent compounds suitable for labeling include, but are not limited to, luciferin and 2,3-dihydropyridazinedione, such as luminol. For a review of various marking systems or signal generating systems that can be used in the methods of the present invention, see U.S. Patent No. 4,391,904.

Means for detecting indicia are well known to those skilled in the art. Thus, for example, when the marker is radioactive, the detection means includes a scintillation counter or photographic film such as in automatic radiography. When labeled as a fluorescent marker, it can be detected by exciting the fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescent light. Fluorescence can be visually detected by using an electronic detector such as a charge coupled device (CCD) or a photomultiplier and the like. Similarly, enzyme labeling can be detected by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. The colorimetric or chemiluminescent label can be detected simply by observing the color associated with the label. Other marking and detection systems suitable for use in the methods of the present invention will be readily apparent to those skilled in the art. Such labeled modulators and ligands can be used to diagnose a disease or condition.

In a preferred embodiment, the method comprises the step of producing a highly hyperphosphorylated lysosomal sulfatase enzyme from a cell line having a defective endosome transport. In a particularly preferred embodiment, the method comprises the step of producing a highly hyperphosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) from a CHO cell line G71 or a derivative thereof. Production of a lysosomal sulfatase such as, but not limited to, GALNS comprises the steps of: (a) producing a co-expressed recombinant human lysosomal sulfatase enzyme (eg, N-acetylgalactosamine-6-sulfatase) GALNS)) a G71 or G71 derivative cell line with recombinant human sulfatase modification factor 1 (SUMF1); (b) a human lysosomal sulfatase and SUMF1 co-expression cell line; and (c) scale up humans The lysosomal sulfatase enzyme and SUMF1 co-express the cell line into the bioreactor to produce lysosomal sulfatase. In a preferred embodiment, human lysosomal sulfatase (eg, N-acetylgalactosamine-6-sulfatase (GALNS)) and human SUMF1 cDNA are subcloned substantially as described herein below. In mammalian expression vectors.

For the production of cell lines, human GALNS mammalian expression vector, human SUMF1 mammalian expression vector and selectable marker gene were co-transfected with G71 or G71S (adapted to G71 pure line grown in serum-free suspension culture), and stable transitions were selected. . After the first round of stable transfection, the cell line was selected using a fluorescent receptor and was specifically designated. The specific ratio of cells (pick product/cell) of G71 or G71S cell lines in a spinner with a microcarrier or in suspension culture was analyzed separately. The best producer of human GALNS is identified and scaled up into a bioreactor to produce preclinical material.

In another embodiment, the invention provides a cell-based assay for measuring the activity of a recombinant human lysosomal enzyme to degrade a natural receptor. The method comprises (a) culturing an isolated human cell lacking a lysosomal enzyme under conditions in which the natural receptor of the lysosomal enzyme is accumulated; (b) contacting the cell with the lysosomal enzyme; (c) dissolving the (d) adding an enzyme to the cell lysate, the enzyme (i) is specific for the natural receptor, and (ii) cleavage of the small oligosaccharide from the natural receptor; (e) Measuring the small oligosaccharides; (f) isolating the labeled small oligosaccharides as appropriate; (g) detecting the labeled small oligosaccharides; and (h) by comparing (i) The amount of labeled small oligosaccharides of the cells contacted by the lysosomal enzyme is (ii) the amount of labeled small oligosaccharides from cells not contacted with the lysosomal enzyme to determine the natural receptor of the lysosomal enzyme degradation. The activity, wherein a decrease in (h)(i) compared to (h)(ii) indicates that the lysosomal enzyme degrades the activity of the natural substrate. In one embodiment, the small oligosaccharide is a monosaccharide, a disaccharide or a trisaccharide. In a related embodiment, the small oligosaccharide is a disaccharide.

In some embodiments, the lysosomal enzyme is selected from the group consisting of arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide/heparin- N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-acetylgalactosamine-6-sulfatase (GALNS). In some embodiments, the lysosomal enzyme is alpha-L-iduronidase (IDU). In some embodiments, the lysosomal enzyme is an acid alpha-glucosidase (GAA). In some embodiments, the lysosomal enzyme is beta-glucuronidase (GUSB). In some embodiments, the lysosomal enzyme is beta-galactosidase (GLB1).

Suitable human cells that can be used in cell-based assays include any lysosomal enzyme deficiency to be tested, thus accumulating any human cells that naturally interfere with the lysosomal enzyme. For example, cells that exhibit complete (100%) or partial deficiency (eg, 30%, 50%, 70%, 80%, 90%, 95%, or 95% reduction in activity) can be used. A cell expressing a mutant enzyme having reduced activity, or a cell derived from a patient suffering from a lysosomal storage disease such as mucopolysaccharidosis may be used. Cells that are recombinantly altered to knock out or reduce lysosomal enzyme activity, for example, by introducing mutations into the coding gene or its promoter or other regulatory regions, can be used. Cells that have been treated to reduce lysosomal enzyme activity (e.g., treated with antisense or RNAi to reduce enzyme performance) can be used.

Suitable enzymes which cleave (digest) small oligosaccharides from carbohydrates and which are "specific" to the natural receptor of the lysosomal enzyme (i.e., primarily digesting such natural receptors) can be selected by the average skilled person. For example, for detecting the activity of GALNS or GLB1 (an enzyme that degrades keratan sulfate), the enzyme of step (d) may be keratanase II or any enzyme that acts primarily on keratan sulfate. As another example, for detecting IDU, ARSB, IDS, or GUSB (an enzyme that degrades dermatan sulfate), the enzyme of step (d) can be chondroitinase ABC or any enzyme that acts primarily on dermatan sulfate. As another example, for detecting IDU, IDS, SGHS, G6S or GUSB (an enzyme that degrades acesulfate heparin), the enzyme of step (d) may be acetaminogen heparinase I or heparanase II or two. By. As another example, for detecting GAA (an enzyme that degrades glycogen), the enzyme of step (d) can be an alpha-amylase or any enzyme that acts primarily on hepatic glucose.

This cell-based method is extremely sensitive in detecting lysosomal enzyme activity. In some embodiments, when the concentration of the lysosomal enzyme is as low as about 10 nM, or about 5 nM, or about 1 nM, or about 0.75 nM, or about 0.5 nM, or about 0.25 nM, or about 0.1 nM, or Lysosomal enzyme activity can be detected at about 0.05 nM, or about 0.01 nM, or about 0.005 nM, or about 1 pM, or about 0.5 pM.

III. Purification of lysosomal sulfatase

The bioreactor material containing recombinant human GALNS was sterile filtered through 0.2 μm and maintained at 4 °C. The bioreactor material is loaded directly onto the capture column or concentrated 10 to 20 times by ultrafiltration and subsequently loaded onto a capture column. The pH of the bioreactor material or the concentrated bioreactor material was adjusted to pH 4.5, followed by loading on a blue-agarose column, followed by 20 mM acetate/phosphate, 50 mM NaCl (pH). 4.5) Wash with 20 mM acetate/phosphate, 50 mM NaCl (pH 6.0), and dissolve with 20 mM acetate/phosphate, 100 mM NaCl (pH 7.0). The blue-agarose gel column was then loaded onto Fractogel SE Hi-Cap with 20 mM acetate/phosphate, 50 mM NaCl (pH 5.0) and 20 mM acetate/phosphate, 50 in sequence. Wash with mM NaCl (pH 5.5) and dissolve with 20 mM acetate/phosphate, 50-350 mM NaCl gradient (pH 5.5). Fractogel SE Hi-Cap was dispensed in 10 mM NaOAc, 1 mM NaH ₂ PO ₄ , 0.005% Tween-80 (Tween-80) (pH 5.5).

Alternatively, the bioreactor material containing recombinant human GALNS is concentrated 20 times by ultrafiltration and subsequently loaded onto a capture column. The pH of the concentrated bioreactor material was adjusted to pH 4.5, and the filtration was then loaded onto a Fractogel SE Hi-Cap column, sequentially using 10 mM acetate/phosphate, 50 mM NaCl (pH 4.5) and 10 mM acetic acid. The salt/phosphate, 50 mM NaCl (pH 5.0) was washed and dissolved with 10 mM acetate/phosphate, 140 mM NaCl (pH 5.0). The Fractogel SE Hi-Cap column was then adjusted to 500 mM NaCl (pH 7.0) and loaded onto a Zn-chelating agarose gel (Zn-IMAC) column with 10 mM acetate/phosphate, 125 mM. Wash with NaCl, 10 mM imidazole (pH 7.0) and dissolve with 10 mM acetate/phosphate, 125 mM NaCl, 90 mM imidazole (pH 7.0). The Zn-chelating agarose gel (Zn-IMAC) column was adjusted to pH 3.5 to achieve low pH virus inactivation, adjusted to 10 mM acetate/phosphate, 2 M NaCl (pH 5.0), and then loaded. It was washed on a ToyoPearl butyl 650 M column with 10 mM acetate/phosphate, 2 M NaCl (pH 5.0), and dissolved in 10 mM acetate/phosphate, 0.7 M NaCl (pH 5.0). ToyoPearl butyl 650M was ultrafiltered and diafiltered in 20 mM acetate, 1 mM phosphate, 150 mM NaCl (pH 5.5), followed by 20 mM acetate, 1 mM phosphate, 150 mM NaCl, 0.01% Formulated in Tween-20 (pH 5.5).

Alternatively, the bioreactor material containing recombinant human GALNS is filtered, concentrated 20 times by ultrafiltration/diafiltration, then filtered through activated carbon, and subsequently loaded onto a capture column. The concentrated bioreactor material was loaded onto a Zn-chelating agarose gel FF (Zn-IMAC) column at a conductivity of approximately 55 ± 5 mS/cm, sequentially using 10 mM acetate/phosphate, 500 mM. Wash with NaCl (pH 7.0) and 10 mM acetate/phosphate, 125 mM NaCl (pH 7.0) (buffer A), followed by 70% buffer A with 30% 10 mM acetate/phosphate, 125 mM NaCl, A mixture of 300 mM imidazole (pH 7.0) (buffer B) was dissolved. The Zn Chelate Sepharose FF (Zn-IMAC) column was adjusted to a conductivity of approximately 6.0 ± 0.5 mS/cm and pH 7.0 and loaded onto a Mustang Q filter to potentially remove the virus. The Mustang Q filtrate was adjusted to pH 4.5 ± 0.1, filtered sequentially through a CUNO 60ZA filter and a 0.2 μm in-line filter, and then loaded onto a Fractogel EMD SE Hi-Cap (M) column, sequentially using 10 mM acetate 1 phosphate. Salt, 50 mM NaCl (pH 4.5) and 80% buffer A (10 mM acetate/phosphate (pH 5.0)) with 20% buffer B (10 mM acetate/phosphate, 250 mM NaCl (pH 5.0) The mixture was washed and dissolved in a linear gradient of 20%-75% Buffer B (in 80%-25% Buffer A). The Fractogel EMD SE Hi-Cap column was then adjusted to pH 3.5 ± 0.1 by the addition of 0.2 M citrate buffer (pH 3.4) to achieve low pH virus inactivation. Fractogel EMD SE Hi-Cap column insoluble solution with low pH virus inactivation was adjusted to 2 M NaCl and pH 5.0 (by adding 0.2 M citrate buffer (pH 6.0)), followed by loading on ToyoPearl butyl 650M tube On the column, wash with 10 mM acetate/phosphate, 2 M NaCl (pH 5.0) (buffer A), followed by 35% buffer A and 65% buffer B (10 mM acetate/phosphate (pH 5.0). The mixture of )) is dissolved. ToyoPearl Butyl 650M Dissolution Buffer was exchanged to 20 mM NaOAc/HOAc, 50 mM NaH ₂ PO ₄ , 30 mM arginine hydrochloride, 2% (w/v) sorbitol (pH 5.4), and Condition was adjusted to the final concentration of 3 mg/mL GALNS. The buffer exchanged and adjusted GALNS was filtered through a virus filter (DV20) and a DNA filter (Mustang Q) to remove any residual virus and DNA. Tween-20 (also known as polysorbate 20 or PS20) was added to a final concentration of 0.01% (w/v) to produce a bulk drug substance (BDS). The BDS is stored at 2-8 ° C or frozen.

Alternatively, the bioreactor material containing recombinant human GALNS is filtered, concentrated 20 times by ultrafiltration/diafiltration, then filtered through activated carbon, and subsequently loaded onto a capture column. The concentrated bioreactor material was loaded onto a Zn-chelating agarose gel FF (Zn-IMAC) column at a conductivity of approximately 50 ± 5 mS/cm, sequentially using 10 mM acetate/phosphate, 500 mM Wash with NaCl (pH 7.0) and 10 mM acetate/phosphate, 125 mM NaCl (pH 7.0) (buffer A), followed by 70% buffer A with 30% 10 mM acetate/phosphate, 125 mM NaCl, A mixture of 300 mM imidazole (pH 7.0) (buffer B) was dissolved. The Zn chelate agarose gel FF (Zn-IMAC) column was adjusted to pH 4.5±0.1 with 1.75 M acetate (pH 4.0) and filtered through a Millipore COHC filter at a ratio of 30:70 (v/v). Blend with 10 mM acetate/phosphate (pH 4.5), then load on a Fractogel EMD SE Hi-Cap (M) column at a conductivity of <7 mS/cm, sequentially using 10 mM acetate/phosphate 50 mM NaCl (pH 4.5) and 80% buffer A (10 mM acetate/phosphate (pH 5.0)) and 20% buffer B (10 mM acetate/phosphate, 250 mM NaCl (pH 5.0)) The mixture was washed and dissolved in a linear gradient of 20%-75% Buffer B (in 80%-25% Buffer A). The Fractogel EMD SE Hi-Cap column was then adjusted to pH 3.5 ± 0.1 by the addition of 0.4 M citrate buffer (pH 3.4) to achieve low pH virus inactivation. Low pH virus inactivated Fractogel EMD SE Hi-Cap column was adjusted to 2 M NaCl and pH 5.05 ± 0.1 (by adding 0.4 M citrate buffer (pH 6.0)), with 10 mM acetate / Phosphate (pH 5) (containing 5 M NaCl to achieve 2 M NaCl concentration) was blended, then loaded onto a ToyoPearl butyl 650 M column, sequentially using 10 mM acetate/phosphate, 2 M NaCl (pH 4.4 ± 0.1) and 10 mM acetate/phosphate, 2.5 M NaCl (pH 5.0) (buffer A), followed by 100% to 32% buffer A and 0% to 68% buffer B (10 mM acetate) A linear gradient of /phosphate (pH 5.0) and a mixture of 32% buffer A and 68% buffer B were dissolved. ToyoPearl Butyl 650M Dissolution Buffer was exchanged to 20 mM NaOAc/HOAc, 50 mM NaH ₂ PO ₄ , 30 mM arginine hydrochloride, 2% (w/v) sorbitol (pH 5.4), and Condition was adjusted to the final concentration of 3 mg/mL GALNS. The buffer exchanged and adjusted GALNS was filtered through a virus filter (DV20) and a DNA filter (Mustang Q) to remove any residual virus and DNA. Tween-20 (also known as polysorbate 20 or PS20) was added to a final concentration of 0.01% (w/v) to produce a bulk drug substance (BDS). The BDS is stored at 2-8 ° C or frozen.

Purification of recombinant human GALNS is detailed below, and the recombinant human GALNS is purified according to the procedure modified from the above protocol, as detailed below.

Recombinant human GALNS enzyme was expressed in G71S cells as described in Example III and purified as described in Example V or Example VI . The purified recombinant human GALNS of the present invention can be compared to other GALNS formulations reported in the literature. Masue et al, J. Biochem . 110:965-970, 1991 describe the purification and characterization of GALNS from human placenta. The purified enzyme was found to have a molecular mass of 120 kDa and consisted of 40 kDa and 15 kDa polypeptides, the latter being shown as glycoproteins. Thus, the Masue et al. GALNS enzyme appears to correspond to the processed form depicted in Figure 5 . Bielicki et al, Biochem. J. 279: 515-520, 1991 describe the purification and characterization of GALNS from human liver. When analyzed by SDS-PAGE, the enzyme has a molecular mass of 70 kDa under non-reducing conditions and a molecular mass of 57 kDa, 39 kDa and 19 kDa under reducing conditions. Bielicki et al, Biochem J. 311: 333-339, 1995 describes the purification and characterization of recombinant human GALNS from Chinese hamster ovary cells. Upon SDS-PAGE, the purified enzyme was found to have a molecular mass of 58-60 kDa under non-reducing conditions and 55-57 kDa, 39 kDa and 38 kDa under reducing conditions. Thus, the Biericki et al. GALNS enzyme appears to correspond to the mixture of the pre-processed (precursor) form of the enzyme depicted in Figure 5 and the processed form. In comparison, the recombinant human GALNS enzyme of the present invention consists almost entirely of the enzyme precursor form (see Figures 9 and 12 ), or predominantly (i.e., at least about 85%) consists of the enzyme precursor form (see figure 10 ).

IV. Lysosomal sulfatase and lysosomal storage diseases

The lysosomal sulfatase enzyme is substantially all or all of the therapeutic activity or biological activity of the full length enzyme or at least the substantial amount of retained enzyme (e.g., at least about 50%, preferably at least about 75%, and more preferably at least about 90%). Any fragment, mutant, variant or derivative of (e.g., sulfatase activity).

In some embodiments, the lysosomal sulfatase enzyme is a lysosomal sulfuric acid that, if not manifested or produced, or that exhibits or substantially reduces, causes disease, including but not limited to lysosomal storage diseases. Esterase. In some embodiments, the lysosomal sulfatase enzyme may not cause disease if it is not manifested or produced, or if manifested or produced substantially, but does not present or exhibit or produce a decrease in disease, including (but Not limited to) lysosomal sulfatase enzymes associated with lysosomal storage diseases. Preferably, the lysosomal sulfatase enzyme is derived from humans or from humans.

Preferably, in the treatment of lysosomal storage diseases, lysosomal sulfatase is an enzyme found in cells which, if not expressed or produced, or exhibits or substantially reduces, will result in lysosomal accumulation. disease. Alternatively, in the treatment of lysosomal storage diseases, lysosomal sulfatase is an enzyme that does not exist or exhibits or produces a substantial decrease in disease-related, but its absence or manifestation or production substantially reduces itself may not Can cause disease. Preferably, the lysosomal sulfatase enzyme is derived from humans or from humans.

Preferably, the enzyme is a lysosomal sulfatase enzyme such as arylsulfatase A (ARSA) (Genbank Accession No. NP_000478 (Isoform A), Genbank Accession No. NP_001078897 (Isoform B) and other variations , arylsulfatase B/N-acetylglucosamine 4-sulfatase (ARSB) (Genbank accession number P15848), iduronic acid-2-sulfatase (IDS) (Genbank deposit number NP_000193 (Isoform a), Genbank accession number NP_006114 (Isoform b), sulfonamide/heparin-N-sulfatase (SGSH) (Genbank accession number NP_000190), N-acetylglucose Amine-sulfatase (G6S) (Genbank Accession No. NP_002067) and galactose 6-sulfatase/N-acetylgalactosamine-6-sulfatase (GALNS) (Genbank Accession No. NP_000503). A table of lysosomal sulfatase enzymes suitable for use as therapeutic agents for lysosomal storage diseases and their absence is as follows:

In a preferred embodiment, the lysosomal sulfatase enzyme is a recombinant human lysosomal sulfatase enzyme produced by a cell line lacking in endosomal acidification. In a more preferred embodiment, the recombinant human lysosomal sulfatase enzyme is active and has a high content of phosphorylated oligosaccharides as specified under "Definitions". In a preferred embodiment, the lysosomal sulfatase enzyme is active highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

Thus, lysosomal storage diseases that can be treated or prevented using the methods of the invention include, but are not limited to, metachromatic leukodystrophy or MLD, Marathi's syndrome or MPS VI, Hunter syndrome or MPS II, St. Philippine A syndrome or MPS IIIa, St. Philippe D syndrome or MPS IIId, and Moquio A syndrome or MPS IVa. In a particularly preferred embodiment, the lysosomal sulfatase enzyme lacks a lysosomal sulfatase enzyme which causes Moquiao A syndrome or MPS IVa. In another particularly preferred embodiment, the lysosomal sulfatase enzyme is a lysosomal sulfatase enzyme which is associated with a human lysosomal storage disease, such as various sulfatase deficiencies or MSD.

