Method for purifying arylsulfatase A

Abstract

To provide methods for purification of arylsulfatase A.SOLUTION: The present invention provides, among other things, improved methods for purifying arylsulfatase A (ASA) protein produced recombinantly for enzyme replacement therapy. The present invention is, in part, based on the surprising discovery that recombinant ASA protein can be purified from unprocessed biological materials, such as an ASA-containing cell culture medium, using a process involving as few as four chromatography columns and only one step of post-chromatographic ultrafiltration / diafiltration.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61 / 750,693, filed January 9, 2013, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by a deficiency of the enzyme arylsulfatase A (ASA). ASA, encoded by the ARSA gene in humans, is an enzyme that breaks down cerebroside 3-sulfate or sphingolipid 3-O-sulfogalactosylceramide (sulfatide) into cerebroside and sulfate. In the absence of the enzyme, sulfatide accumulates in the nervous system (e.g., myelin sheaths, neurons, and glial cells), but to a lesser extent in visceral organs. The result of these molecular and cellular events is progressive demyelination and axonal loss in the CNS and PNS, accompanied by severe motor and cognitive dysfunction.

[0003] The defining clinical feature of this disorder is central nervous system (CNS) degeneration, which leads to cognitive impairment (eg, mental retardation, neuropathy, and blindness).

[0004] MLD may develop in young children (late infantile forms), where affected children typically begin to show symptoms shortly after the first year of life (e.g., about 15-24 months) and generally do not survive past age 5. MLD may develop in children (juvenile forms), where affected children typically show cognitive impairment by about 3-10 years of age, and life spans may vary (e.g., ranging from 10-15 years after onset of symptoms). MLD may develop in adults (adult onset forms), where affected children may present at any age (e.g., typically after age 16), and disease progression may vary widely.

[0005] Enzyme replacement therapy (ERT) is an approved therapy for treating MLD and involves administering exogenous replacement ASA enzyme, specifically recombinant arylsulfatase A (rASA) (e.g., recombinant human arylsulfatase A (rhASA)), to patients suffering from MLD. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides, inter alia, an improved method for purifying recombinantly produced ASA protein for enzyme replacement therapy. The present invention is based, in part, on the surprising discovery that recombinant ASA protein can be purified from unprocessed biological material, such as cell culture media containing ASA, using a process that involves only a single step of post-chromatographic ultrafiltration / diafiltration, resulting in a pharma- ceutically acceptable drug substance directly in the final formulation buffer. As described in the Examples below, this single-step UF / DF process is accomplished by simply pooling the eluates from the chromatography step and adjusting the pH of the pooled eluate to about 6.0. Prior to the present invention, processes for purifying recombinantly produced rASA protein included at least two steps of post-chromatographic ultrafiltration / diafiltration (UF / DF). As described in the Examples section, recombinant ASA protein purified using a one-step UF / DF process according to the present invention has comparable purity, activity, and yield compared to recombinant ASA protein purified using multiple UF / DF steps. For example, recombinant ASA enzymes purified according to the present invention have less than 100 pg / mg of host cell DNA and possess high specific activity (e.g., about 50-140 U / mg) as well as other distinct features that may enhance the bioavailability and / or lysosomal targeting of recombinant ASA protein. Thus, this simplified process is faster, less expensive, and equally effective in purifying recombinant ASA protein. This process is particularly useful when combined with high-load capacity chromatography steps to facilitate large-scale production of recombinant ASA protein.

[0007] Therefore, in one aspect, the present invention provides a method for purifying recombinant arylsulfatase A (ASA) protein, comprising the steps of purifying the recombinant arylsulfatase A (ASA) protein from impure preparations by performing one or more chromatography steps, pooling eluates from the one or more chromatography steps, adjusting the pH of the pooled eluates to a pH of about 5.8, 5.9, or 6.0 or higher, and subjecting the pH-adjusted eluates to ultrafiltration and / or diafiltration. In some embodiments, the pH is adjusted to about 5.8-7.0 (e.g., about 5.8-6.8, about 5.8-6.6, about 5.8-6.4, about 5.8-6.2, about 5.8-6.1, about 5.8-6.0, about 5.9-7.0, about 5.9-6.8, about 5.9-6.6, about 5.9-6.4, about 5.9-6.2, about 5.9-6.1, about 5.9-6.0, about 5.95-6.20, about 5.95-6.15, about 5.95-6.10, or about 5.95-6.05). In some embodiments, the pH is adjusted to about 6.0.

[0008] In some embodiments, the pH is adjusted using a buffer containing sodium phosphate, sodium chloride, and sodium citrate having a pH of 7.0. In some embodiments, the buffer contains about 0.1-0.5 M (e.g., about 0.1-0.4 M, 0.1-0.3 M, 0.2-0.4 M, or 0.2-0.3 M) sodium phosphate, about 0.5-2.5 M (e.g., about 0.5-2.0 M, 0.5-1.5 M, 0.75 M, or 0.8 M) sodium citrate, having a pH of about 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. 0.25M, 0.15M, 0.25M, 0.33M, 0.34M) sodium citrate. In some embodiments, the buffer comprises about 0.25M sodium phosphate, about 1.33M sodium chloride, and about 0.34M sodium citrate, having a pH of about 7.0.

[0009] In some embodiments, virus filtration is performed before the steps of ultrafiltration and diafiltration. In some embodiments, a single step of ultrafiltration and / or diafiltration is performed. In some embodiments, a single step of ultrafiltration and / or diafiltration includes only one diafiltration. In various embodiments, the ultrafiltration is tangential flow ultrafiltration.

[0010] In some embodiments, the one or more chromatography steps include cation exchange chromatography. In some embodiments, the cation exchange chromatography is the last chromatography step, and the eluate from the cation exchange chromatography is pooled before adjusting the pH. In some embodiments, one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed before performing the cation exchange chromatography.

[0011] In some embodiments, the one or more chromatography steps include affinity chromatography. In some embodiments, affinity chromatography is the first chromatography step. In some embodiments, affinity chromatography is the last chromatography step, and the eluates from affinity chromatography are pooled before adjusting the pH. In some embodiments, one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed before performing affinity chromatography. In some embodiments, one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed after performing affinity chromatography.