Thus, according to the above table, for each disease, the lysosomal sulfatase enzyme will preferably comprise a specific active lysosomal sulfatase enzyme that is devoid of disease. For example, for a method involving MPS II, the preferred enzyme is iduronic acid-2-sulfatase. For methods involving MPS IIIA, the preferred enzyme is sulfonamide/heparin-N-sulfatase. For methods involving MPS IIID, the preferred enzyme is N-acetylglucosamine 6-sulfatase. For methods involving MPS IVA, the preferred enzyme is galactose 6-sulfatase/N-acetylgalactosamine-6-sulfatase. For methods involving MPSVI, the preferred enzyme is N-acetylgalactosamine 4-sulfatase. For methods involving metachromatic leukodystrophy (MLD), the preferred enzyme is arylsulfatase A. For methods involving multiple sulfatase deficiency (MSD), the enzyme may be arylsulfatase A, arylsulfatase B/N-acetylglucosamine 4-sulfatase, iduronic acid- 2-sulfatase, sulfonamide/heparin-N-sulfatase, N-acetylglucosamine-sulfatase or galactose 6-sulfatase/N-acetylgalactosamine-6-sulfate Esterase, and a preferred enzyme is galactose 6-sulfatase/N-acetylgalactosamine-6-sulfatase.

V. Type IVA mucopolysaccharidosis (Moqueo syndrome, MPS IVA)

Type IVA mucopolysaccharidosis (Moqueo syndrome, MPS IVa) is a hereditary autosomal recessive disease belonging to the mucopolysaccharide accumulation disease group. The Moquiao syndrome is caused by a lack of lysosomal enzymes required to degrade two glycosaminoglycans (GAG) keratan sulfate (KS) and chondroitin-6-sulfate (C6S). In particular, MPS IVa is characterized by the absence of the enzyme N-acetylgalactosamine-6-sulfatase (GALNS) and KS excretion in the urine. The lack of GALNS results in an abnormally large amount of mucopolysaccharide accumulating in the hyaline cartilage, the main component of bone tissue. All patients have systemic skeletal dysplasia. Other symptoms vary in severity between different patients, and may include, inter alia, hearing loss, cataracts, spinal instability, valvular heart disease, and respiratory problems.

GALNS hydrolyzes the sulfate linkage of galactose-6-sulfate of KS and N-acetylgalactosamine-6-sulfate of C6S. Human GALNS behaves as a 55-60 kDa precursor protein with only two potential aspartate-linked glycosylation sites. Mannose-6-phosphate (M6P) is part of the oligosaccharide present on the GALNS molecule. M6P is recognized by receptors on the surface of lysosomal cells and is therefore essential for efficient uptake of GALNS.

Like all sulfatase enzymes, GALNS requires the processing of formazan-activating enzyme (FGE) encoded by the sulfatase modification factor 1 (SUMF1) gene to obtain activity. Since this activation step involves post-translationally modified to _C α active site cysteine residues - carboxylic acyl glycine (FGly), so that the recombinant esterase sulphate overexpression leads to having a low specific activity of the sulfatase (i.e., a mixture of activated and unactivated sulfatase enzymes) and sulfatase enzymes having low potency (i.e., unactivated sulfatase degradation and/or non-secretion).

It is an object of the present invention to provide a method for treating Moquio syndrome and caused by the deficiency of the enzyme N-acetylgalactosamine-6-sulfatase or with the enzyme N-acetylgalactosamine-6-sulfatase deficiency The activity of other related diseases (eg, multiple sulfatase deficiency (MSD)) is highly phosphorylated by human N-acetylgalactosamine-6-sulfatase. The highly phosphorylated human N-acetylgalactosamine-6-sulfatase enzyme is capable of localizing to tissues accumulated in KS and C6S, has sufficient M6P content for efficient uptake, and has a sufficiently high FGly percentage to achieve enzyme activity, And has a relatively high degree of production.

It will be appreciated that the methods of the invention described herein are suitable for the production of other lysosomal sulfatase enzymes, such as arylsulfatase A (ARSA), arylsulfatase B/N-acetylglucosamine 4-sulfatase (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide/heparin-N-sulfatase (SGSH) and N-acetylglucosamine-sulfatase (G6S), These other lysosomal sulfatase enzymes are useful for treating lysosomal storage diseases caused by or characterized by their deficiency.

VI. Pharmaceutical Compositions and Administration

The lysosomal sulfatase enzyme of the present invention can be administered by a variety of routes. For oral preparations, lysosomal sulfatase enzymes may be used alone or in combination with suitable additives to prepare lozenges, powders, granules or capsules, for example with conventional additives such as lactose, mannitol, corn starch or potato starch With binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; with disintegrants such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants such as talc or hard Magnesium oleate; and if necessary in combination with diluents, buffers, wetting agents, preservatives and flavoring agents.

The lysosomal sulfatase enzyme of the invention may be dissolved, suspended or emulsified in an aqueous or non-aqueous solvent such as vegetable oil or other similar oil, synthetic aliphatic acid glyceride, high carbon aliphatic acid ester or propylene glycol; The lysosomal sulfatase enzyme of the present invention is formulated into an injection preparation, if necessary, together with a conventional additive such as a solubilizer, an isotonic agent, a suspending agent, an emulsifier, a stabilizer, and a preservative.

The lysosomal sulfatase enzyme of the present invention can be utilized in the form of an aerosol formulation to be administered by inhalation. The lysosomal sulfatase enzymes of the invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

Further, the lysosomal sulfatase enzyme of the present invention can be prepared as a suppository by mixing with a variety of substrates such as an emulsifying base or a water-soluble base. The lysosomal sulfatase enzyme of the present invention can be administered rectally via a suppository. Suppositories may include vehicles such as cocoa butter, carbowax, and polyethylene glycol which melt at body temperature but solidify at room temperature.

Unit dosage forms of the lysosomal sulfatase enzymes of the invention for oral or rectal administration, such as syrups, elixirs and suspensions, wherein each dosage unit (eg, one teaspoonful, one tablespoon, lozenge) may be provided Or a suppository) containing a predetermined amount of an active agent comprising a lysosomal sulfatase enzyme. Similarly, unit dosage forms for injection or intravenous administration may comprise a lysosomal sulfatase enzyme in the form of a solution in sterile water, physiological saline or another pharmaceutically acceptable carrier.

In practical use, the lysosomal sulfatase enzyme of the present invention can be combined as an active ingredient with one or more pharmaceutically acceptable carriers, diluents or excipients according to conventional pharmaceutical compounding techniques to form a homogeneous mixture. Things. The carrier, diluent or excipient may take a wide variety of forms depending on the preferred formulation desired for administration, for example, orally or parentally (including intravenously). In the preparation of a lysosomal sulfatase enzyme composition for oral dosage forms, any conventional pharmaceutical medium may be employed, such as water, glycols, oils in the case of oral liquid preparations such as suspensions, elixirs and solutions. , alcohols, flavoring agents, preservatives, coloring agents and the like; or carriers in the case of oral solid preparations such as powders, hard capsules and soft capsules and lozenges, such as starch, sugar, microcrystalline cellulose A diluent, a granulating agent, a lubricant, a binder, a disintegrating agent and the like, wherein the solid oral preparation is superior to the liquid preparation.

The present invention provides a formulation of any of the GALNS enzyme formulations described herein, optionally with a protein concentration of from about 0.1 to 5 mg/mL (or from 0.5 to 1.5 mg/mL), and optionally a pH of from about 5 to about 5.8. The formulation comprises (i) a phosphate buffer effective to reduce the amount of dephosphorylation of the GALNS enzyme; and (ii) one or more stabilizers selected from the group consisting of: amine acid salts, amine groups Acid buffers, surfactants and polyols. In some embodiments, the formulation can include a second buffer. In certain embodiments, the formulation comprises any of the purified recombinant human GALNS enzyme preparations described herein, a phosphate buffer at a concentration between about 25 mM and about 75 mM, at a concentration of between about 10 mM and about 30 An acetate buffer between mM and a stabilizer that reduces protein aggregation. In some embodiments, the formulation comprises arginine or a histidine or buffer, and optionally a nonionic surfactant, and optionally a ternary or trihydric polyol (sugar alcohol). In a particular embodiment, the formulation comprises arginine or buffer, polysorbate (optionally polysorbate 20), and sorbitol. In any of these embodiments, the phosphate buffer can be NaH ₂ PO ₄ . In any of these embodiments, the acetate buffer can be NaOAc/HOAc.

The second buffer can be any agent suitable to maintain the pH within the desired range. Suitable buffers include Tris, citrate, succinate, acetate, gluconate or other organic acid buffers. In some embodiments, the stabilizer is an amino acid salt or buffer, optionally arginine, lysine, glycine, glutamic acid, aspartame, histidine, alanine, bird a salt or buffer of aminic acid, leucine, 2-phenylalanine or glutamic acid. In some embodiments, the stabilizer is a surfactant, optionally a nonionic surfactant. Suitable surfactants include polysorbates (such as polysorbate 20 or polysorbate 80), poloxamers (such as poloxamer 188 or 184), polyoxyethylene derivatives, polyoxygen Propylene derivatives, sodium monolaurate and SDS. Non-limiting examples of known nonionic surfactants include aliphatic primary or secondary linear or branched alcohols having alkylene oxides (typically ethylene oxide and typically 6-30 ethylene oxide groups) or phenol. Other known nonionic surfactants include monoalkyl or dialkyl alkanolamines, alkyl polyglucosides, and polyhydroxy fatty acid decylamines. Non-limiting examples of known anionic surfactants include alkyl sulfuric acid, alkyl ether sulfuric acid, alkylaryl sulfonic acid, alkyl succinic acid, alkyl sulfosuccinic acid, N-alkoxy creatinine, Alkyl phosphoric acid, alkyl ether phosphoric acid, alkyl ether carboxylic acid, and sodium salt, ammonium salt, and monoethanolamine salt, diethanolamine salt, and triethanolamine salt of α-olefin sulfonic acid. Alkyl groups typically contain from 8 to 18 carbon atoms and are unsaturated. The alkyl ether sulfate, alkyl ether phosphate and alkyl ether carboxylate may contain from 1 to 10 ethylene oxide or propylene oxide units per molecule and preferably from 2 to 3 epoxy B per molecule. Alkane unit. Anionic surfactants are known to include sodium lauryl sulfate or ammonium lauryl sulfate and sodium lauryl ether sulfate or ammonium lauryl ether sulfate. Non-limiting examples of amphoteric surfactants are known to include alkylamine oxides, alkylbetaines, alkylguanidinopropylbetaines, alkylsulfobetaines, alkylglycinates, alkylcarboxylates Glycinate, alkyl amphoteric propionate, alkyl guanamidopropyl hydroxy sulfonic acid betaine, decyl tauryl sulphate and decyl glutamate, wherein the alkyl and thiol groups have from 8 to 18 carbon atom.

In some embodiments, the stabilizer is a polyol or a sugar, preferably a ternary or trihydric sugar alcohol. Suitable polyols include erythritol, arabitol, maltitol, cellobiitol, lactitol, mannitol, threitol, sorbitol, xylose Alcohol, ribitol, menoinisitol, galactitol, glycerol and glycerin. Polyols are also known to include alcohols derived from lactose, trehalose and stachyose. Non-reducing sugars are known to include sucrose, trehalose, sorbose, melezitose, and raffinose. Sugars are also known to include xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran.

The invention also provides a method for preventing dephosphorylation of a purified recombinant human GALNS enzyme comprising mixing the GALNS enzyme with a phosphate buffer effective to reduce dephosphorylation, optionally between about 25 mM and about 75 The final concentration of phosphate buffer between mM. In some embodiments, the purified recombinant human GALNS enzyme is also mixed with a second buffer, including any of the agents described above. In some embodiments, the enzyme is also mixed with one or more stabilizers selected from the group consisting of amino acid salts, amino acid buffers, surfactants, and polyols. In an exemplary embodiment, for example, when stored at room temperature (eg, 25 ° C) for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 After the month of testing, the amount of dephosphorylation was reduced compared to the formulation of the same enzyme in 1 mM phosphate buffer. In a particular embodiment, the accelerated stability test is performed after storage for these periods at 40 °C.

In certain embodiments, the reduction in dephosphorylation in the GALNS formulation (relative to 1 mM phosphate buffer) can be at least about 2%, about 3%, about 4%, about 5%, about 10%, About 15%, about 20%, about 30%, about 40% or about 50% or more. In another embodiment, the amount of phosphorylated mannose 7 (BPM7) on the GALNS is from about 25% to about 40%, or from about 30% to about 35%, in the formulation.

In an embodiment of any of the foregoing formulations or methods, the GALNS enzyme can be recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) comprising amino acid 27 of SEQ ID NO: 4 to 522 at least 95% identical amino acid sequence, and (i) having a purity of at least about 95%, as determined by Coomassie blue staining for SDS-PAGE under non-reducing conditions, (ii) having position 53 a conversion of a cysteine residue to at least about 80% of Cα-methionine (FGly), and (iii) a chain of 0.5 to 0.8 diphosphorylated oligomannose chains per monomeric protein chain, wherein At least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5% of the GALNS enzyme is in the form of a precursor, such as SDS-PAGE was performed under reducing conditions by Coomassie blue staining or by SDS-capillary gel electrophoresis (SDS-CGE).

For transdermal routes of administration, methods of transdermal administration of drugs are disclosed in Remington's Pharmaceutical Sciences, 17th Ed. (Gennaro et al., ed. Mack Publishing Co., 1985). A dermal or dermal patch is a preferred means for transdermal delivery of the lysosomal sulfatase enzymes of the invention. The patch preferably provides an absorption enhancer such as DMSO to increase absorption by the lysosomal sulfatase enzyme. Other methods for transdermal drug delivery are disclosed in U.S. Patent Nos. 5,962, 012, 6, 261, 595 and 6, 261, 595 each incorporated herein by reference.

Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are commercially available. In addition, pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusting agents, stabilizers, wetting agents and the like are also commercially available.

In each of these aspects, the lysosomal sulfatase enzyme composition includes, but is not limited to, suitable for oral, rectal, superficial, parenteral (including subcutaneous, intramuscular, and intravenous), transpulmonary The composition (either nasally or buccally inhaled) or administered nasally, but in any given case, the most suitable route will depend, in part, on the nature and severity of the condition being treated and the nature of the active ingredient. Exemplary routes of administration are oral and intravenous routes. The lysosomal sulfatase enzyme composition can be conveniently provided in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

Because lozenges and capsules are easy to administer, they represent the most advantageous oral unit dosage form, in which case solid pharmaceutical carriers will obviously be employed. If desired, the tablet can be coated by standard aqueous or non-aqueous techniques. The percentage of active lysosomal sulfatase enzymes in such compositions may of course vary and is preferably between about 2% and about 60% by weight of the unit.

The lysosomal sulfatase enzyme composition of the invention may be encapsulated in a viral envelope or vesicles or administered in conjunction with or incorporated into a viral envelope or vesicle. The vesicles are micellular granules which are generally spherical and often lipid. Liposomes are vesicles formed from a bilayer membrane. Suitable vesicles include, but are not limited to, monolayer vesicles and multilamellar lipid vesicles or liposomes. Such vesicles and liposomes can be prepared from a variety of lipid or phospholipid compounds, such as phospholipid choline, phosphatidic acid, phospholipids, using standard techniques such as those described in, for example, U.S. Patent No. 4,394,448. Betaine, phospholipid, ethanolamine, sphingomyelin, glycolipid, ganglioside, and the like. Such liposomes or liposomes can be used to administer lysosomal sulfatase enzyme in an intracellular manner and deliver lysosomal sulfatase enzyme to a target organ. Controlled release of the relevant lysosomal sulfatase enzyme can also be achieved using encapsulation (see, e.g., U.S. Patent No. 5,186,941).

Any route of administration that dilutes the lysosomal sulfatase enzyme composition into the bloodstream or preferably at least outside the blood brain barrier can be used. Preferably, the lysosomal sulfatase enzyme composition is administered peripherally, optimally intravenously or by cardiac catheter. Intravenous and intra-arterial injections are also suitable. The lysosomal sulfatase enzyme composition can be administered topically or regionally, such as intraperitoneally, subcutaneously or intramuscularly. In one aspect, the lysosomal sulfatase enzyme composition is administered with one or more pharmaceutically acceptable carriers, diluents or excipients.

Those skilled in the art will readily appreciate that dosages will vary with the particular lysosomal sulfatase enzyme, the severity of the symptoms, and the susceptibility of the individual to side effects. The preferred dosage of a given lysosomal sulfatase enzyme can be readily determined by a variety of means by those skilled in the art, including, but not limited to, dose response and drug administration in patients, test animals, and in vitro. Kinetic evaluation.

The dosage to be administered can also be determined by the individual's needs, the desired effect, the particular lysosomal sulfatase enzyme used, and the route of administration chosen. The dose of lysosomal sulfatase is in the range of from about 0.2 pmol/kg to about 20 nmol/kg, preferably in the range of from 2 pmol/kg to 2 nmol/kg, and particularly preferably at a dose of 2 pmol/ In the range of kg to 200 pmol/kg. Alternatively, the lysosomal sulfatase enzyme may be in the range of 0.01 mg/kg to 1000 mg/kg, preferably in the range of 0.1 mg/kg to 100 mg/kg, and particularly preferably in a dose of 0.1 In the range of mg/kg to 10 mg/kg. Such dosages will be affected by, for example, but not limited to, the particular lysosomal sulfatase enzyme, the form of the pharmaceutical composition, the route of administration, and the site of action of the particular lysosomal sulfatase enzyme.

The lysosomal sulfatase enzymes of the invention are useful in the therapeutic, prophylactic and diagnostic interventions of animals and, in particular, humans. Lysosomal sulfatase enzymes can show preferential accumulation in specific tissues. For diagnostic use, preferred medical indications include, for example, any condition associated with the relevant target organ (e.g., lung, liver, kidney, spleen).

The methods of the invention are useful for treating a variety of different disease conditions. In certain embodiments, particular attention is directed to the use of the methods of the invention in disease conditions in which lysosomal sulfatase having the desired activity has been previously identified, but wherein the lysosomal sulfatase enzyme is not Adequate delivery to the target site, area or compartment does not result in a completely satisfactory treatment outcome. For such lysosomal sulfatase enzymes, the method of the invention for producing an active highly phosphorylated lysosomal sulfatase enzyme can be used to enhance the therapeutic efficacy and therapeutic index of lysosomal sulfatase enzymes.

Treatment is intended to encompass any outcome that is beneficial to the individual in connection with administration of a lysosomal sulfatase enzyme, including a reduced likelihood of developing a disease, preventing the disease, slowing, preventing or reversing the progression of the disease, or ameliorating and afflicting the disease condition of the host. A related symptom, wherein the improvement or benefit is used broadly to refer to at least a decrease in the magnitude of a parameter (e.g., a symptom associated with a pathological condition being treated, such as inflammation and pain associated therewith). Likewise, treatment also includes the following conditions: complete inhibition (e.g., prevention or prevention, e.g., termination) of a pathological condition or at least a symptom associated therewith, such that the host no longer suffers from the pathological condition or at least no longer characterizes the pathology. Learn the symptoms of the disease.

A variety of hosts or individuals are treatable according to the methods of the invention. Such hosts are generally "mammals", where the terms are used to broadly describe organisms belonging to the class mammalia, including carnivores (eg, dogs and cats), rodents (eg, mice, guinea pigs). (guinea pig) and rats) and primates (eg humans, chimpanzees and monkeys). In many embodiments, the host will be a human.

The present invention has been generally described, and the present invention will be more readily understood by reference to the following examples which provide illustrative examples for the production and purification of active highly phosphorylated lysosomal sulfatase enzymes and their treatment Use in lysosomal storage diseases. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (eg, amounts, temperatures, etc.), but of course some experimental errors and deviations should be allowed.

Instance Example I Mammalian expression vector of human sulfatase modification factor 1 (SUMF1) and human N-acetylgalactosamine-6-sulfatase (GALNS)

The goal is to construct a mammalian expression vector suitable for producing a sufficient amount of active lysosomal sulfatase enzyme having a modified degree of phosphorylation in stably transfected cells.

A full-length human sulfatase-modifying factor 1 (SUMF1) cDNA encoding a polypeptide comprising 374 amino acids (see US Patent Application No. US 20005/0123949, published June 9, 2005; and US 2004/0229250 No., published on November 8, 2004, both incorporated herein by reference in their entirety herein to the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire Carlsbad, CA). Efficient transcription termination is ensured by the presence of bovine growth hormone polyadenylation sequences. The zeocin resistance gene was selected for selection under the control of the EM-7 promoter and the SV40 early polyadenylation sequence. The resulting plasmid was called pcDNA4 SUMF1. Sequences of human SUMF1 polynucleotide (SEQ ID NO: 1) and polypeptide (SEQ ID NO: 2) are shown in Figures 1 and 2 , respectively.

Full-length human N-acetylgalactosamine-6-sulfatase (GALNS) cDNA encoding a polypeptide containing 522 amino acids (including a signal peptide containing 26 amino acids) (see Tomatsu et al., Biochem . Biophys) Res . Commun . 181(2): 677-683, 1991) colonized to a human CMV enhancer-promoter and multiple selection sites containing an intron of the rabbit β-globin globin IVS2 The mammalian expression vector pCIN (BioMarin). Efficient transcription termination is ensured by the presence of bovine growth hormone polyadenylation sequences. The neomycin phosphotransferase gene, which is labeled as carrying a point mutation to reduce enzyme efficiency, is selected. The weakened HSV-tk promoter further blocks the attenuated marker. The resulting plasmid is called pCIN 4A. The sequences of human GALNS polynucleotide (SEQ ID NO: 3) and polypeptide (SEQ ID NO: 4) are shown in Figures 3 and 4 , respectively.

To increase the amount of expression of SUMF1 and GALNS, the framework/matrix attachment region (MAR) element (see Mermod et al., U.S. Patent No. 7,129,062) was cloned into the SUMF1 and GALNS expressing plastids.

BMAR SUMF1 was prepared by digesting a P<1_68 X_X NcoI-filled MAR (Selexis) with BamHI and HincII, and then inserting the released MAR fragment into pcDNA4 SUMF1 digested with BglII and NruI.

PMAR SUMF1 was prepared by digesting P <1_68 NcoI-filled (MAR)SV40EGFP (Selexis) with HindIII and XbaI to remove the EGFP gene, followed by insertion of the SUMF1 gene released from pcDNA4 SUMF1 by digestion with HindIII and XbaI.

BMAR 4A was prepared by digesting BMAR SUMF1 with PmeI and SpeI to remove the SUMF1 gene, followed by insertion of the GALNS gene released from pCIN 4A by digestion with PmeI and SpeI.

PMAR 4A was prepared by digesting P <1_68 NcoI-filled (MAR) SV40 EGFP (Selexis) with HindIII and XbaI to remove the EGFP gene, followed by insertion of the GALNS gene released from pCIN 4A by digestion with HindIII and XbaI.

The full-length human GALNS cDNA was also cloned into the mammalian expression vector pcDNA4 (Invitrogen, Carlsbad, CA). pCDNA4 SUMF1 was digested with HindIII and XbaI to remove the SUMF1 cDNA, and pCIN 4A was digested with HindIII and XbaI to isolate the GALNS cDNA. The GALNS cDNA HindIII/XbaI fragment was ligated into the pcDNA4 vector HindIII/XbaI fragment. The resulting plasmid was called pcDNA4-4A.

The integrity of the GALNS gene in the pCIN 4A, BMAR and pCDNA4-4A expression vectors was confirmed by restriction mapping using enzymes obtained from New England Biolabs. The PMAR 4A expression vector was not mapped.

The structure of the fully processed form of human N-acetylgalactosamine-6-sulfatase (GALNS) is depicted in Figure 5 . GALNS appears as a polypeptide containing 522 amino acids having a signal peptide sequence of 26 amino acids. The GALNS polypeptide containing 496 amino acids is secreted in the form of a pre-process (precursor) of the enzyme having a molecular weight of about 55-60 kDa. In active GALNS, the cysteine residue at position 53 of the precursor or fully processed GALNS polypeptide (corresponding to position 79 of the full length GALNS polypeptide) has been converted to C by sulfatase modification factor 1 (SUMF1). _α - methyl acyl glycine (FGly). In lysosomes, GALNS is cleaved after position 325 of the fully processed GALNS polypeptide, resulting in a GALNS peptide fragment of approximately 40 kDa and 19 kDa. These GALNS peptides are linked by a disulfide bridge between the cysteine (C) residues at positions 282 and 393 of the fully processed GALNS polypeptide. There are two typical N-linked glycosylation sites at positions 178 and 397 of the fully processed GALNS polypeptide. Bisphosphorylated mannose 7 (BPM7) containing 2 mannose-6-phosphate residues has been found on N178 but not found on N397.

Example II A total of human sulfatase-modifying factor 1 (SUMF1) and human N-acetylgalactosamine-6-sulfatase (GALNS) G71S cell line

The goal is to produce a cell line that produces an active lysosomal sulfatase enzyme with improved phosphorylation.

G71 cells (Rockford K. Draper) are directly derived from CHO-K1 (ATCC CCL-61). The G71 cell line is a CHO-K1 mutant with endosome acidification temperature sensitivity. It has been observed that at high temperatures, the mutant produces a total protein secretion of several enzymes and a phosphorylation difference on mannose residues (Park et al ., Somat. Cell Mol. Genet. 17(2): 137-150, 1991; Marnell et al, J. Cell. Biol. 99(6): 1907-1916, 1984).

G71 cells were maintained in BioWhittaker UltraCHO medium supplemented with 2.5% fetal calf serum, 2 mM glutamic acid, gentamycin and amphotericin at 34 °C.

In order to allow easier use of cell lines to produce proteins, protocols for adapting fixation-dependent serum-dependent mammalian cells to high-density serum-free suspension culture are used (Sinacore et al ., Mol. Biotechnol. 15(3): 249-257, 2000) Adhesive G71 cells were pre-adapted to serum-free growth medium to produce a serum-free suspension culture-adapted cell line G71S. Alternatively, adherent G71 cells can be adapted to serum-free growth medium after stable transfection as described below, as outlined by Sinacore et al.

Transfection of human SUMF1 with human GALNS expression vector (Example I) in pairs (pcDNA4 SUMF1 plus pCIN4 4A, BMAR SUMF1 plus BMAR 4A or PMAR SUMF1 plus PMAR 4A) was transfected into growth supplements according to the MARtech II protocol as described by Selexis G71S cells in medium with antibiotic-antimycotic solution (100 IU penicillin, 10 mg streptomycin, 25 μg amphotericin B, Cellgro). The transfection pool was in UltraCHO medium (Cambrex) supplemented with 5% gamma-irradiated fetal bovine serum (FBS, JRH), 200 μg/mL G418 (AG Scientific), and 200 μg/mL gibberellin (Invitrogen). Grow and colonize by limiting dilution in the same growth medium in 96-well plates. Pure lineage growth was monitored by cell screening (Cell Screen, Innovatis) imaging. All pure lines were screened against active GALNS using enzyme capture activity ELISA (see Example IV ). Cell yield was calculated by dividing the activity ELISA value of GALNS activity by the amount of cell growth per day (Vi-cell, Beckman Coulter) over a period of 4 days.

The following 202 G71S pure lines were generated and screened for active GALNS: 86 pure lines co-transfected with pcDNA4 SUMF1 plus pCIN 4A, 65 pure lines co-transfected with BMAR SUMF1 plus BMAR 4A, and with PMAR SUMF1 plus PMAR 4A Transfected 51 pure lines. The pure line was initially selected from 96-well tissue culture plates based on high-content active GALNS ( Fig. 6A ). GALNS activity was measured using an enzyme capture activity ELISA and expressed in ng/mL (y-axis). The x-axis shows three co-transfection conditions for SUMF1 and GALNS expression: hCMV promoter, no MAR; hCMV promoter with MAR; and SV40 promoter with MAR. Each bar represents a single pure line from each individual population. Cell density was not indicated in this 96-well pure line screening and not all co-transfected G71S lines are shown in this figure.

Yield analysis was performed on the G71S pure line that produced the highest active GALNS ( Fig. 6B ). Daily cell yield is obtained in picograms per cell per day and is obtained by dividing the GALNS activity by the cell density of the day. This figure shows the fourth day (96 hours) after inoculation with 5 x 10 ⁵ cells per flask. Pure enzyme GALNS (in picograms per cell per day) (y-axis) was assayed using an enzyme capture activity ELISA. The positive control consisted of BHK and CHO pure lines (BioMarin) which express GALNS. Each vertical bar represents a single pure line. Active GALNS were produced from the pure line of pCIN 4A, but only a small amount was above the assay background.

A pure line analysis by 96-well screening and a 4-day yield assay indicated that co-transfection of the expression vector with the MAR element resulted in an increase in the yield of the G71S pure line compared to co-transfection of the expression vector without the MAR element. The pure lineage cells co-transfected with BMAR 4A+BMAR SUMF1 showed rapid production, rapid growth in pure lines, and the ability to produce more than 2 times more active GALNS than the highest-producing PMAR 4A pure line, compared to the CHO 4A and BHK 4A pure lines lacking the MAR element. Increase by up to 10 times.

The G71S-expressing line of GALNS was adapted to serum-free growth medium using the protocol outlined in Sinacore et al , Mol. Biotechnol. 15(3): 249-257, 2000. The entire adaptation process was performed in the presence of selection reagents (200 μg/mL homomycin and 200 μg/mL neomycin). The G71 pure GALNS-expressing G71 was divided as follows: (1) into a 125 mL shaker containing Cambrex UltraCHO medium and 5% FBS (batch 8L2242); (2) to contain JRH 302M medium (production medium) And 5% FBS in a 125 mL shaker; and (3) to T flasks as a backup (UltraCHO, 5% FBS). Once the suspension culture is produced, the adherent cells are discarded and the initiation of the FBS is interrupted. When the growth rate of the 3rd generation returned to >0.5 (1/day) and the activity was >95%, the FBS concentration was reduced by 50%. The cells are allowed to stand at any given FBS concentration for at least 3 generations. Once adapted to grow in 2.5% FBS, the cells are placed directly in serum-free medium. The cells were allowed to accumulate in fresh medium containing 10% (v/v) DMSO. Test the thawing to ensure that the cells survive the freezing process. Two G71S lines (pure lines 4 and 5) from BMAR 4A+BMAR SUMF1 transfected GALNS required approximately 15 generations to be adapted to serum-free suspension culture. Pure line C6 expressing GALNS transfected from pcDNA4 SUMF1 plus pCIN 4A was also isolated and adapted to serum-free culture.

The pairwise combination of human SUMF1 and the human GALNS expression vector ( Example I ) (pcDNA4 SUMF1 plus pCDNA4-4A) was transfected into G71S cells essentially as described above, with the exception of using 200 μg/mL homomycin (Invitrogen) ) Make a choice. Essentially as described above, the six pure lines C2, C5, C7, C10, C11 and C30 representing GALNS were isolated and adapted to serum-free suspension culture.

Example III Large-scale culture of G71S cell line expressing human N-acetylgalactosamine-6-sulfatase (GALNS)

The goal was to measure the enzyme production from the G71S pure line expressing human N-acetylgalactosamine-6-sulfatase (GALNS). A large-scale culture of human SUMF1 and human GALNS-adapted serum-free suspension cultured G71S cell lines and evaluated their active GALNS enzyme production.

Since adaptation to serum-free suspension culture is relatively fast for the G71S host cell line, it was determined that production can be carried out in a WAVE bioreactor operated in perfusion mode. The WAVE bioreactor allows for greater flexibility in inoculation volume, as scaling up can be done directly in the bag, reducing the risk of contamination and accelerating material production. Figure 7 shows a schematic of the WAVE bioreactor configuration. The figure shows the load cell monitoring the volume of medium in the bag in a perfusion mode by measuring the bag weight and adjusting the feed and collection rate to maintain the desired volume. In the 10 L bag, the pH is also controlled by the probe inserted into the bag to the desired set point.

Substances derived from G71S pure lines 4 and 5 which express GALNS are produced on a 1 L scale. The culture pH was not controlled in these operations. The operation of the WAVE bag is limited to a production volume of 3 container volumes per day (VV/day). In order to prevent any inactivation of the substance, the target cell specific perfusion rate (CSPR) was 0.3 liters/cell/day, so that the average residence time of the GALNS enzyme was eight hours. Thus, maintaining the bag per ml of cell density of about 10-12 × 10 ⁶ cells. The growth rates of G71S pure lines 4 and 5 showing GALNS were 0.16 and 0.20, respectively. Bleed directly from the bag to maintain target cell density.

Since GALNS has previously been shown to be stable at pH 6, the pH of the collection fluid was adjusted to a pH between 5.5 and 6.5 to maintain enzyme activity. This was achieved by the rapid addition of 5 vol% pH 4.0 sodium citrate buffer mixed with the collection line exiting the reactor. The conditioned collection fluid was stored at 4 °C followed by downstream processing. The average titer of the two G71S pure lines 4 and 5 showing GALNS was about 4.2 mg/L, and the relative specific yield was about 1.25 pg/cell/day.

The G71S pure lines C2, C5, C6, C7, C10, C11 and C30 expressing GALNS were cultured in a similar manner and evaluated for their active GALNS enzyme production.

Example IV Measurement of the concentration and activity of human N-acetylgalactosamine-6-sulfatase (GALNS)

An enzyme-linked immunosorbent assay (ELISA) was developed to measure the GALNS enzyme concentration and activity of the G71S pure line co-expressing human SUMF1 and human N-acetylgalactosamine-6-sulfatase (GALNS).

Enzyme capture activity ELISA

After capture by an anti-GALNS-specific antibody bound to an ELISA plate, the enzyme capture activity ELISA measures the activity of the GALNS enzyme in the solid phase.

Buffer . Buffer A (carbonate buffer): 3.09 grams of Na ₂ CO ₃ and 5.88 grams of NaHCO _{3 were} dissolved in 900 mL of deionized (DI) H ₂ O followed by a final volume of DI H ₂ O to 1000 mL. Check that the pH is between 9.4 and 9.6, followed by filtration-sterilization. To completely coat a 96-well microplate with 100 μL per well, 19 μL of anti-GALNS antibody was diluted into one tube (12 mL). Buffer B (ELISA blocking buffer and serial dilution buffer): 1 x acidic PBS, 0.05% Tween-20 and 2% BSA, adjusted to pH 6.5 with acetic acid. Buffer B ^W (wash buffer): 100 mM NaOAc and 0.05% Tween-20, adjusted to pH 6.5 with acetic acid. Buffer C (priming buffer): 25 mM sodium acetate, 1 mM NaCl, 0.5 mg/mL desalted BSA, and 0.01% sodium azide, adjusted to pH 4.0 with glacial acetic acid. Buffer D (β-galactosidase buffer): 300 mM disodium hydrogen phosphate, 0.1 mg/mL BSA, 0.01% sodium azide and 0.01% Tween-20, adjusted to pH 7.2 with phosphoric acid. Buffer E (stop buffer): 350 mM glycine and 440 mM carbonate buffer adjusted to pH 10.7 with 6 M NaOH.

Reagents . Anti-GALNS IgG antibody: Multiple rabbit antibodies are protein G purified from serum. Total protein = 3.17 mg/mL (BCA) in D-PBS. Aliquots (19 μL) were stored at -20 ° C and used once each. 4MU-Gal-6-S substrate (solid; 440 MW): 100 mM stock was prepared in DI water and stored at 4 °C. --galactosidase (Sigma G-4155): diluted to 12 μg/mL in buffer D prior to use.

Protocol: Anti-GALNS antibodies were conjugated to plates: Nunc MaxiSorp ELISA plates (Nalge/Nunc International, Fisher No. 12-565-135) were used for anti-GALNS antibody coating with a final protein concentration of 5 μg/mL in buffer A. To prepare this solution, a 19 μL aliquot was thawed and briefly rotated (10 seconds) in a microfuge to collect the liquid. Transfer all 19 μL to 12 mL Buffer A. The mixture was vigorously mixed by inversion, then poured into a reservoir, and then the plate was loaded using a multi-channel pipette (100 μL per well). The plates were covered and incubated overnight at 4 °C. Remove unbound anti-GALNS antibody: by casting with Buffer B ^W plates were washed three times. Blocking: Plates were blocked with Buffer B (320 μL per well), then plated and incubated for 1 hour at 37 °C. Prepare a series of dilutions of purified GALNS standards and test samples (unknown) during the blocking step: Dilute the standards in buffer B to the high end of the linear range of assays (128 ng/mL in column A), This was followed by serial dilution (2 fold) in the BG column on a 96-well plate. Lane H is a buffer blank (ie, no GALNS enzyme). First, 500 μL of a concentration of 128 ng/mL in Buffer B was prepared. Next, 2-fold serial dilution (in 250 μL to 250 μL) was performed in Buffer B until 2 ng/mL was reached. Remove blocking buffer: After the blocking step, buffer B is discarded. GALNS enzyme standards and test samples were combined with anti-GALNS antibodies: plates were loaded with 100 μL of serially diluted standards and test samples (in duplicate) per well. The plates were covered and incubated for 1 hour at 37 °C. Removing GALNS inhibitors: by casting with Buffer B ^W plates were washed three times. Addition of GALNS receptor (first reaction): Prepare enough final solution for loading 100 μL per well (prepared within 1 hour before use). The 4MU-Gal-6-S stock solution (100 mM) was diluted to 1 mM in buffer C. Load 100 μL per well. The plates were covered and incubated for 30 minutes at 37 °C. Addition of β-galactosidase (second reaction): 50 μL of Buffer D containing 12 μg/ml β-galactosidase was added to each well. The plates were covered and incubated for 15 minutes at 37 °C. The reaction was stopped: 100 μL of buffer E (stop buffer) was added to each well to ionize the released 4MU. Transfer to Fluorescent Plate: Transfer (8 wells at a time) 200 μL from 250 μL of each well of the ELISA plate to a black untreated flat-bottomed microtiter plate (fluorescent plate, Costar No. 3915). Fluorescence reading: The plates were read in a Gemini plate reader (Molecular Devices Corporation) using the SOFTmax PRO program (366 nm excitation, 446 nm emission, 435 nm cutoff).

GALNS ELISA

The GALNS ELISA uses a sandwich immunoassay to quantify the concentration of GALNS enzyme in cell culture conditioned medium or other process samples.

Buffer . Buffer A (Carbonate Buffer): A 3.09 g Na ₂ CO ₃ and 5.88 g NaHCO ₃ was dissolved in 900 mL deionized (DI) H ₂ O, followed by adding DIH ₂ O to a final volume of 1000 mL. Check that the pH is between 9.4 and 9.6, followed by filtration-sterilization. To completely coat a 96-well microplate with 100 μL per well, 19 μL of anti-GALNS antibody was diluted into one tube (12 mL). Buffer B (ELISA blocking buffer and serial dilution buffer): 1 x acidic PBS, 0.05% Tween-20 and 2% BSA, adjusted to pH 6.5 with acetic acid. Buffer B ^W (wash buffer): 100 mM NaOAc and 0.05% Tween-20, adjusted to pH 6.5 with acetic acid. Buffer F (stop buffer): 2 NH ₂ SO ₄ : 600 mL total, 100 mL 12 NH ₂ SO ₄ and 500 mL MilliQ water were added.