[0012] In some embodiments, the anion exchange chromatography is Q chromatography. In some embodiments, the anion exchange chromatography comprises a TMAE resin (e.g., FRACTOGEL® TMAE). In some embodiments, the TMAE resin, once loaded with the impure preparation, is washed with a first wash buffer comprising MES-Tris at a pH of about 7.0. In some embodiments, the first wash buffer comprises about 20-75 mM MES-Tris (e.g., about 30-60 mM, 40-70 mM, or 40-60 mM). In some embodiments, the first wash buffer comprises about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, or 75 mM MES-Tris.

[0013] In some embodiments, the TMAE resin is washed with a second wash buffer comprising MES-Tris having a pH of about 7.0 and NaCl. In some embodiments, the second wash buffer comprises about 5-75 mM MES-Tris having a pH of about 7.0 (e.g., about 5-70 mM, 5-60 mM, 5-50 mM, 5-40 mM, 5-30 mM, 10-60 mM, 10-50 mM, 10-40 mM, 10-30 mM, or 15-25 mM) and about 50-150 mM NaCl (e.g., about 50-140 mM, 50-130 mM, 50-120 mM, 50-110 mM, 75-150 mM, 75-140 mM, 75-130 mM, 75-120 mM, or 75-110 mM). In some embodiments, the second wash buffer comprises about 5 mM, 10 mM, 20 mM, 30 MM, 40 mM, 50 mM, 60 mM, or 75 mM MES-Tris having a pH of about 7.0 and about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM NaCl. In some embodiments, the second wash buffer comprises about 20 mM MES-Tris having a pH of about 7.0 and about 100 mM NaCl.

[0014] In some embodiments, the TMAE resin, once loaded with the impure preparation, is eluted with an elution buffer comprising MES-Tris and NaCl having a pH of about 7.0. In some embodiments, the elution buffer comprises about 5-75 mM MES-Tris (e.g., about 5-70 mM, 5-60 mM, 5-50 mM, 5-40 mM, about 5-30 mM, 10-75 mM, 10-60 mM, 10-50 mM, 10-40 mM, or 10-30 mM) having a pH of about 7.0 and about 150-300 mM NaCl (e.g., about 150-250 mM, about 180-260 mM, about 200-280 mM, 200-260 mM, 200-240 mM, 210-300 mM, 210-280 mM, 210-260 mM, or 210-240 mM). In some embodiments, the elution buffer comprises about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or 75 mM MES-Tris having a pH of about 7.0 and about 150 mM, 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM NaCl. In some embodiments, the elution buffer comprises about 20 mM MES-Tris having a pH of about 7.0 and about 220 mM NaCl.

[0015] In some embodiments, the anion exchange chromatography uses a column selected from the group consisting of Q SEPHAROSE™ Fast Flow, Q SEPHAROSE™ High Performance, Q SEPHAROSE™ XL, CAPTO™ Q, DEAE, TOYOPEARL GIGACAP® Q, FRACTOGEL® TMAE, ESHMUNO™ Q, NUVIA™ Q, or UNOSPHERE™ Q.

[0016] In some embodiments, the mixed mode chromatography suitable for the present invention is hydroxyapatite (HA) chromatography.

[0017] In some embodiments, the hydrophobic interaction chromatography suitable for the present invention is phenyl chromatography.

[0018] In some embodiments, anion exchange chromatography (e.g., with a TMAE resin), mixed mode chromatography (e.g., HA chromatography), hydrophobic interaction chromatography (e.g., phenyl chromatography), and cation exchange chromatography (e.g., SP chromatography) are performed in that order.

[0019] In some embodiments, affinity chromatography suitable for the present invention uses an anti-arylsulfatase A antibody (eg, an anti-human arylsulfatase A antibody).

[0020] In some embodiments, a suitable anion exchange chromatography uses a column with a loading capacity of greater than about 4.5 g / L (e.g., greater than about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L). In some embodiments, a suitable anion exchange chromatography uses a column with a loading capacity in the range of about 4.5-20 g / L (e.g., in the range of about 5-20 g / L, 5-19 g / L, 5-18 g / L, 5-17 g / L, 5-16 g / L, 5-15 g / L, 7.5-20 g / L, 7.5-19 g / L, 7.5-18 g / L, 7.5-17 g / L, 7.5-16 g / L, 7.5-15 g / L, 10-20 g / L, 10-19 g / L, 10-18 g / L, 10-17 g / L, 10-16 g / L, or 10-15 g / L). In certain embodiments, a suitable loading capacity of the column is about 10-15 g / L.

[0021] In some embodiments, ultrafiltration of the impure preparation is performed prior to one or more chromatography steps. In some embodiments, the ultrafiltration step suitable for the present invention is tangential flow ultrafiltration. In some embodiments, a suitable ultrafiltration uses a membrane filter comprising a pore size with a molecular weight cutoff of at least about 10 kDA, at least about 20 kDA, at least about 30 kDA, at least about 40 kDA, at least about 50 kDA. In some embodiments, at least 75% of the recombinant ASA is retained in the impure preparation. In some embodiments, at least 75% of the recombinant ASA permeates the filter. In some embodiments, a suitable ultrafiltration step uses a polyethersulfone or cellulose membrane.

[0022] In some embodiments, the clarification of the impure preparation is carried out before the ultrafiltration of the impure preparation. In some embodiments, the clarification step suitable for the present invention is filtration using one or more depth filters. In some embodiments, the filtration of the impure preparation is carried out using a series of depth filters. In some embodiments, the preferred series of depth filters uses membranes that include cellulose, diatomaceous earth, polyethersulfone, or combinations thereof.

[0023] In some embodiments, ultrafiltration is followed by one or more steps of depth filtration and / or virus inactivation.In some embodiments, depth filtration and / or virus inactivation are followed by one or more chromatography steps.In some embodiments, the virus inactivation step comprises adding detergent to the impure preparation.

[0024] In some embodiments, the methods according to the invention may be used to purify recombinant ASA protein produced by mammalian cells cultured in suspension. In some embodiments, the mammalian cells are cultured in a bioreactor. In some embodiments, the medium is serum-based. In some embodiments, the medium is serum-free. In some embodiments, the serum-free medium is a chemically-defined medium.

[0025] In some embodiments, the impure preparation is a feed stream from a perfusion bioreactor. In some embodiments, the impure preparation is prepared from a medium (e.g., a serum-based or serum-free medium) containing recombinant ASA protein secreted from mammalian cells. In some embodiments, the impure preparation is thawed from a frozen medium preparation.

[0026] In some embodiments, the ultrafiltration and / or diafiltration step involves exchanging the purified recombinant ASA protein into a formulation buffer.