Reagents . Anti-GALNS IgG antibody: Rabbit polyclonal antibody is protein G purified from serum. Total protein = 3.17 mg/mL (BCA) in D-PBS. Aliquots (19 μL) were stored at -20 ° C and used once each. HRP-conjugated detection antibody (RIVAH): Finally diluted 1:100 in bound antibody to D-PBS/1% BSA and stored in -20 °C at 120 °L aliquot for use once. TMB EIA Subject Kit (BioRad No. 172-1067).

Program . Anti-GALNS antibodies were conjugated to plates: Nunc MaxiSorp ELISA plates (Nalge/Nunc International, Fisher No. 12-565-135) were coated with an anti-GALNS antibody at a final protein concentration of 5 μg/mL in buffer A. To prepare this solution, a 19 μL aliquot was thawed and briefly rotated (10 seconds) in a microfuge to collect the liquid. Transfer all 19 μL to 12 mL Buffer A. The mixture was vigorously mixed by inversion, then poured into a reservoir, and then the plate was loaded using a multi-channel pipette (100 μL per well). The plates were covered and incubated for 2 hours at 37 ° C (convection incubator). Do not use thermal blocking. Remove unbound anti-GALNS antibody: by casting with Buffer B ^W plates were washed three times. Blocking: Plates were blocked with Buffer B (320 μL per well), then plated and incubated for 1 hour at 37 °C. Prepare a series of dilutions of purified GALNS standards and test samples (unknown) during the blocking step: Dilute the standards in buffer B to the high end of the linear range of assays (40 ng/mL in column A), This was followed by serial dilution (2 fold) in the BG column on a 96-well plate. Lane H is a buffer blank (ie, no GALNS enzyme). First, 500 μL of a concentration of 40 ng/mL in Buffer B was prepared. Next, 2-fold serial dilution (in 250 μL to 250 μL) was performed in Buffer B until 0.625 ng/mL was reached. Remove blocking buffer: After the blocking step, buffer B is discarded. GALNS enzyme standards and test samples were combined with anti-GALNS antibodies: plates were loaded with 100 μL of serially diluted standards and test samples (in duplicate) per well. The plates were covered and incubated for 1 hour at 37 °C. Washing: by casting with Buffer B ^W plates were washed three times. Binding detection antibody conjugate: An aliquot (120 μL) of antibody RIVAH was thawed and briefly rotated (10 seconds) in a microfuge to collect the liquid. 120 μL was all diluted into 11.9 mL of Buffer B and the tubes were vigorously inverted for mixing. Pour into the reservoir and add 100 μL per well with a multichannel pipette. The plates were covered and incubated for 30 minutes at 37 °C. Washing: by casting with Buffer B ^W plates were washed three times. TMB substrate: The final substrate was prepared by mixing 1.2 mL of Solution B with 10.8 mL of Solution A. Pour into the reservoir and add 100 μL per well with a multichannel pipette. The plates were covered and incubated for 15 minutes at 37 °C. Stop solution: Pipette 12 mL of 2 NH ₂ SO ₄ stop solution into the reservoir and add 100 μL per well using a multichannel pipette. Gently tap to mix. Read A450: Read the board in the board reader.

GALNS specific activity assay

The GALNS specific activity assay uses GALNS-specific mass-measured GALNS enzyme activity in solution.

Buffer. MilliQ H ₂ O was used for all buffers. Dilution Buffer (DB): For 1 L DB, 1.74 mL of acetic acid, 0.75 g of sodium acetate, 233.6 mg of NaCl, 2 mL of 50% Tween-20, and 10 mL of 1% sodium azide were dissolved in MilliQ H ₂ O. If the pH is less than 3.95, use 0.1 M NaOH. If the pH is greater than 4.05, adjust the pH to 4.0 +/- 0.5 with 0.1 M acetic acid. Final concentrations were: 19.5 mM acetic acid, 5.5 mM sodium acetate, 1 mM NaCl, 0.1% Tween-20, and 0.01% sodium azide. Phosphate Buffer (PB): For 1 L PB, 13.9 g of NaH ₂ PO ₄ -H ₂ O and 55 g of NaHPO ₄ -7H ₂ O were dissolved in MilliQ H ₂ O and the pH was adjusted to 7.2. The final concentration is 300 mM NaPi. Stop Buffer (SB): For 1 L SB, 26.2 g of glycine and 46.6 g of sodium carbonate were dissolved in MilliQ H ₂ O and the pH was adjusted to 10.6 with NaOH. Assay Buffer (AB): 4MU-Gal-6S stock (2 mM final) was diluted 1 :50 in DB. --galactosidase buffer (βGB): 25 μg/mL β-galactosidase in 300 mM NaPi, pH 7.2.

Reagents . 4MU-Gal-6S: 100 mM in H ₂ O (Toronto Research Chemicals catalog number M334480). --galactosidase: Sigma G-4155. 4-methylumbellone (4MU standard): Sigma M-1381 (10 mM stock solution in DMSO).

Program. The GALNS enzyme was serially diluted. For purified and formulated GALNS (approximately 1.5 mg/ml), samples were diluted 1:10,000 in a low protein adhesion microcentrifuge tube containing DB (USA Scientific catalog number 1415-2600) followed by a 1:1 serial dilution. 100 μL of DB was placed in a low protein binding 96-well plate. In the first column, pipette 100 μL of GALNS sample. At this point, serial dilution (1:1) was performed along the plate down (A-G on a 96-well plate). No sample (blank) was added to the hole H. The linear range for this assay is 1-75 ng/mL. The same procedure was used to prepare a 4MU standard curve. A 10 mM 4 MU DMSO stock solution was diluted 1:100 in DB. The 4MU standard curve was initiated by adding 50 μL of 50 μM 4MU to the first well, followed by serial dilution. 50 μL of the substrate diluted in AB (2 mM 4MU-galactose-6S in DB) was added to a 96-well fluorescent plate. The substrate was pre-incubated for 10 minutes at 37 °C. Add 50 μL of serial dilutions of 100 μL of GALNS and 4MU standards to 50 μL of the substrate containing B. Incubate at 37 ° C for 30 minutes (this first reaction removes sulfate from the substrate), quench the first reaction and add 50 μL of β-galactosidase (diluted β-galactosidase reserve in βGB) The second reaction was initiated to 25 μg/mL. Phosphate inhibits GALNS and an increase in pH also terminates the GALNS response. The resulting pH is now in the optimal pH range for beta-galactosidase. This second reaction was incubated at 37 ° C for 15 minutes. The released 4MU was ionized by the addition of 100 μL SB. The Ex355 Em460 was read on a 96-well fluorometer reader. Enzyme activity calculation (in pH 4.0 buffer at 37 ° C): 1 unit = μmol released 4 MU / min; activity = μmol 4 MU / min / mL; specific activity = μmol 4 MU / min / mg. Protein concentration calculation: The extinction coefficient of GALNS (1 mg/mL = 1.708 absorbance units at 280 nm) was used.

Example V Purification of human N-acetylgalactosamine-6-sulfatase (GALNS)

The goal is to obtain a large amount of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS). G71 cells stably expressing human SUMF1 and human GALNS stably transfected were grown under bioreactor culture conditions, and the active GALNS enzyme was purified from the cell culture medium.

Liquid chromatography apparatus. Amersham Pharmacia Biotech AKTA explorer 900 system, using Unicorn control software.

Protein analysis methods . Standard procedures for SDS-PAGE, Coomassie Blue staining (B101-02-COOM), Western blotting, and Bradford protein assays were followed. Purification procedures were assessed by activity generation and the purity of the GALNS product was visually assessed by SDS-PAGE. The presence of processed impurities is detected by Western blotting using anti-GALNS antibodies. Protein concentration was quantified using a Bradford protein assay. By using an extinction coefficient of 1.708 for A ₂₈₀ measurement to measure the final concentration of the purified GALNS was performed proteins.

Chromatography resin . Blue agarose gel 6 FF (GE Healthcare, lot 306346) and Fractogel SE Hi-Cap (Merck KgaA, FC040894449).

GALNS enzyme activity assay . The specific activity of GALNS was determined using a small fluorescent dye 4-methylumbelliferin-6-S-GAL (4-MU-6-S-GAL). The GALNS specific activity assay involves a two-step reaction in which β-galactosidase must be added after incubation of the GALNS with the substrate for a certain time to release the fluorescent tag. Measurements were made using a fluorescent plate reader.

The 10DG desalting column (Bio-RAD) was equilibrated with equilibration buffer (EQB, 50 mM NaOAc, 10 mM NaCl (pH 5.8)). MilliQ H ₂ O was used for all buffers. Three (3) mL of purified GALNS (0.5-2 mg/mL) was loaded onto a desalting column, lysed and collected in separate tubes using EQB in 4 mL aliquots. Protein concentration was calculated using the extinction coefficient of GALNS (1 mg/mL = 1.708 absorbance units at 280 nm).

The desalted GALNS samples were serially diluted (1:1) in dilution buffer (DB, 50 mM NaOAc, 1 mM NaCl (pH 4.0) + 0.5 mg/mL BSA). Prior to use by the 50 mg / mL BSA stock material (not more than 5% CV) to the previously loaded onto the BSA stock was desalted using G25 column of milliQ H ₂ O equilibrium. 100 μL of the desalted GALNS sample was pipetted into the first column of the low protein-bound 96-well plate, and serially diluted GALNS samples were pipetted down the plate (AG column on the 96-well plate). 100 μL of DB was pipetted into the last well (H). The linear range of this assay has a top end of 200 ng/mL and a linear range of 3-200 ng/mL. The same procedure was followed to prepare a standard curve with 4-methylumumone (4 MU) (Sigma M-1381, 10 mM stock in DMSO). Transfer 50 μL of 100 μL GALNS and 4MU serial dilutions to a new 96-well fluorescent plate (black plate). 50 μL of 2 mM 4MU-galactose-6S (in milliQ H ₂ O) was added to the sample to be assayed and incubated at 37 ° C for 30 minutes. This first reaction was quenched and incubated by adding 50 μL of β-galactosidase (Sigma G-4155, stock diluted to 12 μg/mL in 300 mM NaPi (pH 7.2)) and incubated at 37 °C. 15 minutes. The released 4MU was ionized by the addition of 100 μL of Stop Buffer (glycine/carbonate (pH 10.6)). Plates were read on a 96-well fluorescence plate reader (excitation 355 nm, emission 460 nm). 1 unit is defined as 1 μmol of released 4 MU/min, enzyme activity is given in μmol 4 MU/min/mL, and specific activity is given in μmol 4 MU/min/mg, all at 37 ° C in pH 4.0 buffer. .

The first purification process . The first purification process includes an ultrafiltration (UF) step followed by a 2-column purification process.

1. Collection Filtration (HF): The bioreactor material was sterile filtered through 0.2 μm.

2. Ultrafiltration (UF): The bioreactor material was concentrated 10-20 times by ultrafiltration through a 30 kD Sartocon membrane.

3. pH 4.5 adjustment: The concentrated bioreactor material (UF (20 times)) was adjusted to pH 4.5 with a pH adjustment buffer (1.75 M NaOAc (pH 4.0)) at room temperature and sterile filtered, then loaded Blue agarose gel on the column.

4. Blue Sepharose 6-speed flow (FF): UF (20 times) adjusted to pH 4.5 was loaded on a blue agarose gel column and dissolved as shown in Table 1 and Figure 9A . GALNS protein.

*CV: Column volume. Flow rate = 92 cm hr ^-1

5. Fractogel SE Hi-Cap: The eluate from the blue agarose gel column was adjusted to pH 4.3 and loaded on a Fractogel SE Hi-Cap column and dissolved in the GALNS protein as shown in Table 2 and Figure 9B . .

*CV: Column volume. Flow rate = 150 cm hr ^-1

The GALNS protein in the eluate was collected by fractionation, discarding the "pre-elution shoulder" before dissolution and the post-elution tail after dissolution.

6. Final UF/HF: As described above, the eluate from the Fractogel SE Hi-CAP column was concentrated by ultrafiltration and sterile filtered.

Provisioning . The purified GALNS protein was formulated in 10 mM NaOAc, 1 mM NaH ₂ PO ₄ , 0.005% Tween-80 (pH 5.5).

Stability study . The stability of the final formulated purified GALNS over time was monitored by storing small aliquots of GALNS samples at 4 ° C and -70 ° C at various temperatures. At some time points, aliquots of frozen samples were quickly thawed in a 37 °C water bath and the activity was measured. Figure 8 shows the period of time during which the purified GALNS was stable in the formulation buffer for at least 79 days at 4 °C and -70 °C.

The first purification process results . Table 3 shows the purified yields of the three GALNS protein preparations produced by G71S pure line 4 in the suspension culture bioreactor. In all cases, the purity was estimated to be about 95% by SDS-PAGE visual inspection.

Figure 9 shows SDS-PAGE of GALNS protein isolated by (A) blue agarose gel 6-speed flow chromatography followed by (B) Fractogel SE Hi-CAP chromatography. The gel was stained with Coomassie blue (left) or anti-GALNS antibody (right). For the western blot method, the anti-GALNS rabbit antibody was diluted 1:5000, and the secondary antibody was an anti-alkaline phosphatase rabbit antibody. The GALNS protein has an apparent molecular weight of about 55-60 kDa on SDS-PAGE, which is associated with the lack of a signal peptide containing 26 amino acid residues and a secretory processing (precursor) lacking cleavage after the 325 position. The expected size of the form is consistent.

N-terminal characterization . The N-terminus of the purified GALNS protein was determined by LC/MS. The N-terminal sequence is APQPPN, which corresponds to the predicted N-terminus of the secreted form of the signal peptide lacking 26 amino acid residues of GALNS (compare the human GALNS polypeptide sequences in Figures 4 and 5 ).

The second purification process . The second purification process includes an ultrafiltration/diafiltration (UF/DF) step followed by a 3-column purification process.

1. Ultrafiltration (UF/DF): The bioreactor material was concentrated 20-fold by ultrafiltration/diafiltration through a 30 kD Sartocon membrane at pH 5.5.

2. pH 4.5 adjustment: The concentrated bioreactor material (UF/DF (20 times)) was adjusted to pH 4.5 at room temperature with pH adjustment buffer (1.75 M NaOAc (pH 4.0)) and sterile filtered. Loaded on a Fractogel EMD SE Hi-Cap column.

3. Fractogel EMD SE Hi-Cap: UF/DF (20 times) adjusted to pH 4.5 was loaded onto a Fractogel EMD SE Hi-Cap column with 10 mM acetate/phosphate, 50 mM NaCl (pH). 4.5) and 10 mM acetate/phosphate, 50 mM NaCl (pH 5.0) were washed, and the GALNS protein was eluted with 10 mM acetate/phosphate, 140 mM NaCl (pH 5.0).

5. Zn Chelate Agarose Gel FF: The eluate from the Fractogel EMD SE Hi-Cap column was adjusted to 500 mM NaCl (pH 7.0) and loaded on a Zn chelate agarose gel FF (Zn-IMAC) tube. On the column, it was washed with 10 mM acetate/phosphate, 125 mM NaCl, 10 mM imidazole (pH 7.0), and the GALNS protein was eluted with 10 mM acetate/phosphate, 125 mM NaCl, 90 mM imidazole (pH 7.0).

6. pH 3.5 adjustment: The GALNS protein-containing eluate from the Zn chelate agarose gel FF column was adjusted to pH 3.5 to achieve low pH virus inactivation, followed by adjustment to 10 mM acetate/phosphate, 2 M NaCl (pH 5.0).

7. ToyoPearl Butyl 650M: The solution from the Zn chelate agarose gel FF column adjusted to a low pH was loaded onto a ToyoPearl Butyl 650M column with 10 mM acetate/phosphate, 2 M The solution was washed with NaCl (pH 5.0), and the GALNS protein was eluted with 10 mM acetate/phosphate, 0.7 M NaCl (pH 5.0).

8. Final UF/HF: The eluate from ToyoPearl Butyl 650M was filtered and diafiltered through 20 mM acetate, 1 mM phosphate, 150 mM NaCl (pH 5.5).

Provisioning . Purified GALNS protein was formulated in 10 mM NaOAc/HOAc, 1 mM NaH ₂ PO ₄ , 150 mM NaCl, 0.01% Tween-20 (pH 5.5).

The second purification process results . Table 4 shows the recovery of GALNS protein produced from G71S pure line C2 in a suspension culture bioreactor using a second purification process. The purity of the formulated GALNS enzyme (i.e., the precursor together with the mature or processed form) was about 98% as determined by C3 RP-HPLC. The percentage of the precursor form of the GALNS enzyme was about 85% as determined by SDS-capillary gel electrophoresis.

Figure 10 shows SDS-PAGE of GALNS enzyme isolated by ultrafiltration/diafiltration (UF/DF), Fractogel SE Hi-CAP chromatography, Zn chelate agarose gel FF chromatography and ToyoPearl butyl 650 M chromatography. . The gel was stained with Coomassie blue (top left), anti-GALNS antibody (top right), anti-Cathepsin L (bottom left), and anti-CHO protein (CHOP, bottom right). For the western blot method, 1:5000 anti-GALNS rabbit polyclonal antibody was diluted, and the secondary antibody was anti-rabbit AP conjugate; 1:1000 diluted anti-cathepsin L goat polyclonal antibody, and the secondary antibody was anti-goat HRP binding And 1:1000 diluted anti-CHOP rabbit polyclonal antibody, and the secondary antibody is an anti-rabbit HRP conjugate. The precursor GALNS enzyme has an apparent molecular weight of about 55-60 kDa on SDS-PAGE, and the mature or processed form of the GALNS enzyme has an apparent molecular weight of about 39 kDa and about 19 kDa on SDS-PAGE.

An overview of the first purification process . The standard has been used since the improvement group of columns (standard train) purification train (purification train) (see Table 5) was purified GALNS enzyme. The bioreactor collection material was sterile filtered through 0.2 μm and maintained at 4 ° C, then loaded onto a blue agarose gel capture column. The filtered bioreactor material is loaded directly or concentrated by ultrafiltration up to 15 times. Since the downstream purification step (i.e., SP agarose gel chromatography followed by phenyl sepharose chromatography) does not produce sufficiently pure GALNS, the purification set must be improved. Separation of the two downstream purification columns was used to generate a 2-column purification process using SEHi-Cap chromatography, the purity of the final material was significantly improved, and the total GALNS recovery was significantly increased from about 22% to about 80%. The purity of the GALNS enzyme (consisting essentially of the precursor form, see Figure 9 ) was roughly estimated to be >95% as determined by C4-RP chromatography, and the purified GALNS enzyme was at 4 °C and at -70 °C. Both remain stable in the formulation buffer for more than 79 days.

*This step is optional depending on the situation.

An overview of the second purification process . Also GALNS enzyme was purified using a second set of column purified (see Table 6). The total GALNS recovery was about 70% and the purity of the GALNS enzyme (including both precursor and mature or processed forms, see Figure 10 ) was roughly estimated to be about 97% as determined by C4-RP chromatography.

These assays indicate that the above-described protocol for the preparation of recombinant lysosomal sulfatase provides a highly efficient production of a large number of highly purified enzymes (specifically, human N-acetylgalactosamine-6-sulfatase (GALNS). The method of the former (precursor) form.

Instance VI Purification of human N-acetylgalactosamine-6-sulfatase (GALNS) with minimal shear

Control of proteolytic digestion of recombinant enzymes is a concern in the production and formulation of protein-based therapeutics. The goal is to obtain a large amount of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) in the form of a secreted pre-process (precursor). A method of producing a large-scale preparation of human GALNS under the condition that the shear caused by a protease (specifically, cathepsin L) is minimized has been developed.