[0027] In some embodiments, the recombinant ASA protein has an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the recombinant ASA protein has an amino acid sequence that is identical to SEQ ID NO:1.

[0028] In some embodiments, purified recombinant ASA protein according to the present invention contains less than about 150 ng / mg, 140 ng / mg, 130 ng / mg, 120 ng / mg, 110 ng / mg, 100 ng / mg, 90 ng / mg, 80 ng / mg, 70 ng / mg, 60 ng / mg, 50 ng / mg, 40 ng / mg, 30 ng / mg, 20 ng / mg, or 10 ng / mg of host cell protein (HCP).

[0029] In some embodiments, the purified recombinant ASA protein has no new bands with an intensity greater than 1.0% of the assay control when subjected to SDS-PAGE with Coomassie blue staining.

[0030] In some embodiments, the purified recombinant ASA protein contains less than about 150 pg / mg, 140 pg / mg, 130 pg / mg, 120 pg / mg, 110 pg / mg, 100 pg / mg, 90 pg / mg, 80 pg / mg, 70 pg / mg, 60 pg / mg, 50 pg / mg, 40 pg / mg, 30 pg / mg, 20 pg / mg, or 10 pg / mg of host cell DNA.

[0031] In some embodiments, the present invention provides a method for purifying a recombinant arylsulfatase A (ASA) protein from an impure preparation by performing one or more chromatography steps, the first chromatography step using a column having a loading capacity of about 4.5 g protein / L resin or more (e.g., greater than about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, or 20 g / L), pooling the eluates from the one or more chromatography steps, adjusting the pH of the pooled eluates to a pH of about 6.0 or more, and subjecting the pH-adjusted eluates to ultrafiltration and / or diafiltration.

[0032] In some embodiments, performing one or more chromatography steps comprises performing, in order, a first ion exchange chromatography, a mixed mode chromatography, a hydrophobic interaction chromatography, and a second ion exchange chromatography.

[0033] In some embodiments, the one or more chromatography steps include affinity chromatography. In some embodiments, affinity chromatography is performed before the first ion exchange chromatography step. In some embodiments, affinity chromatography is performed after the second ion exchange chromatography step. In some embodiments, affinity chromatography is performed between the first and second ion exchange chromatography steps.

[0034] In some embodiments, performing one or more chromatography steps comprises performing anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and cation exchange chromatography, in that order.

[0035] In some embodiments, performing one or more chromatography steps comprises performing affinity chromatography anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and cation exchange chromatography, in that order.

[0036] In some embodiments, performing one or more chromatography steps comprises performing anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, cation exchange chromatography, and affinity chromatography, in that order.

[0037] In some embodiments, the anion exchange chromatography uses a column using TMAE resin. In some embodiments, the TMAE column has a loading capacity of about 5-20 g protein / L resin (e.g., about 5-19 g / L, 5-18 g / L, 5-17 g / L, 5-16 g / L, 5-15 g / L, 7.5-20 g / L, 7.5-19 g / L, 7.5-18 g / L, 7.5-17 g / L, 7.5-16 g / L, 7.5-15 g / L, 10-20 g / L, 10-19 g / L, 10-18 g / L, 10-17 g / L, 10-16 g / L, or 10-15 g / L).

[0038] In some embodiments, a single step of ultrafiltration and / or diafiltration is performed after one or more chromatography steps.

[0039] In particular, the present invention provides recombinant arylsulfatase A (ASA) protein purified by the methods of the invention described herein and pharmaceutical compositions containing same.

[0040] In some embodiments, the invention provides compositions comprising purified recombinant arylsulfatase A (ASA) having an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, wherein the purified recombinant ASA has a specific activity of at least about 50 U / mg, and further, the purified recombinant ASA contains less than 150 ng / mg host cell protein (HCP) and / or less than 150 pg / mg host cell DNA (HCD). In some embodiments, the purified recombinant ASA has an amino acid sequence identical to SEQ ID NO:1. In some embodiments, the purified recombinant ASA contains less than about 140ng / mg, 130ng / mg, 120ng / mg, 110ng / mg, 100ng / mg, 90ng / mg, 80ng / mg, 70ng / mg, 60ng / mg, 50ng / mg, 40ng / mg, 30ng / mg, 20ng / mg, or 10ng / mg of host cell protein (HCP). In some embodiments, the purified recombinant ASA contains less than about 140pg / mg, 130pg / mg, 120pg / mg, 110pg / mg, 100pg / mg, 90pg / mg, 80pg / mg, 70pg / mg, 60pg / mg, 50pg / mg, 40pg / mg, 30pg / mg, 20pg / mg, or 10pg / mg of host cell DNA.

[0041] In some embodiments, the purified recombinant ASA has a specific activity of at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg. In some embodiments, the purified recombinant ASA has a concentration in the range of about 50-200 U / mg (e.g., about 50-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-140 U / mg, 60-130 U / mg, 60-120 U / mg, 60-110 U / mg, 60-100 U / mg, 70-140 U / mg, 70-130 U / mg, 70-120 U / mg, 70-110 U / mg, g, 70-100 U / mg, 80-140 U / mg, 80-130 U / mg, 80-120 U / mg, 80-110 U / mg, 80-100 U / mg, 90-140 U / mg, 90-130 U / mg, 90-120 U / mg, 90-110 U / mg, 90-100 U / mg, 100-140 U / mg, 100-130 U / mg, 100-120 U / mg, 100-110 U / mg, 110-140 U / mg, 110-130 U / mg, 110-120 U / mg, 120-140 U / mg, 120-130 U / mg, or 130-140 U / mg).

[0042] In some embodiments, the invention provides a composition comprising a purified recombinant arylsulfatase A (ASA) having an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the purified ASA is characterized by a glycan map comprising one or more peak groups selected from peak groups representing neutral (Peak Group 1), monosialylated (Peak Group 2), capped mannose-6-phosphorylated (Peak Group 3), disialylated (Peak Group 4), mono-mannose-6-phosphorylated (Peak Group 5), hybrid (Peak Group 6), and di-mannose-6-phosphorylated (Peak Group 7). In some embodiments, the purified ASA is characterized by a glycan map comprising at least two or more, three or more, four or more, five or more, six or more, or seven or more of Peak Groups 1-7. In some embodiments, the purified recombinant ASA has an amino acid sequence identical to SEQ ID NO:1.

[0043] In some embodiments, the present invention provides a formulation comprising a composition described herein and a physiologically acceptable carrier. In some embodiments, the formulation is suitable for intravenous administration. In some embodiments, the formulation is suitable for intrathecal administration. In some embodiments, the formulation is suitable for subcutaneous administration.

[0044] In particular, the present invention relates to a method for treating metachromatic leukodystrophy comprising administering to a subject in need of treatment a purified recombinant ASA protein, pharmaceutical composition, or formulation described herein.

[0045] As used herein, "ASA protein," "ASA," "ASA enzyme," or grammatical equivalents, unless specifically indicated otherwise, refer to a preparation of recombinant ASA protein molecules.

[0046] As used in this application, the terms "about" and "approximately" are used as equivalents. Any numbers used in this application, whether about / approximately or not, are intended to include any normal variations understood by one of ordinary skill in the relevant art.

[0047] Other characteristics, objects, and advantages of the present invention will become apparent in the following detailed description. It should be understood, however, that this detailed description, while indicating embodiments of the present invention, is given by way of example only, and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for purifying a recombinant arylsulfatase A (ASA) protein, comprising: purifying the recombinant arylsulfatase A (ASA) protein from impure preparations by performing one or more chromatography steps; pooling the eluates from said one or more chromatography steps; and adjusting the pH of the pooled eluate to a pH of about 6.0 or greater; and subjecting the pH adjusted eluate to ultrafiltration and / or diafiltration. (Item 2) 2. The method of claim 1, wherein the pH is adjusted to about 6.0. (Item 3) 3. The method according to claim 1 or 2, wherein the pH is adjusted using a buffer comprising sodium phosphate, sodium chloride, and sodium citrate having a pH of 7.0. (Item 4) 4. The method according to item 3, wherein the buffer solution comprises about 0.1 to 0.5 M sodium phosphate having a pH of 7.0, 0.5 to 2.5 M sodium chloride, and 0.1 to 0.6 M sodium citrate. (Item 5) 5. The method according to any of items 1 to 4, wherein viral filtration is carried out prior to said steps of ultrafiltration and diafiltration. (Item 6) 6. The method according to any of items 1 to 5, wherein a single step of ultrafiltration and / or diafiltration is performed. (Item 7) 6. The method according to item 5, wherein said single step of ultrafiltration and / or diafiltration comprises only one diafiltration. (Item 8) 8. The method according to any one of items 1 to 7, wherein the ultrafiltration is tangential flow ultrafiltration. (Item 9) 9. The method according to any of items 1 to 8, wherein the one or more chromatography steps comprises cation exchange chromatography. (Item 10) 10. The method of claim 9, wherein the cation exchange chromatography is the final chromatography step and the eluates from the cation exchange chromatography are pooled before adjusting the pH. (Item 11) 11. The method of claim 10, wherein one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed prior to performing the cation exchange chromatography. (Item 12) 12. The method of claim 11, wherein the anion exchange chromatography is Q chromatography. (Item 13) 12. The method of claim 11, wherein the anion exchange chromatography comprises a TMAE resin. (Item 14) 14. The method of claim 13, wherein the TMAE resin, once loaded with the impure preparation, is washed with a first wash buffer comprising MES-Tris having a pH of 7.0. (Item 15) Item 15. The method according to item 14, wherein the first wash buffer comprises about 20 to 75 mM MES-Tris. (Item 16) 16. The method of any one of items 13 to 15, wherein the TMAE resin is washed with a second wash buffer comprising MES-Tris having a pH of 7.0 and NaCl. (Item 17) 17. The method according to item 16, wherein the second wash buffer comprises about 5 to 75 mM MES-Tris having a pH of 7.0 and about 50 to 150 mM NaCl. (Item 18) 18. The method according to any one of items 13 to 17, wherein the TMAE resin, once loaded with the impure preparation, is eluted using an elution buffer comprising MES-Tris having a pH of 7.0 and NaCl. (Item 19) 20. The method according to item 18, wherein the elution buffer comprises about 5 to 75 mM MES-Tris having a pH of 7.0 and about 150 to 300 mM NaCl. (Item 20) The anion exchange chromatography may be performed using Q Sepharose™ Fast Flow, Q Sepharose™ High Performance, Q Sepharose™ XL, Capto™ Q, DEAE, TOYOPEARL 12. The method of claim 11, wherein the column is selected from the group consisting of GigaCap® Q, Fractogel® TMAE, Eshmuno™ Q, Nuvia™ Q, or UNOsphere™ Q. (Item 21) 21. The method according to any one of items 11 to 20, wherein the mixed mode chromatography is hydroxyapatite (HA) chromatography. (Item 22) 22. The method according to any one of items 11 to 21, wherein the hydrophobic interaction chromatography is phenyl chromatography. (Item 23) 23. The method according to any one of items 9 to 22, wherein performing one or more chromatography steps comprises performing anion exchange chromatography using a TMAE resin, HA chromatography, phenyl chromatography, and cation exchange SP chromatography, in that order. (Item 24) 24. The method according to any one of items 9 to 23, wherein the anion exchange chromatography comprises using a column having a loading capacity of greater than about 4.5 g / L. (Item 25) 25. The method according to item 24, wherein the loading capacity of the column is about 5 to 20 g / L. (Item 26) 26. The method according to any one of items 1 to 25, wherein ultrafiltration of the impure preparation is carried out prior to the one or more chromatography steps. (Item 27) 27. The method of claim 26, wherein the ultrafiltration is tangential flow ultrafiltration. (Item 28) 28. The method according to item 26 or 27, wherein the ultrafiltration uses a membrane filter comprising a pore size having a molecular weight cut-off of at least 10 kDA. (Item 29) 28. The method according to item 26 or 27, wherein the ultrafiltration uses a membrane filter comprising a pore size having a molecular weight cut-off of at least 30 kDA. (Item 30) 28. The method according to item 26 or 27, wherein the ultrafiltration uses a membrane filter comprising a pore size having a molecular weight cut-off of at least 50 kDA. (Item 31) 28. The method of claim 26 or 27, wherein at least 75% of the recombinant ASA is retained in the impure preparation. (Item 32) 28. The method of claim 26 or 27, wherein at least 75% of the recombinant ASA permeates the filter. (Item 33) 28. The method of claim 26 or 27, wherein the ultrafiltration comprises a polyethersulfone or cellulose membrane. (Item 34) 34. The method according to any one of items 26 to 33, wherein the ultrafiltration is followed by one or more steps of depth filtration and / or viral inactivation. (Item 35) 35. The method of claim 34, wherein the one or more steps of depth filtration and / or viral inactivation are followed by the one or more chromatography steps. (Item 36) 36. The method according to item 34 or 35, wherein the step of viral inactivation comprises adding a detergent to the impure