As used herein, "minimum shear" means, for example, by performing SDS-PAGE under reducing conditions, followed by Coomassie blue staining, or by SDS-capillary gel electrophoresis (SDS-CGE), in the final The GALNS in the purified formulation is at least 98%, at least 98.5%, at least 99%, or at least 99.5% intact.

During the development of a method for the production of GALNS on a large scale, it was found that the about 55 kDa secretory precursor form of the enzyme is susceptible to proteolytic degradation by a protease (specifically, cathepsin L) active at acidic pH. Protease degradation of GALNS produces a mature splicing form of GALNS that can be observed on SDS-PAGE under reducing conditions as two bands of approximately 40 kDa and approximately 19 kDa. This proteolytic cleavage is exacerbated by the low pH (i.e., 4.5 to 5.0) required for the cation exchange column capture step. This pH range provides conditions that facilitate the activity of an acidic protease (such as cathepsin) present in the cell culture collection.

Changes are made in the GALNS recovery and purification process to minimize the presence and/or activity of proteases that can cleave GALNS secreted into the cell culture medium.

A flow chart describing an exemplary recovery and purification process for the large scale preparation of GALNS is shown in FIG . The process on the left shows a recovery and purification process similar to that described in Example V above, used in the Phase I/II process, which produces a variable amount of shear due to cathepsin L activation, at about 6- Within the range of 30% peptide chain, as determined by operating SDS-PAGE under reducing conditions followed by Coomassie blue staining (see Figure 12 , lane 3); or GALNS in the range of 65.3% to 93.7% full precursor form, as judged by SDS-CGE (see table 8). The SDS-PAGE method provides visual but more qualitative information on protein degradation, while the SDS-CGE method provides more quantitative information on the % of intact protein present in the final purified formulation.

The process on the right in Figure 11 shows the recovery and purification process used in the Stage III process, which produces a minimum shear, GALNS intact precursor form in the range of 98% to 99.6%, such as by operating SDS under reducing conditions. - PAGE, followed by Coomassie blue staining (see FIG. 12, lane 5), or by SDS-CGE (see table 8) is determined.

An overview of process changes . In order to reduce the extent and variability of GALNS shear, two factors are considered: (i) significantly less protease activity at neutral pH; and (ii) cation exchange chromatography and fixed metal affinity chromatography (IMAC). The steps all separate the protease from GALNS. The Phase III process (see Figure 11 , right) takes advantage of these factors and helps reduce the shear of GALNS during the purification process. This was achieved by switching the order of the first two column steps and capturing it on the Zn-IMAC column at pH 6.5 to 7.0. The two columns before conversion also result in a higher pH (ie pH) compared to the more acidic pH (ie pH 5.5) used in the Phase I/II process (see Figure 11 , left). 6.5) It is convenient to collect and store cell culture collections. Storing a cell-free collection at pH 6.5 reduces the likelihood of activation of a protease (eg, cathepsin) present in the cell culture fluid, thereby preventing or reducing the shear of GALNS.

Phase III process . The steps in the exemplary Phase III process recovery and purification of GALNS are summarized below.

1. Cell-free collection (1 x). G71 cells stably co-expressing human SUMFl and human GALNS were grown under bioreactor culture conditions as described in Example III . The bioreactor material containing GALNS (i.e., cell culture fluid) was collected at pH 6.5 and filtered using a filter train of CUNO30SP02A, CUNO 90ZA08A and 0.2 μm CUNO BioAssure filters in sequence.

2. UF/DF (20×). Cell culture fluid is subjected to ultrafiltration/diafiltration (UF/DF) to conductivity 7 mS/cm in 10 mM phosphate/acetate, 50 mM NaCl (pH 6.5). The UF/DF step was performed using a Sartocon 30 kDa Hydrosart cassette. The cell culture fluid was concentrated 20 times. For comparison, the Phase I/II process UF/DF step was performed at pH 5.5. The higher pH in the Phase III process reduces the likelihood of GALNS being cleaved by proteases present in the cell-free collection.

3. Charcoal filter. UF/DF (20x) material was filtered through Zeta Plus R55 activated carbon (Z1274) followed by sterile filtration using a 0.2 μm filter followed by storage at 2-8 °C. The charcoal filter significantly reduces the pressure after loading the subsequent Zn-IMAC column.

4. Fixed metal affinity chromatography (IMAC). The Zn fixed metal affinity chromatography (Zn-IMAC) column was equilibrated with 10 mM phosphate/acetate, 500 mM NaCl (pH 7.0), and was filtered with charcoal at a conductivity of about 55 ± 5 mS/cm. /DF (20x) material (loaded by adding 50 mM phosphate buffer (pH 9.2) containing 2.5 M NaCl maintained at pH 7.0 ± 0.1). The loaded column was washed sequentially with 10 mM phosphate/acetate, 500 mM NaCl (pH 7.0) and 10 mM phosphate/acetate, 125 mM NaCl (pH 7.0) (buffer A). GALNS was lysed from the column with a mixture of 70% buffer A and 30% buffer B (10 mM phosphate/acetate, 125 mM NaCl, 300 mM imidazole, pH 7.0).

5. Mustang Q filter. The Zn-IMAC column was adjusted to a conductivity of about 6.0 ± 0.5 mS/cm, pH 7.0, and loaded onto a Mustang Q filter to remove the virus.

6. pH adjustment and filtration. The Mustang Q filtrate was adjusted to pH 4.5 ± 0.1, filtered sequentially using a CUNO 60ZA filter and a 0.2 μm in-line filter, and then loaded onto a cation exchange column.

7. Cation exchange chromatography. The Fractogel SE HiCap cation exchange column was equilibrated with 10 mM phosphate/acetate, 50 mM NaCl (pH 4.5) and filtered to a filtered Mustang Q filtrate adjusted to pH 4.5 ± 0.1 at a conductivity of <7 mS/cm. load. The loaded column was sequentially treated with 10 mM phosphate/acetate, 50 mM NaCl (pH 4.5); and 10 mM phosphate/acetate (pH 5.0) (buffer A) with 10 mM phosphate/acetate, 250 80% of mM NaCl (pH 5.0) (buffer B): 20% mixture washed. The GALNS was lysed from the column with a linear gradient of 20 to 75% Buffer B in 80% to 25% Buffer A (i.e., 50 to 190 mM NaCl).

8. Maintain a low pH to achieve virus inactivation. Fractogel SE HiCap Dissolution was acidified to pH 3.5 ± 0.1 by the addition of 0.2 M citrate buffer (pH 3.4) to achieve virus inactivation, maintained at low pH for about 1 hour, by adding 0.2 M citrate The buffer (pH 6.0) was adjusted to pH 5.0 ± 0.1 and loaded onto a hydrophobic interaction chromatography (HIC) refining column.

9. Hydrophobic interaction chromatography (HIC). The ToyoPearl Butyl 650M HIC column was equilibrated with 10 mM phosphate/acetate, 2 M NaCl (pH 5.0) and loaded with a low pH virus inactivated Fractogel SE HiCap solution adjusted to 2 M NaCl (pH 5.0). . The loaded column was washed with 10 mM phosphate/acetate, 2 M NaCl (pH 5.0) (buffer A). GALNS was lysed from the column with a mixture of 35% buffer A and 65% buffer B (10 mM phosphate/acetate pH 5.0).

10. Buffer exchange and adjustment of rhGALNS to 3 mg/mL. ToyoPearl Butyl 650M HIC Dissolution Buffer was exchanged to 20 mM acetate, 50 mM phosphate, 30 mM arginine, 2% (v/v) sorbitol (pH 5.4), then adjusted in the same buffer Final GALNS concentration to 3 mg/mL.

11. Remove virus and DNA by filtration. The buffer exchanged ToyoPearl Butyl 650M HIC dissolvate was filtered using a virus filter (DV20) and a DNA filter (Mustang Q) to remove any residual virus and DNA.

12. Add PS20 to 0.01%. The virus and DNA filtered buffer exchanged ToyoPearl Butyl 650M HIC was adjusted to 0.01% (v/v) polysorbate 20 (PS20 or Tween-20).

13. BDS is stored at 2-8 ° C or frozen. The final formulation of purified GALNS, ie bulk drug substance (BDS), is stored at 2-8 ° C or frozen.

The result . As seen in the SDS-PAGE results (see Figure 5, lane 5), the apparent molecular mass of the backbone is about 55 kDa, which is consistent with the expected value for the GALNS monomer. The band migrated at the apparent molecular mass of about 40 kDa and about 19 kDa in batch number AP400802 (lane 3) produced by the Phase I/II process is produced by proteolytic cleavage between G348 and G349 by GALNS. Degradation products. This shear is greatly reduced in batch number BMN110-0110-001 (lane 5) produced using the Phase III process. Sub-bands with slightly slower migration than about 40 kDa cleavage products were present in both formulations.

An overview of other process changes. An improved Phase III process was developed to address certain challenges: (i) precipitation in concentrated collections; (ii) loss of GALNS in the Zn-IMAC capture step; (iii) GALNS in the wash fraction during the ToyoPearl butyl step Loss; and (iv) the presence of high levels of CHOP impurities in the solubilization of the ToyoPearl butyl step.

Improved Phase III process. The steps in the exemplary Phase III process recovery and purification of GALNS are summarized below.

1. Cell-free collection (1 x). G71 cells stably co-expressing human SUMFl and human GALNS were grown under bioreactor culture conditions as described in Example III . The bioreactor material containing GALNS (i.e., cell culture fluid) was collected at pH 6.5 and filtered using a filtration train of Millipore DOHC, Millipore XOHC and 0.2 μm Millipore SHC filters.

2. UF/DF (20×). Cell culture fluid is subjected to ultrafiltration/diafiltration (UF/DF) to conductivity 7 mS/cm in 10 mM phosphate/acetate, 50 mM NaCl (pH 6.5). The UF/DF step was performed using a Sartocon 30 kDa Hydrosart cassette. The cell culture fluid was concentrated 20 times.

3. Charcoal filter. UF/DF (20x) material was filtered through Zeta Plus R55 activated carbon (Z1274) followed by sterile filtration using a 0.2 μm filter followed by storage at 2-8 °C.

4. Fixed metal affinity chromatography (IMAC). The Zn-immobilized metal affinity chromatography (Zn-IMAC) column was rinsed with 50 mM acetate buffer (pH 5.0), charged with 50 mM ZnSO ₄ , rinsed with 50 mM acetate buffer (pH 5.0), and then contained. 5 mM imidazole was equilibrated with 10 mM phosphate/acetate, 500 mM NaCl (pH 7.0). The equilibrated Zn-IMAC is charcoal-filtered UF/DF (20x) material at a conductivity of about 50 ± 5 mS/cm (by loading 75:25 (v/ during the loading of the Zn-IMAC column) v) Loading was carried out in line with a 50 mM phosphate buffer (pH 9.2 ± 0.1) containing 2.5 M NaCl maintained at pH 7.0 ± 0.1). The loaded column was washed sequentially with 10 mM phosphate/acetate, 500 mM NaCl (pH 7.0) and 10 mM phosphate/acetate, 125 mM NaCl (pH 7.0) (buffer A). GALNS was lysed from the column with a mixture of 70% buffer A and 30% buffer B (10 mM phosphate/acetate, 125 mM NaCl, 300 mM imidazole, pH 7.0).

5. pH adjustment and filtration. The Zn-IMAC column was adjusted to pH 4.5 ± 0.1 with 1.75 M acetate (pH 4.0), followed by filtration using a Millipore COHC filter. During the loading of the cation exchange column, the filtered material was blended in-line with 10 mM phosphate/acetate (pH 4.5) at a ratio of 30:70 (v/v).

6. Cation exchange chromatography. The Fractogel SE HiCap cation exchange column was equilibrated with 10 mM phosphate/acetate, 50 mM NaCl (pH 4.5) and was adjusted to pH 4.5 and filtered Zn-IMAC column at <7 mS/cm conductivity. Dissolve loading. The loaded column was sequentially treated with 10 mM phosphate/acetate, 50 mM NaCl (pH 4.5); and 10 mM phosphate/acetate (pH 5.0) (buffer A) with 10 mM phosphate/acetate, 250 80% of mM NaCl (pH 5.0) (buffer B): 20% mixture washed. GALNS was lysed from the column with a linear gradient of 20% to 75% Buffer B in 80% to 25% Buffer A (i.e., 50 to 190 mM NaCl).

7. Maintain a low pH to achieve virus inactivation. Fractogel SE HiCap Dissolution was acidified to pH 3.5 ± 0.1 by the addition of 0.4 M citrate buffer (pH 3.4) to achieve virus inactivation, maintained at low pH for approximately 1 hour (at a temperature of 12-23 ° C) ), adjusted to pH 5.0 ± 0.1 by adding 0.4 M citrate buffer (pH 6.0), blended with 10 mM phosphate/acetate buffer (pH 5) containing 5 M NaCl to achieve 2 M NaCl The final concentration was then filtered through a 0.2 μm filter and subsequently loaded onto a hydrophobic interaction chromatography (HIC) refining column.

8. Hydrophobic interaction chromatography (HIC). The ToyoPearl Butyl 650M HIC column was equilibrated with 10 mM phosphate/acetate, 2 M NaCl (pH 4.4), and the Fractogel was inactivated with a filtered low pH virus adjusted to 2 M NaCl (pH 4.3-4.4). SE HiCap dissolvate loading. The loaded column was washed sequentially with 10 mM phosphate/acetate, 2 M NaCl (pH 4.4); and 10 mM phosphate/acetate, 2.5 M NaCl (pH 5.0) (buffer A). Linear gradient of 100% to 32% buffer A in 0 to 68% buffer B (10 mM phosphate/acetate (pH 5.0)) (ie 2.5 to 0.8 M NaCl); and 32% buffer A mixture of A and 68% Buffer B (i.e., 0.8 M NaCl) dissolves GALNS from the column.

9. Buffer exchange, removal of DNA and virus by filtration, and adjustment of rhGALNS to 3 mg/mL. ToyoPearl Butyl 650M HIC Dissolution was buffer exchanged into 20 mM sodium acetate, 50 mM sodium phosphate, 30 mM arginine hydrochloride, 2% (w/v) sorbitol (pH 5.4). The buffer exchanged ToyoPearl Butyl 650M HIC dissolvate was filtered using a DNA filter (Mustang Q) and a virus filter (DV20) to remove any residual DNA and virus. The filtered and buffer exchanged ToyoPearl Butyl 650M HIC eluate was then adjusted to a final GALNS concentration of 3 mg/mL in the same buffer as above.

10. Add PS20 to 0.01%. The DNA and virus filtered buffer exchanged ToyoPearl Butyl 650M HIC solution was adjusted to 0.01% (v/v) polysorbate 20 (PS20 or Tween-20).

11. BDS is stored at 2-8 ° C or frozen. The final formulation of purified GALNS, ie bulk drug substance (BDS), was passed through a 0.2 μm Millipak 200 filter into a final storage container and stored or frozen in a bag at 2-8 °C.

result. After this improved Stage III process, the combination of GALNS with the Zn-IMAC and ToyoPearl butyl columns was improved and the CHOP impurities in the ToyoPearl butyl solution were reduced. The GALNS purified by this modification of the Stage III process is similar to the enzyme purified by the above Stage III process in terms of all properties tested (e.g., properties in Table 7 below).

Characterization of rhGALNS prepared by the Stage III recovery and purification process. The GALNS purified using the Phase III process was compared to the enzyme purified by the Phase I/II process. The characterization results are given in Table 7 . Although the two GALNS formulations appeared to be similar in all of the properties tested, the enzymes purified by the Phase III process showed significantly less shear than the enzymes purified by the Phase I/II process.

*CHOP: Chinese hamster ovary host cell protein contaminant

** SEC-HPLC data from other rhGALNS batches purified from the same 200 L cMFG reactor using Phase III process showed >99% intact protein.

Table 8 compares the percentage of intact GALNS in the final purified formulation in the batch prepared using the Phase I/II process (65.3-93.7%) or the Phase III process (98-99.6%) using the SDS-CGE method. The values obtained here support the results obtained from the SDS-PAGE method and show that the degree of shear is significantly reduced due to improvements in the purification process.

These assays indicate that the above-described protocol for preparing recombinant lysosomal sulfatase enzymes with minimal shear provides a highly efficient production of a large number of highly purified enzymes (specifically human N-acetylgalactosamine-6-sulfatase) (GALNS) method of secretory pre-process (precursor) form.

Example VII Characterization of purified human N-acetylgalactosamine-6-sulfatase (GALNS)

The G71 cell line produces a protein with a high degree of mannose phosphorylation greater than the high mannose phosphorylation noted in normal mammalian cell lines and a correspondingly low phosphorylated high mannose oligosaccharide content (eg lysozyme) Body enzyme). A lysosomal sulfatase enzyme comprising a high level of diphosphorylated high mannose oligosaccharide as defined herein (eg, recombinant human N-acetylgalactosamine-6-sulfatase (GALNS)) and Canfield et al, USA Molecules obtained in Patent 6,537,785 which do not comprise complex oligosaccharides and which only exhibit high mannose oligosaccharides are compared.

To determine the amount of unphosphorylated high mannose on lysosomal sulfatase enzymes, those skilled in the art can use exoglycosidase sequencing ("FACE sequencing") to release oligosaccharides. Determine the percentage of un-phosphorylated high mannose oligosaccharide chains. On a normal batch-release FACE profile gel, unphosphorylated high mannose co-migrates with specific complex oligosaccharides such as oligomannose 6 and fully sialylated bi-tactate complexes. The unphosphorylated high mannose and other oligosaccharides are then distinguished by enzymatic sequencing.

To determine whether a purified lysosomal sulfatase enzyme (eg, recombinant human N-acetylgalactosamine-6-sulfatase (GALNS)) expressed in G71S cells exhibits increased phosphorylation, determination of lysosomal sulfate The amount of mannose-6-phosphate (M6P) on the enzyme and the ability of the enzyme to bind to the M6P receptor (MPR).

The recombinant human GALNS enzyme expressed in G71S cells and purified was analyzed by fluorescence assisted carbohydrate electrophoresis (FACE) and by MPR-Sepharose resin chromatography. The FACE system uses polyacrylamide gel electrophoresis to separate, quantify, and measure the sequence of oligosaccharides released from glycoproteins. The relative intensity of the oligomannose 7-bisphosphate (O7P) band on FACE (Hague et al ., Electrophoresis 19(15): 2612-20, 1998) and the percentage of activity retained on the MPR column (Cacia et al ., Biochemistry 37). (43): 15154-61, 1998) gives a reliable measure of the degree of phosphorylation per mole of protein.

Specific activity . Recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) was assayed at 37 ° C using a small fluorescent 4-methylumbellone fluorenyl-6-S-GAL (4MU-Gal-6S) ) specific activity. Using this assay, the specific activity of purified GALNS was 165 μmol/min/mg (0.165 U/mg).

Human serum stability . The in vitro serum stability of GALNS was determined. Human serum (Sigma H-4522) was sterilized by filtration through a 0.2 μm PES filter, and 4 mL of filter-sterilized human serum was pre-incubated in a T-25 cell culture flask at 37 ° C for 1 hour in a 10% CO ₂ atmosphere ( At this time, the pH was 7.4 ± 0.1). 0.4 mL of desalted purified GALNS (2 mg/mL purified GALNS desalted into PBS using a Bio-RAD 10DG column) was added to pre-incubated human serum or PBS control containing 0.5 mg/L BSA. Take 100 μL of sample at the indicated time points (eg 0, 1, 3.5, 7.5 and 26 hours) and add to 900 mL of quenching buffer (QB, 50 mM NaOAc (pH 5.6) + 150 mM NaCl + 0.5 mg/mL BSA) +0.001% Tween-80). Samples were stored at 4 °C until the GALNS enzyme activity was prepared for measurement.

GALNS enzyme activity was measured using an enzyme capture activity ELISA. The ex vivo serum half-life of purified GALNS was estimated to be 217 hours by extrapolating the exponential decay curve of % residual GALNS enzyme activity.

Ingested into synovial cells (chondrocytes) . The ability of GALNS to be absorbed into synovial cells (chondrocytes) was measured.

At 37 [deg.] C in 5% CO ₂ in 12 well plates in growth medium (Ham's (Ham's) F12 + 10% FBS) were cultured cartilage cells (ATCC No. CRL-1832). A 4 x 12 well plate was required for the uptake analysis of the three samples. Purified GALNS samples and GALNS reference were diluted to 1 μM in acPBS/BSA (acidic PBS + 200 μg/mL BSA). Uptake dilution curve for GALNS samples and reference preparations from 1 μM stock: 50.5 μL (1 μM rhASB) to 5 mL uptake assay dilution (UAD, DMEM + 2 mM L-glutamic acid + 0.5 mg/mL BSA In order to produce a 10 nM GALNS sample and reference material, which was further serially diluted to 5, 2.5, 1.25, 0.62, 0.31 and 0.16 nM by continuous 2-fold dilution in UAD. Aspirate the growth medium from the 12-well plate of confluent chondrocytes, add 1 mL of UAD (blank) or GALNS sample or serial dilution of the reference to the well, and incubate for 4 hours at 37 ° C in a 10% CO ₂ incubator . The ingestion medium was aspirated, the plates were completely aspirated, and each well was washed once with 1 mL of PBS. The PBS was aspirated and the chondrocytes were detached by adding 0.5 mL trypsin/EDTA (0.25% trypsin/0.1% EDTA (Mediatech 25-053-CI, lot 25053025)) per well. After detachment from the plate, the chondrocytes were aliquoted into Eppendorf tubes pre-cooled on ice (total of 30 tubes). The trypsin-treated chondrocytes were cooled and then agglomerated at a low speed (4000 rpm, 3 minutes) in a microfuge. Trypsin was completely aspirated, the cells were agglomerated with 1 mL of PBS, and the microcentrifugation and aspiration steps were repeated once. 200 μL of cell lysis buffer (CLB, 50 mM sodium acetate (pH 5.6) + 0.1% Triton X-100) was added to each tube. The cells were resuspended by pulse vortexing three times. After resuspension, the cell lysate mixture was stored overnight at -80 °C or directly analyzed.

The cell lysate was thawed at room temperature and transferred to ice when thawed. The cell lysate was vortexed to resuspend any visible solid material, followed by spinning in a microfuge at 4 ° C for 10 minutes at 14 K rpm to collect the insoluble material. The supernatant was transferred to a set of fresh tubes and the agglomerates were discarded. The supernatant was then subjected to GALNS activity assay. A seven point dilution curve (starting at 10 nM and ending at 0.16 nM with two consecutive dilutions) is typically included, which includes a fairly uniform expected K _uptake on both sides. The molar concentration of the starting sample was calculated by using a protein-only molecular weight.

Based on single site ligand binding, the Kd uptake by purified GALNS into synovial cells was 4.9 nM.

Mannose-6-phosphate (M6P) receptor plate binding assay . The ability of GALNS to bind to the mannose-6-phosphate (M6P) receptor was determined in a plate binding assay. FluoroNunc high binding plates were coated with 4 μg/mL M6P receptor. The coated plates were washed twice with 250 μL of wash buffer (WB, TBS + 0.05% Tween 20) per well, and 200 μL of blocking and dilution buffer per well (BDB, Pierce SuperBlock buffer, batch number CA 46485) Block non-specific binding. The plates were incubated for 1 hour at room temperature (RT). During this blocking step, purified GALNS samples (0.5-2 mg/mL, stored at 4 °C for 2 weeks) were diluted to 10 nM in BDB, followed by dilution buffer (DB, 50 mM NaOAc, 1 mM NaCl) Serial dilutions (250 μL + 250 μL) to 5, 2.5, 1.25, 0.62, 0.31, and 0.16 nM (pH 4.0) + 0.5 mg/mL BSA). The blocked plates were washed with WB as above, and the diluted GALNS samples were dispensed into wells in duplicate at 100 μL per well and incubated for 1 hour at room temperature. During this incubation step, 2 mM active substrate was prepared by diluting 0.1 mL of 100 mM 6S-galactose-4 MU stock (stored in H ₂ O, -20 ° C) to 5 mL DB, and at 37 Preheat in a °C water bath. After incubation, the plates were washed twice with WB as above, and 100 μL of the diluted substrate was added and the GALNS specific activity was determined.

Using this assay, the Kd bound to the M6P receptor by purified GALNS was 2.4 nM based on single site binding.

Mannose-6-phosphate (M6P) receptor column binding . The ability of GALNS to bind to the mannose-6-phosphate (M6P) receptor was determined in a column binding assay. The M6P receptor column was prepared according to the manufacturer's instructions. The M6P receptor was from Peter Lobel Laboratories, and the column resin was NHS activated resin (Bio-RAD Affi-Gel 15) with a column size of 0.7 mL. Use 10 column volume (CV) equilibration buffer (EQ, acidic PBS (pH 6.0) containing 5 mM β-glycerophosphate, 0.05% Tween 20, 5 mM glucose-1-phosphate and 0.02% NaN ₃ ) The M6P receptor column was equilibrated at a flow rate of 0.25 ml/min. 6 μg of purified GALNS (per 200 μl) was loaded onto the M6P receptor column at a flow rate of 0.1 ml/min. Unbound GALNS was washed out of the column at a flow rate of 0.25 mL/min at 10 CV EQ. Use 0-100% dissolution buffer (EL, acidic PBS (pH 6.0) containing 5 mM β-glycerophosphate, 0.05% Tween 20, 5 mM mannose-6-phosphate and 0.02% NaN3) gradient (10 CV) Then, 100% of the 2 CV EL dissolves the combined GALNS out of the column. Re-equilibrate the column with 3 CV EQ.

The percentage of purified GALNS bound to the M6P receptor was determined to be 56% using the GALNS ELISA.

Total oligosaccharide analysis was performed by capillary electrophoresis (CE) . To determine the degree of mannose-6-phosphorylation on GALNS, total oligosaccharides on GALNS were determined by capillary electrophoresis (CE) as described in Ma et al., Anal. Chem. 71(22): 5185-5192, 1999. N-linked carbohydrate profile. This method uses PNGase F to cleave aspartate N-linked oligosaccharides. The cleaved oligosaccharides were separated and derivatized with a fluorescent dye and applied to a G10 rotating column to remove excess dye. The purified fluorescently labeled oligosaccharides were separated by electrophoresis, and then the peaks were quantified using MDQ-CE software (32 Karat version 7.0).

Using this assay, the amount of purified GALNS diphosphorylated mannose 7 (BPM7), monophosphorylated mannose 6 (MPM6), and oligosaccharide-containing sialic acid was 0.58 mol per mol enzyme, per mol enzyme. 0.08 moles and undetectable. The percentage of GALNS protein containing BPM7 was estimated to be 29%.

Characterization of Bis7 oligosaccharides . The position of the double phosphorylated mannose 7 (BPM7) oligosaccharide on GALNS was determined. The aspartic acid (Asn) residue at position 178 is N-linked glycosylated to BPM7. The Asn residue at position 397 was not N-linked glycosylated to BPM7, but was found to be predominantly oligomannose type sugar.

Hydroxyapatite affinity . Develop an in vitro skeletal model that determines whether GALNS has the ability to target bone. A 4 mg/mL HTP-DNA grade hydroxyapatite (Bio-RAD) suspension was prepared and equilibrated in DBS + 50 μg/mL BSA (pH 7.4). After addition of 50 μg/mL BSA, the purified GALNS was desalted in DBS (pH 7.4). The desalted GALNS was serially diluted in DBS + 50 μg/mL BSA (pH 7.4) in a 96-well plate at a final concentration of approximately 2 mg/mL. 50 μL of serially diluted GALNS was transferred to a 96-well filter plate (Millipore No. MSGVN2210, hydrophilic PVDF, low protein binding, 22 μm pore size). 50 μL of hydroxyapatite suspension was added to the wells of the filter plate containing serially diluted GALNS and incubated for 1 hour at 37 ° C with moderate shaking. The plate was vacuum filtered.

The vacuum filtration supernatant was analyzed by HPLC or GALNS enzyme activity as described above. The Kd of the purified GALNS to hydroxyapatite was 3-4.0 μM.

Expression of human sulfatase modifying produce a higher amount of activating factor 1 (SUMF1) of G71S cell line (i.e., the active site cysteine residue to C α _- conversion of p-acyl glycine (FGIy) of) the Lysosomal sulfatase.

To determine whether purified recombinant lysosomal sulfatase (eg, human N-acetylgalactosamine-6-sulfatase (GALNS)) co-expressed with SUMF1 in G71S cells exhibits increased activation, assays are purified and purified. The amount of conversion of the active site cysteine residue to FGly on the enzyme sulfatase.

GALNS activation . The percentage of activation of GALNS, i.e., the percent conversion of the active site cysteine (Cys) residue to C _? -methionine (FGly), was determined by LC/MS (TFA). The TIC/1000 of Cys, FGly and Gly were 39, 1840 and 183, respectively, indicating that approximately 90% of the purified GALNS lines were active (i.e., FGly).

Overview . Table 9 shows an overview of the characterization of recombinant GALNS expressed in G71S elite 4 cells. Table 10 shows an overview of the characterization of recombinant GALNSs expressed in G71S pure line C2 cells.

These results demonstrate that purified recombinant human GALNS has a high degree of activation and a high degree of mannose 6-phosphate phosphorylation. Thus, G71S cells that express both SUMF1 and lysosomal sulfatase (ie, GALNS) efficiently produce active hyperphosphorylated lysosomal sulfatase. An increase in the content of high mannose residues on these lysosomal sulfatase enzymes results in increased MPR uptake by the cells.

Example VIII Uptake and activity of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) in ex vivo Moqueo chondrocytes

To evaluate the uptake of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) by lysosomes of ex vivo autologous chondrocytes and the ability of GALNS to degrade keratan sulfate (KS).

Chondrocytes from patients suffering from type IVa mucopolysaccharidosis (MPS IVa, Moquio syndrome) have reduced GALNS activity and exhibit KS lysosomal accumulation. An in vitro model of MPS IVa was established using chondrocytes isolated from the iliac crest biopsy of MPS IVa patients. However, primary chondrocytes de-differentiate in culture and lose their chondrocyte characteristics. Therefore, culture conditions were established to induce in vitro differentiation of chondrocytes.

Chondrocytes isolated from MPS IVa patients in alginate beads in the presence of IGF-1, TGF-β, transferrin, insulin and ascorbic acid (chondrocyte growth medium, Lonza No. CC-3225), called MQCH . The medium was changed twice a week for a duration of 6 to 15 weeks of the experiment. These culture conditions induce phenotypic expression and differentiation of chondrocytes. These MQCH cells exhibit chondrocyte markers, including sex determining regions Y-box 9 (Sox 9), collagen II, collagen X, cartilage oligomeric matrix proteins, and aggrecan mRNA, as used by culture from MQCH cells. The isolated RNA was quantified by quantitative RT-PCR analysis. These cultured MQCH cells also produce an extracellular matrix.

Confocal microscopy was performed to confirm the accumulation of KS by MQCH cells. The MQCH cells in the 8-week culture were trypsinized, cytospun on a glass slide, fixed in acetone, and frozen until use. After thawing, the cells were rehydrated and stained with primary and secondary antibodies using anti-KS monoclonal antibody (Chemicon) and Alexa-488 (green)-conjugated goat anti-rabbit antibody, respectively. MQ-CH cells showed punctate intracellular staining consistent with lysosomal KS accumulation.

To determine whether purified recombinant human GALNS can be taken up into lysosomes by MQCH cells and degrade KS, 6 weeks of MQCH cell culture was incubated with 10 nM recombinant human GALNS for 9 weeks twice a week. GALNS uptake and KS clearance were measured by confocal microscopy. The primary antibodies used were: (a) anti-GALNS rabbit polyclonal antibody and anti-lysosomal associated membrane protein-1 (LAMP-1) monoclonal antibody, or (b) anti-KS monoclonal antibody and anti-LAMP-1 polyclonal antibody . The secondary antibodies used were: Alexa-488 (green)-conjugated antibody to detect anti-GALNS or anti-KS antibodies, or Alexa-555 or Alexa-594 (red)-conjugated antibodies to detect anti-LAMP-1 antibodies) . The MQCH cell preparation was fixed in a mountant containing DAPI staining the nuclei.

It was observed that GALNS enzyme and KS were significantly co-localized with lysosomal marker LAMP-1 in GALNS-treated MQCH cells. After exposure of MQCH to recombinant human GALNS, the amount of intracellular KS is reduced.

GALNS enzyme capture ELISA and GALNS specific activity ELISA (both described in Example IV above) were also used to measure GALNS uptake. Normal human chondrocytes (NHKC) expressing GALNS were used as positive controls. As shown in Tables 11 and 12 , untreated MQCH cells did not have detectable GALNS enzyme or activity, whereas MQCH cells treated with 10 nM GALNS for 9 weeks had significant GALNS enzyme and activity.

^a not detected; ^b ng GALNS antigen / μg total protein

^a not detected; ^b GALNS activity / ng antigen

These results demonstrate that purified recombinant human GALNS is in vitro absorbed by Moqueo chondrocytes into lysosomes and degrades lysosomal KS. These Moqueo chondrocytes are useful as in vitro efficacy models to test for lysosomal sulfatase enzymes that degrade KS, such as GALNS.

Example IX Recombinant human lysosomal enzyme degrading natural receptor activity in in vitro cell-based assay

In vitro cell-based assays were developed to measure the activity of recombinant human lysosomal enzymes (eg, lysosomal sulfatase) to degrade natural receptors.

The enzymatic activity of recombinant human lysosomal enzymes (e.g., lysosomal sulfatase) is typically measured by cell-free in vitro assays using artificial fluorescent receptors (see Example 4 for GALNS). However, the measured enzyme activity depends on the size of the artificial receptor (i.e., the number of monosaccharide units). In addition, the activity of the enzyme in an environment that does not reflect the in vivo condition is measured. Thus, cell-free in vitro assays do not take into account the ability of lysosomal enzymes to cleave natural receptors or their ability to be absorbed into target cells and localized to lysosomes.

Development of a cell-based in vitro assay to measure two recombinant human lysosomal enzymes (α-L-iduronidase (IDU) and arylsulfatase B (ARSB)) to degrade their natural receptors (also That is, the activity of the dermatan sulfate (DS) in the cells is contained. DS contains a variable sulfated iduronic acid g(1-3)-N-ethylidene-galactosamine beta (1-4) disaccharide unit.

ARSB-deficient GM00519 human fibroblasts or IDU-deficient GM01391 human fibroblasts were cultured in confluence in 12-well plates, and the cultures were maintained for 3-6 weeks after confluence to allow intracellular DS accumulation.

Confluent GM00519 or GM01391 cells were then exposed to a saturating dose of recombinant human ARSB (10 nM) or recombinant human IDU (25 nM) for 4-5 days, respectively. Untreated and lysosomal sulfatase treated cells were collected, dissolved and centrifuged.

The lysosomal enzyme activity in the cell lysate is measured by measuring the residual DS content of the cells, and the assay comprises the steps of: (1) lysing the cells; (2) using chondroitin ABC lyase (EC) in the cell lysate 4.2.2.4) specifically digesting the DS-containing substance into the disaccharide; (3) labeling the DS disaccharide with a fluorescent dye (eg 2-amino-acridone, AMAC); (4) isolating the DS disaccharide ( For example, by capillary zone electrophoresis, CZE); and (5) detection of labeled DS disaccharides (eg, by laser induced fluorescence, LIF). Such methods are described, for example, in Zinellu et al, Electrophoresis 2: 2439-2447, 2007 and Lamari et al, J. Chromatogr . B 730: 129-133, 1999 (reviewed in Volpi et al, Electrophoresis 29: 3095-3106, In 2008).

Table 13 shows the percent degradation of DS when using ARSB-treated GM00519 cells, such as the amount of disaccharide (4S disaccharide) containing N-acetylgalactosamine-4-sulfate as the main DS disaccharide by measurement. Measured. Similar results were obtained using IDU-treated GM01391 cells.

^The percentage of degradation is calculated by measuring the area under the curve of the 4S disaccharide in the lysate of the ARSB-treated cells compared to the untreated cells detected in the CZE-LIF scan.

The above assays indicate that the target cells are absorbing recombinant human ARSB and IDU, which are then localized to lysosomes, where they degrade their native-derived intracellular DS.

A dose-finding experiment was performed to determine the concentration at which the IDU became a rate limit in this cell-based assay. GM01391 cells were cultured in 12-well plates. At 4 weeks after confluence, cells were exposed to various concentrations (0.8 nM to 25 nM) of IDU for 6 or 26 hours. Cell lysates are prepared and processed as described above. It has been determined that the IDU does not become a rate limit below 1 nM.

In the second dose search experiment, GM01391 cells were exposed to various concentrations (0.01 nM to 0.2 nM) of IDU for 2 days at 3 weeks after confluence. Cell lysates are prepared and processed as described above. In this experiment, a known amount of the internal standard monosaccharide GlcNAc-6S was incorporated into the cell lysate to control recovery during processing. As shown in Figure 13, GM01391 cells were observed in the IDU DS treated by the amount of dose-dependent decrease in quality.

In a similar dose-finding experiment, GM00519 cells were exposed to various concentrations (0.001 nM to 0.06 nM) of ARSB for 5 days at 3 weeks after confluence. Cell lysates are prepared and processed as described above. In this experiment, a known amount of the internal standard monosaccharide GlcNAc-6S was incorporated into the cell lysate to control recovery during processing. As shown in FIG. 14, it was observed GM00519 cells treated by the ARSB DS amount dose-dependent reduction in the quality of receiving.

A cell-based in vitro assay was developed to measure the activity of recombinant human lysosomal sulfatase GALNS to degrade its natural receptor (i.e., the receptor containing keratan sulfate (KS) in cells).

GALNS-deficient MQCH cells were cultured as described in Example 8 above and treated with 1 nM or 10 nM recombinant human GALNS. After treatment, MQCH cell lysates were prepared and digested with keratan sulfate II (EC 3.2.1) which cleaves the larger KS oligosaccharides into KS disaccharides. As described above for the DS disaccharide, the KS disaccharide was labeled with AMAC, separated by CZE and detected by LIF. GlcNAc-6S, a KS monosaccharide, was incorporated as an internal standard into cell lysates to control recovery during processing. The amount of two characteristic KS disaccharides (Gal6S-GlcNAc6S and Gal-GlcNAc6S) was measured, and the obtained data was corrected from the amount of GlcNAc6S recovered. Table 14 shows the percent degradation of KS when using GALNS-treated MQCH cells as determined by measuring the amount of two characteristic KS disaccharides.

^{a, b} degradation percentage by measuring the area under the curve of Gal6S-GlcNAc6S and Gal-GlcNAc6S in the lysates of GALNS-treated cells compared to untreated MQCH cells detected in the CZE-LIF scan And calculated for adjusting the area under the curve of the control GlcNAc6S.

The above assays indicate that the target cells are absorbing recombinant human GALNS, which is then localized to lysosomes, where GALNS degrades its native-derived intracellular KS.

Taken together, these results demonstrate that the activity of recombinant human lysosomal enzymes ARSB, IDU and GALNS to degrade their natural receptors can be measured and quantified in cell-based in vitro assays. It will be appreciated that this cell-based in vitro assay can be readily modified to quantify and quantify the activity of other lysosomal sulfatase enzymes as well as various recombinant lysosomal enzymes.

Example X Transfer of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) to specific tissues

Evaluation of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), which is expressed in G71 cells and delivered to mice, is delivered to mice that are affected by GALNS deficiency or associated with GALNS deficiency. ability.