preparation. (Item 37) 37. The method according to any one of items 1 to 36, wherein the recombinant ASA protein is produced by mammalian cells cultured in suspension. (Item 38) 38. The method of claim 37, wherein the mammalian cells are cultured in a bioreactor. (Item 39) 39. The method of claim 37 or 38, wherein the medium is serum-free. (Item 40) 40. The method of claim 39, wherein the serum-free medium is a chemically defined medium. (Item 41) 41. The method according to any one of items 37 to 40, wherein the mammalian cell is a human cell. (Item 42) 42. The method according to any one of items 1 to 41, wherein the impure preparation is a feed stream from a perfusion bioreactor. (Item 43) 42. The method according to any one of items 1 to 41, wherein the impure preparation is prepared from the serum-free medium containing recombinant ASA protein secreted from the mammalian cells. (Item 44) 44. The method of claim 43, wherein the impure preparation is thawed from a frozen medium preparation. (Item 45) The method according to any one of claims 1 to 44, wherein the step of ultrafiltration and / or diafiltration comprises exchanging the purified recombinant ASA protein into a formulation buffer. 46. ​​The method according to any one of items 1 to 45, wherein the recombinant ASA protein has an amino acid sequence that is at least 70% identical to SEQ ID NO: 1 (corresponding to the wild-type human ASA protein). (Item 47) 47. The method according to any one of items 1 to 46, wherein the recombinant ASA protein has an amino acid sequence identical to SEQ ID NO:1. (Item 48) 48. The method according to any one of items 1 to 47, wherein the purified recombinant ASA protein contains less than 100 ng / mg HCP. (Item 49) 49. The method according to any one of items 1 to 48, wherein the purified recombinant ASA protein contains less than 60 ng / mg HCP. (Item 50) 50. The method of any one of items 1 to 49, wherein the purified recombinant ASA protein has no new bands having an intensity greater than 1.0% of the assay control when subjected to SDS-PAGE using Coomassie blue staining. (Item 51) 51. The method according to any one of items 1 to 50, wherein the purified recombinant ASA protein contains less than 100 pg / mg of host cell DNA. (Item 52) 52. The method according to any one of items 1 to 51, wherein the purified recombinant ASA protein contains less than 50 pg / mg of host cell DNA. (Item 53) 1. A method for purifying a recombinant arylsulfatase A (ASA) protein, comprising: purifying recombinant arylsulfatase A (ASA) protein from an impure preparation by performing one or more chromatography steps, the first chromatography step using a column having a loading capacity of about 4.5 g protein / L resin or more; pooling the eluates from said one or more chromatography steps; and adjusting the pH of the pooled eluate to a pH of about 6.0 or greater; and subjecting the pH adjusted eluate to ultrafiltration and / or diafiltration. (Item 54) 54. The method of claim 53, wherein said step of performing one or more chromatography steps comprises performing, in order, anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. (Item 55) 55. The method of claim 54, wherein the anion exchange chromatography uses a column containing a TMAE resin. (Item 56) Item 56. The method of item 55, wherein the TMAE column has a loading capacity of about 5-20 g protein / L resin. (Item 57) 57. The method according to any one of items 53 to 56, wherein a single step of ultrafiltration and / or diafiltration is performed after the one or more chromatography steps. (Item 58) A recombinant arylsulfatase A (ASA) protein purified by the method according to any one of items 1 to 57. (Item 59) A pharmaceutical composition comprising the recombinant ASA protein according to item 58. (Item 60) 60. A method for treating metachromatic leukodystrophy, comprising administering to a subject in need of treatment the pharmaceutical composition of item 59. (Item 61) A composition comprising a purified recombinant arylsulfatase A (ASA) having an amino acid sequence at least 70% identical to SEQ ID NO:1, the purified recombinant ASA has a specific activity of at least about 50 U / mg; Further, the purified recombinant ASA contains less than 150 ng / mg host cell protein (HCP) and / or less than 150 pg / mg host cell DNA (HCD). (Item 62) 62. The composition of claim 61, wherein the purified recombinant ASA contains less than 100 ng / mg HCP. (Item 63) 63. The composition of claim 61 or 62, wherein the purified recombinant ASA contains less than 60 ng / mg HCP. (Item 64) 64. The composition according to any one of items 61 to 63, wherein the purified recombinant ASA contains less than 100 pg / mg HCD. (Item 65) 65. The composition of any one of items 61 to 64, wherein the purified recombinant ASA contains less than 50 pg / mg HCD. (Item 66) 66. The composition of any one of items 61 to 65, wherein the purified recombinant ASA has a specific activity of at least about 70 U / mg. (Item 67) 67. The composition of any one of items 61 to 66, wherein the purified recombinant ASA has a specific activity of at least about 100 U / mg. (Item 68) 68. The composition of any one of items 61 to 67, wherein the purified recombinant ASA has a specific activity of at least about 120 U / mg. (Item 69) 69. The composition according to any one of items 61 to 68, wherein the purified recombinant ASA has a specific activity in the range of about 50 to 200 U / mg. (Item 70) 70. The composition according to any one of items 61 to 69, wherein the purified recombinant ASA has a specific activity in the range of about 50 to 140 U / mg. (Item 71) A composition comprising a purified recombinant arylsulfatase A (ASA) having an amino acid sequence at least 70% identical to SEQ ID NO:1, The purified ASA is characterized by a glycan map comprising seven or fewer peak groups selected from peak groups representing neutral (Peak Group 1), monosialylated (Peak Group 2), capped mannose-6-phosphorylated (Peak Group 3), disialylated (Peak Group 4), mono-mannose-6-phosphorylated (Peak Group 5), hybrid (Peak Group 6), and di-mannose-6-phosphorylated (Peak Group 7). (Item 72) 72. The composition of claim 71, wherein the purified recombinant ASA has an amino acid sequence that is at least 80% identical to SEQ ID NO:1. (Item 73) 73. The composition of claim 71 or 72, wherein the purified recombinant ASA has an amino acid sequence that is at least 90% identical to SEQ ID NO:1. (Item 74) 74. The composition of any one of items 71 to 73, wherein the purified recombinant ASA has an amino acid sequence that is at least 95% identical to SEQ ID NO:1. (Item 75) 75. The composition according to any one of items 71 to 74, wherein the purified recombinant ASA has an amino acid sequence identical to SEQ ID NO:1. (Item 76) 76. A formulation comprising the composition according to any one of items 71 to 75 and a physiologically acceptable carrier. (Item 77) 77. The formulation of item 76, wherein the formulation is suitable for intravenous administration. (Item 78) 77. The formulation according to item 76, wherein the formulation is suitable for intrathecal administration. (Item 79) 77. The formulation of item 76, wherein the formulation is suitable for subcutaneous administration. [Brief description of the drawings]

[0048] The figures described below taken together with the drawings are intended for purposes of illustration only and not limitation.

[0049] [Figure 1] 1 shows a generalized flow diagram of an exemplary purification process. [Diagram 2] FIG. 1 shows a generalized flow diagram of an exemplary purification process that includes pooling the eluates from a cation exchange column and adjusting the pH of the pooled eluates. [Diagram 3] Exemplary results are presented showing the correlation between feed flow rate, transmembrane pressure (TMP), and permeate flux for each membrane. [Figure 4]An exemplary SDS-PAGE (silver) gel is shown showing similar band profiles for both Fractogel and Q FF throughout the entire process. [Diagram 5] Exemplary results are shown in which feasibility runs and control runs including a single UFDF or UFDFDF step showed similar banding patterns as the reference standard (lane 3), indicating that drug substances from all runs showed relatively low levels of HCPs. [Figure 6] Exemplary results are shown showing comparability between experimental (feasibility) and control runs against a reference standard, as well as no additional banding patterns were detected by SDS-PAGE (Coomassie). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms, as well as other terms, are set forth throughout the specification.

[0051] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more subject values, refers to a value similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a value within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise specified or otherwise clear from the context (except where such number exceeds 100% of the possible value).

[0052] Biologically active: As used herein, the phrase "biologically active" refers to the characteristic of any agent that has activity in a biological system, particularly in an organism. For example, an agent that, when administered to an organism, has a biological effect on the organism is considered to be biologically active. In certain embodiments in which a protein or polypeptide is biologically active, a portion of the protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a "biologically active" portion.

[0053] Cation-independent mannose-6-phosphate receptor (CI-MPR): As used herein, the term "cation-independent mannose-6-phosphate receptor (CI-MPR)" refers to a cellular receptor that binds the mannose-6-phosphate (M6P) tag on acid hydrolase precursors in the Golgi apparatus that are destined for transport to lysosomes. In addition to mannose-6-phosphate, CI-MPR also binds other proteins, including IGF-II. CI-MPR is also known as "M6P / IGF-II receptor", "CI-MPR / IGF-II receptor", "IGF-II receptor", or "IGF2 receptor". These terms and their abbreviations are used interchangeably herein.

[0054] Chromatography: As used herein, the term "chromatography" refers to a technique in the separation of mixtures. Typically, the mixture is dissolved in a liquid called the "mobile phase" and transported through a structure that holds another material, called the "stationary phase." Column chromatography is a separation technique in which the fixed bed is within a tube, or column.

[0055] Diluent: As used herein, the term "diluent" refers to a pharma- ceutically acceptable (e.g., safe and non-toxic for human administration) diluent substance useful for the preparation of a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0056] Elution: As used herein, the term "elution" refers to the process of extracting one material from another by washing with a solvent. For example, in ion exchange chromatography, elution is the process of washing a loaded resin to remove trapped ions.

[0057] Elution: As used herein, the term "elution" refers to the combination of the mobile phase "carrier" and analytical material that emerges from chromatography, typically as a result of elution.

[0058] Enzyme replacement therapy (ERT): As used herein, the term "enzyme replacement therapy (ERT)" refers to any therapeutic strategy that corrects an enzyme deficiency by providing the missing enzyme. Once administered, the enzyme is taken up by cells and transported to lysosomes, where it acts to remove material that has accumulated in the lysosome due to enzyme deficiency. Typically, for lysosomal enzyme replacement therapy to be effective, the therapeutic enzyme is delivered to lysosomes in appropriate cells in the target tissue where the storage defect is evident. The purification process described herein can be used to purify and formulate recombinant arylsulfatase A as a drug substance for ERT of MLD.

[0059] Equilibrate or equilibration: As used herein, the term "equilibrate" or "equilibrium" in reference to chromatography generally refers to the process of effecting balance of a first liquid (e.g., a buffer) with another to achieve a stable and equitable distribution of the liquid's (e.g., a buffer) components. For example, in some embodiments, a chromatography column may be equilibrated by passing one or more column volumes of a desired liquid (e.g., a buffer) through the column.

[0060] Improve, increase, or reduce: As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a baseline measurement, e.g., a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control individual (or control individuals) in the absence of a treatment described herein. A "control individual" is an individual suffering from the same type of lysosomal storage disease as the individual to be treated, and who is approximately the same age as the individual to be treated (to ensure that the stage of disease in the treated and control individual(s) is comparable).

[0061] Impurity: As used herein, the term "impurity" refers to a substance in a confined volume of a liquid, gas, or solid that is different from the chemical composition of a target material or compound. Impurities are also referred to as contaminants.

[0062] Loading: As used herein, the term "loading" refers to the addition of a liquid or solid containing a sample to a column in chromatography. In some embodiments, certain components of the sample loaded onto the column are captured as the loaded sample passes through the column. In some embodiments, certain components of the sample loaded onto the column are not captured by the column or "flow through" as the loaded sample passes through the column.

[0063] Polypeptide: As used herein, a "polypeptide" is generally a chain of at least two amino acids linked together by peptide bonds. In some embodiments, a polypeptide may contain at least 3-5 amino acids, each of which is linked to the others by at least one peptide bond. It will be understood by those of skill in the art that polypeptides sometimes include "unnatural" amino acids or other entities that may still optionally be incorporated into a polypeptide chain.

[0064] Pooling: As used herein, the term "pooling" in reference to chromatography refers to combining the liquid of one or more fractions that have been passed through a column together. For example, in some embodiments, one or more fractions (e.g., "peak fractions") that contain a desired component of a sample that has been separated by chromatography may be "pooled" together to form a single "pooled" fraction.