The highly specific distribution of keratan sulfate produces a characteristic phenotype of type Ia mucopolysaccharidosis (MPS IVA) or Moquiao syndrome. Keratin sulfate is mainly found in cartilage (joint bone growth plate, heart valve, larynx and nasal septum) and cornea, and these tissues exhibit keratan sulfate accumulation in patients with MPS IVA. Therefore, for the lack of N-acetylgalactosamine-6-sulfatase (GALNS) in MPS IVA or Moquiao syndrome, it is important to show GALNS enzymes to long bone growth plates, heart valves, corneas, larynx and nose. Pass. To examine these specific tissues as targets of poor vascularization, the delivery of fluorescent GALNS was studied in mice.

Two immunohistochemical staining methods were tested in mice: (1) human GALNS in combination with Alexa 488 and (2) unconjugated human GALNS. Binding of human GALNS to Alexa 488 was performed using a Molecular Probes Alexa Fluor 488 C ₅ maleimide labeling kit (A-10254). The maleimide binding chemistry yields a ratio of 1:1 label to protein.

To confirm that the fluorescent tag did not interfere with the uptake of GALNS, cultured synoviocytes (ATCC No. CRL-1832) were used for immunocytochemistry experiments. Standard unincorporated assays were used to compare unbound GALNS with bound GALNS (GALNS-A488 or GALNS-A555). The cells were incubated with the GALNS enzyme for 4 hours and subsequently followed by α-L-iduronidase (IDU) for 2 hours. The results show that Alexa 488 binding does not interfere with cellular uptake. Figure 15 shows the estimated Kd values for GALNS, GALNS-A488 and GALNS-A555. Since the label does not activate the enzyme, the uptake is measured by an antigen ELISA of cell lysate rather than enzymatic activity. The Kd values of the unbound and bound GALNS enzymes were determined to be approximately equal.

To determine the stability of the fluorescent tag, once the GALNS enzyme is incorporated into the cell, immunostaining is performed on the unbound and bound GALNS. The primary antibody used for staining was protein G purified anti-GALNS rabbit antibody at a concentration of 1 μg/mL. All images were acquired on a Leica IRE2 wide field epi-fluorescent microscope using the MetaMorph software. Because of the presence of out-of-plane light, Deconvolution of the image stack is required to measure co-localization in such images. The software is observed using AutoQuant/AutoDeblur, and the theoretical point spread function (blind algorithm) is used for deconvolution.

Immunostaining showed a fairly good overlap with the signal amplified by the GALNS-A488 material. The observed increase in sensitivity is due to the fact that both the primary antibody and the secondary antibody are multiple antibodies.

To determine if the GALNS enzyme targets lysosomes, cultured synoviocytes are immunostained with Molecular Probes Lysotracker or another enzyme localized to lysosomes. Lysotracker appears to show some degree of overlap with the GALNS-488 enzyme; however, the staining is not uniform. Tracking with recombinant human N-acetylgalactosamine-6-sulfatase (rhASB) (a lysosomal enzyme) for 2 hours did show a degree of colocalization with GALNS.

The above experiments show that the GALNS-A488 enzyme is taken up by cells and localized to lysosomes and can be used to determine in vivo biodistribution.

Conduct two in vivo studies. The first preparatory study was a single dose (10 mg/kg) of rapid injections in the tail vein of normal Balb/c mice, followed by multiple times (5 times) every other day in the tail vein of normal Balb/c mice. A second study was performed by injecting 10 mg/kg. Tables 15 and 16 respectively describe the experimental plans for the first and second studies.

In the first preliminary study, heart, liver, and amine/femoral joints were collected at 2 hour and 24 hour time points. In the second study, the heart, kidney, liver, and bones with quadriceps and soleus muscles were collected at 2, 4, and 8 hour time points. For both studies, the heart, kidney, and liver were immersed in 4% paraformaldehyde (PFA) for 4 days, embedded in paraffin, and then sectioned to a thickness of 7 μm. In the second study, muscle bones were immersed in 4% PFA for 8 days, decalcified, embedded in paraffin, and sectioned to a thickness of 7 μm.

Images of GALNS-A488 injected mice were obtained using a Zeiss laser scanning confocal microscope. For the analysis in the first preliminary study, each heart valve and liver sample obtained a confocal stack and was used for volume analysis. Each growth plate sample obtained two confocal stacks and was used for volume analysis. In the second study, each heart valve, kidney, and liver sample obtained a confocal stack and was used for volume analysis; each growth plate and zone of rest cartilage (zrc) sample obtained two confocal stacks. And for volume analysis.

The conclusions from confocal microscopy studies are: (1) it is possible to detect fluorescent GALNS in vivo; (2) the signal is specific (no background) and localized as lysosome; (3) the sinus in the liver GALNS is present in sinusoidal cells; (5) in the heart, GALNS is present in the septum and atria, but more importantly it is clearly visible at the heart valve level, where it is distributed after multiple injections Deeper; (6) At the femoral/tibia junction, the GALNS enzyme is present in the mineralized part of the bone (骺) and in the bone marrow. GALNS is present in the growth plate. More specifically, GALNS is enriched in the chondrocytes of the resting zone (or reserve cartilage zone), is present at the beginning of the proliferative zone, and is largely reproduced in the ossification zone at the end of the growth plate. Although it is difficult to quantify the cumulative effect of multiple injections, the second study appears to show a broader distribution. Table 17 shows an overview of confocal microscopy studies.

For secondary staining, the initial step is to optimize the GALNS primary antibody. Various tissues were stained with a 1:100 to 1:400 dilution of anti-GALNS rabbit antibody purified with protein G. The results in the first preliminary study indicated that the dilution of 1:100 was superior. This result was confirmed in the second study. The remaining slides were processed with a 1:100 primary antibody dilution and a 1:1000 secondary antibody dilution.

When stained with protein G purified anti-GALNS antibody, the signal from Balb/c mice administered with GALNS exceeded the signal from the control (i.e., mice administered with PBS-Cys). To confirm that the GALNS enzyme was localized in lysosomes, the sections were stained with anti-LAMP1 antibody. LAMP1 is a marker of lysosomes. The image shows an overlap between the anti-LAMP1 antibody and the anti-GALNS antibody, indicating that the GALNS enzyme is localized in the lysosome.

In summary, two in vivo studies indicate that GALNS biodistribution is associated with angiogenesis, ie, more vascularized tissue contains more fluorescent signals. More importantly, studies have demonstrated the presence of GALNS in the accumulation of keratan sulfate in the Moquiao syndrome, even if these sites are poorly vascularized.

Example XI Formulation of human N-acetylgalactosamine-6-sulfatase (GALNS)

The objective was to investigate the effect of various excipients (e.g., buffers, isotonic agents, and stabilizers) on the activity and structure of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) in the formulations of the present invention.

GALNS enzyme was prepared as described in Example V.

The GALNS enzyme was characterized as described in Example VII.

In Example V, purified recombinant human GALNS enzyme was formulated in 10 mM NaOAc/HOAc, 1 mM NaH ₂ PO ₄ , 150 mM NaCl, and 0.005% or 0.01% Tween-20 (pH 5.5). It should be noted that the GALNS enzyme undergoes dephosphorylation after storage in a low concentration phosphate buffer. Therefore, the phosphate buffer concentration was increased to 100 mM NaH ₂ PO ₄ . No significant dephosphorylation of the GALNS enzyme was observed after storage in high concentration phosphate buffer. However, soluble GALNS aggregates were observed after storage at 5 ° C, 25 ° C or 40 ° C, and insoluble GALNS aggregates were observed after storage at 40 °C.

In the first study, the effect of stabilizer concentration and pH on the stability of recombinant GALNS was assessed. The purified recombinant GALNS enzyme was formulated in 20 mM NaOAc/HOAc, 50 mM NaH ₂ PO ₄ , 0.01% Tween-20, and 4% sucrose or 2% sorbitol. Stabilizers tested: 15 mM or 30 mM arginine hydrochloride (arginine HCl); and 15 mM or 30 mM NaCl. Tested pH values: 5.0, 5.4 and 5.8. The pH 5.8 formulation was determined to be in the range of pH 5.8 to 6.0. After the enzyme formulation was stored at 5 ° C, 25 ° C or 40 ° C for up to 2 months, the GALNS enzyme was analyzed using the various assays described in Example VI.

The formation of soluble aggregates in various formulations was determined by profiling the GALNS enzyme by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). The presence of both 15 mM and 30 mM arginine hydrochloride inhibited the growth of soluble aggregates as determined by SEC-HPLC ( Figure 16 ).

The stability of GALNS enzyme activity in the formulation was determined by in vitro enzyme activity assay. At 5 ° C, GALNS enzyme activity is stable in the presence of arginine hydrochloride or NaCl; GALNS enzyme activity is slightly reduced in the presence of arginine hydrochloride or NaCl at 25 ° C; and at 40 ° C The GALNS enzyme activity in the formulation containing NaCl exhibited minimal stability ( Fig. 17 ).

After digesting the enzyme with PNGase F to cleave aspartate N-linked oligosaccharides, the dephosphorylation of GALNS enzyme in the formulation was investigated by capillary electrophoresis (CE) measuring the percentage of diphosphorylated mannose 7 (BPM7). Chemical. After 2 months at 5 ° C, 25 ° C or 40 ° C, the glycosylation profile of the GALNS enzyme in all enzyme formulations in BPM 7% is similar to the reference batch GALNS used in Phase I clinical formulations. Glycosylation profile ( Figure 18 ).

The purity of the GALNS enzyme in the formulation was determined by profiling the GALNS enzyme by reverse phase high performance liquid chromatography (RP-HPLC). After 2 months at 5 ° C or 25 ° C, the formulation did not exhibit any peak area change; at 40 ° C, the formulation containing arginine hydrochloride did not exhibit any peak area at pH 5.0 and pH 5.4. The change, but formulations containing NaCl exhibited a decrease in peak area at pH 5.0 and pH 5.4 and all formulations at pH 5.8 ( Figure 19 ). In the RP-HPLC chromatogram, a post-peak shoulder was observed in the formulation. The GALNS formulation containing 30 mM arginine hydrochloride exhibited the least significant peak back shoulder.

Example XII Exemplary formulations of human N-acetylgalactosamine-6-sulfatase (GALNS)

The following examples provide guidelines regarding the parameters to be used in the formulation of a composition comprising recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active fragment, mutant, variant or analog thereof, These compositions are useful for the treatment of Moquia syndrome or MPS IVa. Parameters to be used in the formulation of the GALNS compositions of the present invention include, but are not limited to, pH-maintaining buffers, isotonicity adjusting agents, absence or presence of stabilizers, and the absence or presence of other excipients, vehicles , diluents and their analogues.

In Example XI, recombinant GALNS was formulated at a concentration of about 1 mg/mL. Preferably, GALNS is formulated at a concentration in the range of from about 0.1 to 10 mg/mL, preferably from about 0.5 to 5 mg/mL, and more preferably from about 0.5 to 1.5 mg/mL. In one embodiment, the formulation of the GALNS composition of the invention has a GALNS enzyme concentration of about 1 +/- 0.5 mg/mL.

In Example XI, recombinant GALNS enzyme was formulated in 20 mM NaOAc/HOAc, 50 mM NaH ₂ PO ₄ at pH 5.0, 5.4, and 5.8. Preferred buffers are NaOAc/HOAc or its equivalent; and NaH ₂ PO ₄ or its equivalent, wherein the NaOAc/HOAc concentration ranges from about 5 to 100 mM, preferably from about 5 to 50 mM, and more preferably. 10 to 30 mM, and the NaH ₂ PO ₄ concentration ranges from about 5 to 100 mM, preferably from about 25 to 100 mM, and more preferably from about 25 to 75 mM. In an exemplary embodiment, the formulation of the GALNS composition of the invention has a NaOAc/HOAc buffer concentration of about 20 +/- 10 mM and a NaH ₂ PO ₄ buffer concentration of about 50 +/- 25 mM.

The preferred pH of the formulation is from about pH 4.5 to 6.5, preferably from about pH 5.0 to 6.0, and more preferably from about pH 5.0 to 5.8. In one embodiment, the formulation of the GALNS composition of the invention has a pH of about pH 5.4 +/- 0.4.

In Example XI, the recombinant GALNS enzyme was formulated in 15 mM or 30 mM arginine hydrochloride or NaCl. A preferred stabilizing agent is arginine hydrochloride or an equivalent thereof, wherein the concentration ranges from about 5 to 200 mM, preferably from about 10 to 100 mM, and more preferably from about 10 to 50 mM. In an exemplary embodiment, the formulation of the GALNS compositions of the invention has a arginine hydrochloride concentration of about 30 +/- 20 mM.

In Example XI, the recombinant GALNS enzyme was formulated in 0.01% Tween-20. A preferred stabilizer is Tween-20 (also known as polysorbate-20) or an equivalent thereof, wherein the concentration ranges from about 0.001 to 1.0% (w/v), preferably from about 0.005 to 0.2% (w/ v), and more preferably from about 0.005 to 0.015% (w/v). In one embodiment, the formulation of the GALNS composition of the invention has a Tween-20 concentration of about 0.01% +/- 0.005% (w/v).

In Example XI, the recombinant GALNS enzyme was formulated in 4% sucrose or 2% sorbitol. Preferably, the stabilizing/low temperature protectant/tensioning agent is sorbitol or an equivalent thereof, wherein the concentration ranges from about 0.1 to 10% (w/v), preferably from about 0.5 to 5% (w/v), and more Good about 1.0 to 3.0% (w/v). An exemplary formulation of a GALNS composition of the invention has a sorbitol concentration of about 2.0% +/- 1.0% (w/v).

Thus, one exemplary formulation of a GALNS enzyme composition of the invention is shown in Table 18 .

*pH is adjusted by titration with glacial acetic acid (HOAc) or 1 N sodium hydroxide (NaOH).

Example XIII Effect of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and other lysosomal sulfatase enzymes in mice lacking lysosomal sulfatase activity

Evaluation of the active hyperphosphorylated human lysosomal sulfatase enzyme of the present invention (eg, recombinant human N-acetylgalactosamine-6-sulfatase (GALNS)) in mice lacking lysosomal sulfatase activity effect.

Recombinant human GALNS protein was expressed in G71S cells and purified. Other recombinant human lysosomal sulfatase enzymes can be expressed and purified essentially according to the methods described herein or by procedures known in the art.

Several mouse models of human lysosomal sulfatase deficiency have been described, including: metachromatic leukodystrophy (MLD) (arylsulfatase A deficiency) (Hess et al, Proc. Natl. Acad. Sci. USA 93:14821-14826, 1996), Type VI mucopolysaccharidosis (MPS VI) or Malayan's syndrome (arylsulfatase B deficiency) (Evers et al, Proc. Natl. Acad. Sci USA 93:8214-8219, 1996), Type II mucopolysaccharidosis (MPS II) or Hunter syndrome (iduronic acid-2-sulfatase deficiency) (Muenzer et al., Acta Paediatr. Supplement 91 (439): 98-99, 2002; Cardone et al, Hum. Mol. Genet. 15: 1225-1236, 2006), type IIIa mucopolysaccharidosis (MPS IIIa) or St. Philippe A syndrome (sulfonamide) Enzyme/acetyl heparin-N-sulfatase deficiency) (Bhaumik et al, Glycobiology 9(12): 1389-1396, 1999), type IVa mucopolysaccharidosis (MPS IVa) or Moquiao A syndrome (N) - acetaminosamine-6-sulfatase deficiency) (Tomatsu et al., Hum. Mol. Genet. 12: 3349-3358, 2003) and various sulfatase deficiency (MSD) (sulfatase modification factors) 1 deficiency) (Settembre et al, Proc. Natl. Acad. Sci. USA 104: 4506-4511, 2007). A mouse model of type IIId (MPS IIId) mucopolysaccharidosis or St. Philippe D syndrome (N-acetylglucosamine-6-sulfatase deficiency) has yet to be described.

A mouse model of human lysosomal sulfatase deficiency can be used to assess the feasibility of enzyme replacement therapy (ERT) as a means of treating lysosomal storage disorders. For example, MPS IVa knockout mice ( GALNS ^-/- mice; Tomatsu et al, Hum. Mol. Genet. 12: 3349-3358, 2003) do not have detectable GALNS enzyme activity and show increased urine Glycosaminoglycans (GAG) (ie, keratin sulfate and chondroitin-6-sulfate) and GAG in multiple tissues and organs (eg liver, kidney, spleen, heart, brain, bone marrow, and cartilage) In the accumulation of). However, GALNS ^-/- mice do not show skeletal abnormalities associated with human disease. Another MPS IVa mouse model has been developed which exhibits inactive human GALNS and mutant inactive endogenous mouse GALNS ( GALNS ^{tm (hC79S.mC76S) slu} mice; Tomatsu et al., Hum. Mol. Genet. 14: 3321-3335, 2005). In GALNS ^{tm (hC79S.mC76S) slu} mice without detectable GALNS activity, urinary GAG excretion increased, and GAG was found in multiple tissues including visceral organs, brain, cornea, bone, ligament, and bone marrow. Cumulative, lysosomal storage is significant in multiple tissues and bone storage is evident. The pathological changes in GALNS ^{tm (hC79S.mC76S) slu} mice differed from those observed in GALNS ^-/- mice. However, similar to GALNS ^-/- mice, GALNS ^{tm (hC79S.mC76S) slu} mice did not show skeletal abnormalities associated with human disease. Therefore, GALNS ^-/- or GALNS ^{tm (hC79S.mC76S) slu} mice can be used to study the effect of administration of recombinant human GALNS on the accumulation of increased urinary GAG and GAG in tissues.

Each dose of recombinant human GALNS (eg 0.1, 0.1 ^{) was administered} weekly to 4 weeks old GALNS ^-/- , GALNS ^{tm (hC79S.mC76S) slu} or wild type mice (n = at least 6 or 8 mice per group). 0.3, 1, 3, 10 mg/kg) or vehicle control up to 16-20 weeks of age, followed by sacrifice for histological examination. Urine urine was collected and urine GAG excretion was determined as described (Tomatsu et al, Hum. Mol. Genet. 12: 3349-3358, 2003). Pathological examination of various tissues was performed as described (Tomatsu et al., Hum. Mol. Genet. 12: 3349-3358, 2003).

Using GALNS ^-/- or GALNS ^{tm (hC79S.mC76S) slu} mice, it is expected that recombinant human GALNS of the invention will be shown to reduce: (1) urinary GAG excretion; (2) GAG in multiple tissues (eg, internal organs, Accumulation in brain, cornea, bone, ligament, and bone marrow; (3) storage of lysosomes in multiple tissues; and (4) bone storage.

To study the effect of recombinant human GALNS in a mouse model of multiple sulfatase deficiency (MSD) ( SUMF1 ^-/- mice; Settembre et al, Proc. Natl. Acad. Sci. USA 104: 4506-4511, 2007) . Since SUMF1 ^-/- mice were shown to die frequently in early life, these mice were initially injected with recombinant human GALNS earlier than the time described above for GALNS ^-/- mice.

Other recombinant human lysosomal sulfatase enzymes (ie, arylsulfatase A, arylsulfatase B, iduronic acid-2-sulfatase, sulfonamide) were studied following procedures known in the art. Aminase/acetyl heparin-N-sulfatase and N-acetylglucosamine-6-sulfatase) in MLD ( ASA ^-/- mice; Hess et al, Proc. Natl. Acad. Sci. USA) 93:14821-14826, 1996), MPS VI ( As1 - s ^-/- mice; Evers et al, Proc. Natl. Acad. Sci. USA 93:8214-8219, 1996), MPS II ( ids ^y/- Mouse; Cardone et al, Hum. Mol. Genet. 15: 1225-1236, 2006), MPS IIIa (Bhaumik et al, Glycobiology 9(12): 1389-1396, 1999 ) and MSD ( SUMF1 ^{- /-} mice) The effect in the mouse model of Settembre et al, Proc. Natl. Acad. Sci. USA 104: 4506-4511, 2007).