[0065] Replacement enzyme: As used herein, the term "replacement enzyme" refers to any enzyme that can act to at least partially replace an enzyme that is deficient or missing in a disease to be treated. In some embodiments, the term "replacement enzyme" refers to any enzyme that can act to at least partially replace an enzyme that is deficient or missing in a lysosomal storage disease to be treated. In some embodiments, the replacement enzyme (e.g., rASA) can reduce stored material in mammalian lysosomes or rescue or ameliorate one or more symptoms of lysosomal storage disease (e.g., MLD). Replacement enzymes suitable for the present invention include both wild-type or modified lysosomal enzymes and can be produced using recombinant and synthetic methods or purified from natural sources. Replacement enzymes can be recombinant, synthetic, gene-activated, or natural enzymes.

[0066] Solubility: As used herein, the term "soluble" refers to the ability of a therapeutic agent to form a homogeneous solution. In some embodiments, the solubility of a therapeutic agent in the solution in which it is administered and in which it is transported to the target site of action is sufficient to allow delivery of a therapeutically effective amount of the therapeutic agent to the target site of action. Several factors can affect the solubility of a therapeutic agent. For example, relevant factors that can affect protein solubility include ionic strength, amino acid sequence, and the presence of other co-solubilizing agents or salts (e.g., calcium salts). In some embodiments, a therapeutic agent according to the present invention is soluble in its corresponding pharmaceutical composition.

[0067] Stability: As used herein, the term "stability" refers to the ability of a therapeutic agent (e.g., a recombinant enzyme) to maintain its therapeutic efficacy (e.g., all or most of its intended biological activity and / or physicochemical integrity) over an extended period of time. The stability of a therapeutic agent, as well as the ability of a pharmaceutical composition to maintain the stability of such a therapeutic agent, can be evaluated over an extended period of time (e.g., at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). In the context of a formulation, a stable formulation is one in which the therapeutic agent therein essentially retains its physical and / or chemical integrity and biological activity during storage and processing (e.g., freezing / thawing, mechanical mixing and lyophilization). With respect to protein stability, it can be measured by the formation of high molecular weight (HMW) aggregates, loss of enzymatic activity, generation of peptide fragments, and shifts in charge profile.

[0068] Virus Treatment: As used herein, the term "virus treatment" refers to "virus removal" (e.g., virus filtration), where viruses are simply removed from a sample, or "virus inactivation," where viruses remain in the sample but in a non-infectious form. In some embodiments, virus removal may use nanofiltration and / or chromatography techniques, among others. In some embodiments, virus inactivation may use solvent inactivation, detergent inactivation, pasteurization, acidic pH inactivation, and / or ultraviolet inactivation, etc.

[0069] Detailed Description The present invention provides, inter alia, improved methods for purifying recombinantly produced ASA protein for enzyme replacement therapy. In some embodiments, the present invention provides methods for purifying recombinant ASA protein from impure preparations (e.g., unprocessed biomaterials, e.g., cell culture media containing ASA) using a process that involves only a single step of post-chromatographic ultrafiltration / diafiltration. In some embodiments, this single-step UF / DF process is accomplished by pooling the eluates from the chromatography step and adjusting the pH of the pooled eluate to about 6.0 or higher. In some embodiments, this simplified process is combined with a high-load capacity chromatography step to facilitate large-scale production of recombinant ASA protein.

[0070] Various aspects of the invention are described in further detail in the following subsections. The use of the subsections is not meant to limit the invention. Each subsection may apply to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise specified.

[0071] Arylsulfatase A Arylsulfatase A (ASA, ARSA, or cerebroside-sulfatase) is an enzyme that degrades cerebroside 3-sulfate (or sulfatide) to cerebrosides and sulfate. Specifically, galactosylsulfatide is normally metabolized by hydrolysis of the 3-O-sulfate bond to form galactocerebroside through the combined action of the lysosomal enzyme arylsulfatase A (EC 3.1.6.8) (Austin et al. Biochem J. 1964, 93, 15C-17C) and a sphingolipid activator protein called saposin B. Arylsulfatase A deficiency occurs in all tissues of patients with late infantile, juvenile, and adult forms of metachromatic leukodystrophy (MLD). As used herein, arylsulfatase A protein is referred to as "ASA" or "ARSA" and saposin B is referred to as "Sap-B."

[0072] Arylsulfatase A is an acidic glycoprotein with a low isoelectric point. Above pH 6.5, the enzyme exists as a monomer with a molecular weight of approximately 100 kDa. Under acidic conditions (pH ≤ about 5.0), ASA exists as an octamer of 480 kDa, but dissociates into dimers at neutral pH levels. In human urine, the enzyme consists of two unequal subunits of 63 and 54 kDa (Laidler PM et al. Biochim Biophys Acta. 1985, 827, 73-83). Arylsulfatase A purified from human liver, placenta, and fibroblasts also consists of two subunits of slightly different sizes that vary between 55 and 64 kDa (Draper RK et al.Arch Biochemica Biophys.1976,177,525-538; Waheed A et al.Hoppe Seylers Z Physiol Chem.1982,363,425-430; Fujii T et al.Biochim Biophys Acta.1992,15 1122,93-98). As with other lysosomal enzymes, arylsulfatase A is synthesized on membrane-bound ribosomes as a glycosylated precursor. It then passes through the endoplasmic reticulum and Golgi, where its N-linked oligosaccharides are processed with the formation of complex phosphorylated and sulfated oligosaccharides (Waheed A et al.Biochim Biophys Acta. 1985, 847, 53-61, Braulke T et al. Biochem Biophys Res Commun. 1987, 143, 178-185). In normal cultured fibroblasts, a 62 kDa precursor polypeptide is produced and transported to acidic pre-lysosomal endosomes via mannose-6-phosphate receptor binding (Braulke T et al. J Biol Chem. 1990, 265, 6650-6655) (Kelly BM et al. Eur J Cell Biol. 1989, 48, 71-78).

[0073] The methods described herein can be used to purify arylsulfatase A from any source, for example, from tissues or cultured cells, such as human cells (e.g., fibroblasts) that recombinantly produce arylsulfatase A. Arylsulfatase A from any source can be produced by the methods described herein, including, but not limited to, humans and other animals, e.g., mammals.