Example XIV Treatment of type IVA mucopolysaccharidosis (or Moquiao syndrome) or other lysosomal sulfates with recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and other lysosomal sulfatase enzymes Human patient with enzyme deficiency

Recombinase (ie human N-acetamidine) is expected to be present in human patients, such as the clinical phenotype of lysosomal sulfatase deficiency in patients diagnosed with type IVA mucopolysaccharidosis (MPS IVa or Moquio syndrome) Galactosamine-6-sulfatase (GALNS) is used for enzyme replacement therapy. All patients with lysosomal sulfatase deficiency have clinical signs of accumulation or accumulation of lysosomes in the visceral and soft tissues, such as varying degrees of accumulation with specific lysosomes. Related dysfunction or worsening health conditions appear. All MPS IVa patients showed some of the following clinical signs: skeletal malformations, short stature and gait abnormalities, and/or glycosaminoglycan (GAG) accumulation in the blood or urine, and varying degrees of dysfunction.

Preferably, the enzyme content is monitored in patients suffering from lysosomal sulfatase deficiency to confirm the absence or reduced activity of lysosomal sulfatase enzymes in their tissues. A patient whose lysosomal enzyme activity is 10% or less, preferably 5% or less, more preferably 2% or less, and even more preferably 1% or less of another normal individual is a candidate suitable for treatment with an appropriate lysosomal sulfatase enzyme. . Data can be collected before, during, and after the implementation of the therapy to determine the patient's lysosomal sulfatase activity.

Efficacy was determined by measuring the percentage decrease in urinary excretion of the lysosomal sulfatase enzyme (i.e., glycosaminoglycan (GAG)) over time. The urinary GAG content of patients suffering from lysosomal sulfatase deficiency and the content of normal excretion and/or the content of untreated patients suffering from the same lysosomal sulfatase deficiency and/or the same patient in the implementation of lysozyme The content before the body sulfatase treatment was compared. A reduction in excretion of undegraded GAG after lysosomal sulfatase therapy is more than a 25% reduction, preferably greater than 50%, is an effective means of measuring an individual's response to therapy.

Efficacy can also be determined based on signs and symptoms of lesions associated with lysosomal storage diseases. Efficacy can be determined by tissue biopsy and examination of cells and/or lysosomes for measuring the degree of reduction in GAG in lysosomes, cells or tissues. Efficacy can be determined by functional assessment, which can be objective or subjective (eg, reduction in pain or dysfunction, increase in muscle strength or energy, increase in cardiac output, exercise durability, weight change, height, or Appearance changes and similar functional assessments).

The pharmaceutical composition comprises purified recombinant human GALNS expressed in G71S cells and formulated according to procedures known in the art. Preferably, the pharmaceutical composition of the present invention is administered intravenously.

The basic design of an initial clinical study investigating the effect of administering recombinant human GALNS to MPS IVa patients involves an open-label dose-increasing safety/efficacy study in which various doses of enzyme are administered intravenously to patients at regular intervals (eg (but Not limited to) weekly enzymatic injection).

For patients with MPS IVa, by measuring, for example, reduced blood or urine GAG (which may be observed within weeks of ERT), increased durability in heart, lung, and/or motor function tests (which may be in ERT) Efficacy was determined by observations and/or bone changes and/or body growth (which may have been observed within several years of ERT) within a few months.

Urine GAG measurements are useful for determining the appropriate dosage regimen and for determining efficacy by measuring the percentage decrease in urinary GAG excretion over time.

Various endurance tests can be used including, for example, but not limited to, walking tests (distances traveled within 6 or 12 minutes), stair climbing (steps/minutes), and lung/respiratory functions, including cardiac function (ECG, supersonic) Echocardiogram, lung function (FVC, FEV ₁ , peak flow).

For younger patients who have been treated for long periods of time, growth (height) can be measured.

The lysosomal storage diseases associated with the lack of lysosomal sulfatase activity that can be treated or prevented by the method of the present invention are: metachromatic leukodystrophy (MLD), type VI mucopolysaccharidosis (MPS VI) or horse Lad's syndrome, type II mucopolysaccharidosis (MPS II) or Hunter's syndrome, type IIIa mucopolysaccharidosis (MPS IIIa) or St. Philippe A syndrome, type IIId mucopolysaccharidosis (MPS IIId) or holy Philippe D syndrome, type Ia mucopolysaccharidosis (MPS IVa) or Moquiao A syndrome, or multiple sulfatase deficiency (MSD). For each lysosomal storage disease, the recombinant lysosomal sulfatase enzyme will comprise a specific lysosomal sulfatase enzyme.

For methods involving MLD, the preferred lysosomal sulfatase enzyme is arylsulfatase A. For methods involving MPS VI, the preferred lysosomal sulfatase enzyme is arylsulfatase B. For methods involving MPS II, the preferred lysosomal sulfatase enzyme is iduronic acid-2-sulfatase. For methods involving MPS IIIA, the preferred lysosomal sulfatase enzyme is sulfonamide/acetyl heparin-N-sulfatase. For methods involving MPS IIID, the preferred lysosomal sulfatase enzyme is N-acetyl glucosamine-6-sulfatase. For methods involving MPS IVA, the preferred lysosomal sulfatase enzyme is N-acetylgalactosamine-6-sulfatase. For methods involving MSD, the preferred lysosomal sulfatase enzyme is N-acetylgalactosamine-6-sulfatase.

*****

Numerous modifications and variations of the present invention as set forth in the foregoing illustrative examples are contemplated. Therefore, the present invention should be construed as having only such limitations as found in the appended claims.

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Figure 1 depicts the nucleotide sequence of human sulfatase modifying factor 1 (SUMF1) (SEQ ID NO: 1).

Figure 2 depicts the amino acid sequence of human sulfatase modifying factor 1 (SUMF1) (SEQ ID NO: 2).

Figure 3 depicts the nucleotide sequence of human N-acetylgalactosamine-6-sulfatase (GALNS) (SEQ ID NO: 3).

Figure 4 depicts the amino acid sequence of human N-acetylgalactosamine-6-sulfatase (GALNS) (SEQ ID NO: 4). There is no N-terminal signal peptide containing 26 amino acids in the processed GALNS.

Figure 5 depicts the structure and characteristics of processed human N-acetylgalactosamine-6-sulfatase (GALNS) (SEQ ID NO: 5). 1) The molecular weight of the enzyme before processing is about 55 kDa, 2) the conversion of cysteine to C _α -methionine at position 53, 3) the processed form in the lysosome is linked by a disulfide bridge. 4) 2N-linked glycosylation sites are at positions 178 and 397, and 5) BisP is found on Asn178 rather than Asn397.

Figure 6 shows the performance of human N-acetylgalactosamine-6-sulfatase (GALNS) from G71S cells co-transfected with human sulfatase modifying factor 1 (SUMF1) and human GALNS expression vector. (A) G71S pure line screening for active GALNS in 96 wells. (B) G71S pure GALNS yield in picograms per cell per day.

Figure 7 illustrates a schematic representation of a WAVE bioreactor controller for large scale production of G71S cells expressing human N-acetylgalactosamine-6-sulfatase (GALNS) and variants thereof.

Figure 8 shows the stability of purified human N-acetylgalactosamine-6-sulfatase (GALNS) enzymatic activity after storage at 4 ° C (diamonds) or -70 ° C (triangles).

Figure 9 shows human N-acetylgalactosamine-6-sulfatase (GALNS) by (A) blue agarose gel 6-speed flow chromatography and (B) Fractogel SE Hi-CAP chromatography. purification. Purity was determined by Coomassie blue staining (left) by SDS-PAGE and Western blotting (right) by using anti-GALNS (IVA) antibody.

Figure 10 shows human N-acetylgalactosamine by ultrafiltration/diafiltration (UF/DF), Fractogel SE Hi-Cap chromatography, Zn chelate agarose gel chromatography and ToyoPearl butyl 650M chromatography. Purification of 6-sulfatase (GALNS). Purity was performed by Coomassie blue staining (upper left) by SDS-PAGE and by anti-GALNS antibody (top right), anti-Cathepsin L antibody (bottom left) and anti-CHOP (Chinese hamster ovary cell protein) (bottom right) Western blotting method is used to determine.

Figure 11 shows a flow diagram of the human N-acetylgalactosamine-6-sulfatase (GALNS) recovery and purification process for Phase I/II processes (left) and Phase III processes (right).

Figure 12 shows a comparison of human N-acetylgalactosamine-6-sulfatase (GALNS) purified according to Phase I/II process (lane 3) or phase III process (lane 5). 5 μg (5 μg) of purified GALNS was isolated by SDS-PAGE under reducing conditions, and the gel was stained with Coomassie blue. Lane 1 corresponds to the 15 μL SeeBlue Plus2 marker. The molecular weight in kDa is indicated on the left side of the stained gel.

Figure 13 shows a dose-dependent decrease in the amount of dermatan sulfate receptor observed in IDU-treated GM01391 cells.

Figure 14 shows a dose-dependent decrease in the amount of dermatan sulfate receptor observed in ARSB-treated GM00519 cells.

Figure 15 shows the uptake of human N-acetylgalactosamine-6-sulfatase (GALNS) by uncultured synovial cells to unlabeled (circles) or bound to A488 (squares) or A555 (triangles).

Figure 16 shows the stability of purified human N-acetylgalactosamine-6-sulfatase (GALNS) after storage for 1 or 2 months at 5 ° C, 25 ° C or 40 ° C, as in the presence of 15 mM spermine The formulation of the hydrochloride, 30 mM arginine hydrochloride, 15 mM NaCl or 30 mM NaCl (designated as 51, 52, 54 or 55, respectively) at pH 5.0, pH 5.4 or pH 5.8 (page respectively) Specified as indicated under A, B, or C). Stability was measured by the percentage of peak area (%) of GALNS aggregates in the formulation after storage, as determined by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC).

Figure 17 shows the stability of purified human N-acetylgalactosamine-6-sulfatase (GALNS) enzymatic activity after storage for 2 months at 5 ° C, 25 ° C or 40 ° C, as in containing 15 mM spermine The formulation of the hydrochloride, 30 mM arginine hydrochloride, 15 mM NaCl or 30 mM NaCl (designated as 51, 52, 54 or 55, respectively) at pH 5.0, pH 5.4 or pH 5.8 (page respectively) Specified as indicated under A, B, or C).

Figure 18 shows a glycosylation profile of purified human N-acetylgalactosamine-6-sulfatase (GALNS) after storage for 2 months at 5 ° C, 25 ° C or 40 ° C, as included, as indicated A formulation of 15 mM arginine hydrochloride, 30 mM arginine hydrochloride, 15 mM NaCl or 30 mM NaCl is indicated at pH 5.0, pH 5.4 or pH 5.8. After the GALNS enzyme was digested with PNGase F to cleave aspartate N-linked oligosaccharides, the percentage of diphosphorylated mannose 7 (BPM7) was measured by capillary electrophoresis (CE). The reference indicates the percentage of BPM7 of the reference batch GALNS stored at 5 ° C, 25 ° C or 40 ° C for 2 months, as indicated in the formulation containing 100 mM phosphate buffer.

Figure 19 shows the stability of purified human N-acetylgalactosamine-6-sulfatase (GALNS) after storage for 2 months at 5 ° C, 25 ° C or 40 ° C, as in containing 15 mM arginine The formulation of the acid salt, 30 mM arginine hydrochloride, 15 mM NaCl or 30 mM NaCl (designated as 51, 52, 54 or 55 respectively) at pH 5.0, pH 5.4 or pH 5.8 (designated as Indicated under A, B or C). The stability of the GALNS enzyme was measured by the percentage (%) of the peak area of the GALNS enzyme in the formulation after storage, as determined by reverse phase high performance liquid chromatography (RP-HPLC).

(no component symbol description)

Claims (13)

A method for purifying recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) comprising an amino acid sequence at least 95% identical to amino acid 27 to 522 of SEQ ID NO: 4, Wherein the GALNS enzyme: (i) has a purity of at least 95% as determined by Coomassie blue staining on SDS-PAGE under non-reducing conditions, (ii) a cysteine residue having a position 53 conversion of C α _- carboxylic acyl glycine (FGIy) of at least 50% conversion, and (iii) per monomeric protein chain having from 0.5 to 0.8 bis-phosphorylated oligomannose chains, and wherein at least 98% of the The GALNS enzyme system is in the form of a precursor, which is determined by SDS-CGE, and comprises: a) filtering secretion of a mammalian cell line containing the self-expressing human sulfatase modifying factor 1 (SUMF1) and the recombinant human GALNS enzyme. The medium of the GALNS enzyme is ultrafiltered and/or diafiltered to the filtered medium, whereby the ultrafiltration and/or diafiltration medium is concentrated 20 times, and the charcoal is filtered and concentrated 20 times by ultrafiltration and/or Or diafiltered medium; b) loading the charcoal-filtered 20-fold ultrafiltration and/or diafiltration medium of step a) on Zn chelate agar The gel FF captures the column, washes the column under the condition that the GALNS enzyme remains on the column, and dissolves the GALNS enzyme from the column; c) if necessary, the Zn in step b) Chelation of the agarose gel FF capture column of the solution is filtered by a filter to remove the virus; d) adjusting the Zn chelate agarose gel FF of the step b) to capture the column of the solution or the filtrate of step c) pH to pH 4.5 ± 0.1, and filtering the Zn chelate agarose gel FF trap column adjusted to pH 4.5 ± 0.1 or the filtrate adjusted to pH 4.5 ± 0.1; e) The solution of the Zn chelate FF trap column adjusted to pH 4.5±0.1 from step d) or the filtrate adjusted to pH 4.5±0.1 is loaded on a Fractogel EMD SE Hi-Cap cation exchange column. Washing the column under conditions that retain the GALNS enzyme on the column, and dissolving the GALNS enzyme from the column; f) adjusting the solution of the Fractogel EMD SE Hi-Cap cation exchange column of step e) pH to pH 3.5 ± 0.1 to make the virus inactive; g) adjust the pH of the solution of step f) 3.5 ± 0.1 virus inactivated solution to pH 5.0, and then adjust the loading to a virus inactivated at pH 5.0 is applied to a ToyoPearl butyl 650M purified column, the column is washed under conditions such that the GALNS enzyme remains on the column, and the GALNS enzyme is dissolved from the column; The buffer of the ToyoPearl butyl 650M purified column of step g) was buffer exchanged to contain 20 mM NaOAc/HOAc, 50 mM NaH ₂ PO ₄ , 30 mM arginine hydrochloride, 2% (w/v) Yamanashi a sugar alcohol and a pH 5.4 formulation, and adjusting the concentration of the GALNS enzyme in the formulation to 3 mg/mL; i) filtering the buffer exchange in step h) by DV20 filter and Mustang Q filter Formulation to remove any residual virus and/or DNA; and j) Add polysorbate 20 (PS20) to a final concentration of 0.01% (w/v) in the formulation in step i). A formulation comprising: (a) recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) purified according to the method of claim 1, the GALNS enzyme comprising an amino group of SEQ ID NO: Acids 27 to 522 having at least 95% identical amino acid sequence, wherein the GALNS enzyme: (i) has a purity of at least 95% as determined by Coomassie blue staining on SDS-PAGE under non-reducing conditions (ii) conversion of the cysteine residue having position 53 to at least 50% conversion of C _α -methionine (FGly), and (iii) 0.5 to 0.8 pairs of each monomeric protein chain Phosphorylated oligomannose chains, and wherein at least 98% of the GALNS enzymes are in the form of a precursor, which is determined by Coomassie blue staining on SDS-PAGE under reducing conditions, and (b) one or more A pharmaceutically acceptable carrier, diluent or excipient consisting essentially of (i) NaOAc/HOAc wherein the concentration of NaOAc/HOAc is 20 +/- 10 mM; (ii) NaH ₂ PO ₄ wherein the concentration of the NaH ₂ PO ₄ is 50 +/- 25 mM; (iii) arginine hydrochloride, Tween-20 and sorbitol as stabilizers, wherein the concentration of the arginine hydrochloride is 30 +/- 20m M, the concentration of the Tween-20 is 0.01% +/- 0.005% (w/v), and the concentration of the sorbitol is 2.0% +/- 1.0% (w/v); and (iv) the pH is 5.4. +/- 0.4. . A formulation comprising: (a) recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) purified according to the method of claim 1, the GALNS enzyme comprising an amino group of SEQ ID NO: Acids 27 to 522 are at least 95% identical amino acid sequence, and (i) have a purity of at least 95% as determined by Coomassie blue staining on SDS-PAGE under non-reducing conditions, (ii) The cysteine residue having position 53 is converted to at least 50% conversion of C _α -methionine (FGly), and (iii) 0.5 to 0.8 diphosphorylated oligomannose per monomeric protein chain a sugar chain wherein at least 98% of the GALNS enzyme is in the form of a precursor, as determined by SDS-CGE, and (b) one or more pharmaceutically acceptable carriers, diluents or excipients, It comprises: (i) a phosphate buffer effective to reduce the amount of dephosphorylation of the GALNS enzyme, wherein the phosphate buffer is at a concentration of 25 mM to 75 mM NaH ₂ PO ₄ ; and (ii) a stable amount as follows Stabilizer: arginine or buffer, wherein the concentration of the arginine or buffer is 10 mM to 50 mM; polysorbate and ternary or trivalent sugar alcohol; wherein the blending The pH of the substance is 5.0-5.8. The formulation of claim 3, wherein the formulation further comprises acetate A buffering agent wherein the acetate buffer is at a concentration of 10 mM to 30 mM NaOAc/HOAc. The formulation of claim 3, wherein the arginine or buffer is a spermine hydrochloride, the polysorbate 20 (Tween-20), and the ternary or trivalent or higher The sugar alcohol is sorbitol. The formulation of claim 3, wherein the GALNS enzyme has a purity of at least 95% as determined by RP-HPLC. The formulation of claim 3, wherein 50% to 80% of the GALNS enzyme binds to the mannose-6-phosphate receptor column. The formulation of claim 3, wherein the GALNS enzyme exhibits a specific uptake (Kuptake) of 1 to 5 nM in the fibroblast. The formulation of claim 3, wherein the GALNS enzyme exhibits a specific uptake (Kuptake) of 1 to 3.5 nM in the fibroblast. The formulation of claim 3, wherein the concentration of the GALNS enzyme is from 0.5 to 1.5 mg/mL. The formulation of claim 5, wherein the concentration of the Tween-20 is from 0.005% to 0.015% (w/v), and the concentration of the sorbitol is from 1.0% to 3.0% (w/v). A formulation comprising: (a) recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) purified according to the method of claim 1, the GALNS enzyme comprising an amino group of SEQ ID NO: Acids 27 to 522 are at least 95% identical amino acid sequence, and (i) have a purity of at least 95% as determined by Coomassie blue staining on SDS-PAGE under non-reducing conditions, (ii) The cysteine residue having position 53 is converted to at least 50% conversion of C _α -methionine (FGly), and (iii) 0.5 to 0.8 diphosphorylated oligomannose per monomeric protein chain a sugar chain, wherein at least 98% of the GALNS enzyme is in the form of a precursor, which is determined by Coomassie blue staining on SDS-PAGE under reducing conditions, and (b) one or more pharmaceutically acceptable a carrier, diluent or excipient comprising: (i) NaOAc/HOAc and NaH ₂ PO ₄ as a buffer, wherein the NaOAc/HOAc concentration is 20 +/- 10 mM, and the NaH ₂ PO ₄ Concentration 50 +/- 25 mM; (ii) arginine hydrochloride, Tween-20 and sorbitol as stabilizers, wherein the concentration of the arginine hydrochloride is 30 +/- 20 mM, the Tween-20 The concentration is 0.01% + /- 0.005% (w/v), and the concentration of the sorbitol is 2.0% +/- 1.0% (w/v); and (iii) the pH is 5.4 +/- 0.4. Use of a formulation according to claim 3 for the manufacture of a medicament for the treatment of an individual suffering from a mucopolysaccharidosis Iva (MPS Iva) or Moquio A syndrome.