[0074] The length of the signal peptide of human arylsulfatase A (18 amino acids) is based on the consensus sequence and the specific processing site for the signal sequence. Thus, from the putative human ASA cDNA (EMBL GenBank accession numbers J04593 and X521151), the signal peptide is cleaved in all cells after residue number 18 (Ala), resulting in the mature form of human arylsulfatase A. As used herein, recombinant arylsulfatase A is abbreviated as "rASA". The mature form of arylsulfatase A, including the mature form of human arylsulfatase A, is referred to as "mASA", and mature recombinant human ASA is referred to as "mrhASA".

[0075] Multiple forms of arylsulfatase A have been identified in human urine (Luijten JAFM et al. al.J Mol Med.1978,3,213), leukocytes (Dubois et al.Biomedicine.1975,23,116-119, Manowitz P et al.Biochem Med Metab Biol.1988,39,117-120), platelets (Poretz et al.Biochem J.1992,287,979-983), cultured fibroblasts (Waheed A et al.Hoppe Seylers Z Physiol Chem.1982,363,425-430, Stevens RL et al.Biochim Biophys Acta.1976,445,661-671, Farrell DF et al. al.Neurology.1979,29,16-20), and liver (Stevens RL et al.Biochim Biophys This has been demonstrated by electrophoresis and isoelectric focusing of enzyme preparations from arylsulfatase A (Farrell DF et al. Neurology. 1979, 29, 16-20; Sarafian TA et al. Biochem Med. 1985, 33, 372-380). Treatment with endoglycosidase H, sialidase, and alkaline phosphatase reduced the molecular size and complexity of the electrophoretic pattern, suggesting that much of the charge heterogeneity of arylsulfatase A is due to variations in the carbohydrate content of the enzyme.

[0076] The active site of arylsulfatase A contains an essential histidine residue (Lee GD and Van Etten RL, Arch Biochem Biophys. 1975, 171, 424-434) and contain two or more arginine residues (James GT, Arch Biochem Biophys. 1979, 97, 57-62). Many anions are inhibitors of enzymes at concentrations in the submillimolar range.

[0077] The human arylsulfatase A gene structure has been described. As used herein, this gene is referred to as "ARSA". However, "ARSA" may also refer to the arylsulfatase A protein in some cases. The ARSA gene is located near the end of the long arm of chromosome 22 (22ql3.31-qter), spans 3.2 kb (Kreysing et al. Eur J Biochem. 1990, 191, 627-631), and consists of eight exons that specify a 507 amino acid enzyme unit (Stein et al. J Biol Chem. 1989, 264, 1252-1259). Messenger RNAs of 2.1, 3.7, and 4.8 kb were detected in fibroblasts, with the 2.1 kb message apparently responsible for the majority of the active arylsulfatase A produced by the cells (Kreysing et al. Eur J Biochem. 1990, 191, 627-631). The ARSA sequence has been deposited in EMBL GenBank with the accession number X521150. Differences between the published cDNA and the coding portion of ARSA were described by Kreysing et al. (Eur J Biochem. 1990, 191, 627-631). The cDNA sequence originally described by Stein et al. (J Biol Chem. 1989, 264, 1252-1259) and by Kreysing et al. (Eur J Biochem. 1990, 191, 627-631) have been deposited in EMBL GenBank with the accession numbers J04593 and X521151, respectively.

[0078] Several polymorphisms and over 40 disease-associated mutations have been identified in the ARSA gene (Gieselmann et al. Hum Mutat. 1994, 4, 233-242; Barth et al. Hum Mutat. 1995, 6, 170-176; Draghia et al. Hum Mutat. 1997, 9, 234-242). Disease-associated mutations in the ARSA gene can be classified into two broad groups that correlate fairly well with the clinical phenotype of MLD. One group (I) shows no active enzyme, no immunoreactive protein, and no ASA activity when introduced into cultured animal cell lines. The other group (A) produces small amounts of cross-reactive material and low levels of functional enzyme in cultured cells. Individuals who are homozygous for group (I) mutations or who have two different mutations from this group are described as having late-infantile MLD. Most individuals with one group (I) and one group (A) mutation develop early-onset MLD, while those with two group (A) mutations generally develop adult MLD. Some of the mutations are found relatively frequently, while others are only detected in a single family. Although it is possible to trace a particular mutation through many family members, general carrier screening is still not feasible.

[0079] In addition to the above-mentioned disease-related mutations, several polymorphisms have been identified in the ARSA gene. Very low ASA activity has been found in some clinically normal patients with MLD and in the general population. This so-called pseudodeficiency ASA is associated with a common polymorphism in the ARSA gene (Gieselmann et al. Dev Neurosci. 1991, 13, 222-227).

[0080] Recombinant ASA protein As used herein, the term "recombinant ASA protein" refers to any molecule or portion of a molecule that can replace at least partial activity of naturally occurring arylsulfatase A (ASA) protein or rescue one or more phenotypes or symptoms associated with ASA deficiency. As used herein, the terms "recombinant ASA enzyme" and "recombinant ASA protein" and grammatical equivalents are used interchangeably. In some embodiments, the present invention is used to purify recombinant ASA protein, which is a polypeptide having an amino acid sequence substantially similar or identical to the mature human ASA protein.

[0081] Typically, human ASA is produced as a precursor molecule that is processed to a mature form. This process generally occurs by removing an 18 amino acid signal peptide. Typically, the precursor form is also referred to as a full-length precursor or full-length ASA protein, which contains 507 amino acids. The N-terminal 18 amino acids are truncated, resulting in a mature form that is 489 amino acids long. Therefore, it is generally contemplated that the N-terminal 18 amino acids are not required for ASA protein activity. The amino acid sequences of the mature form (SEQ ID NO: 1) and full-length precursor (SEQ ID NO: 2) of a typical wild-type or naturally occurring human ASA protein are shown in Table 1. [Table 1]

[0082] Thus, in some embodiments, the recombinant ASA protein purified according to embodiments of the invention is a mature human ASA protein (SEQ ID NO: 1). In some embodiments, the recombinant ASA protein purified according to embodiments of the invention may be a homolog or analog of the mature human ASA protein. For example, a homolog or analog of the mature human ASA protein may be a modified mature human ASA protein that includes one or more amino acid substitutions, deletions, and / or insertions compared to a wild-type or naturally occurring ASA protein (e.g., SEQ ID NO: 1), but retains substantial ASA protein activity. Thus, in some embodiments, the recombinant ASA protein purified according to embodiments of the invention is substantially homologous to the mature human ASA protein (SEQ ID NO: 1). In some embodiments, the recombinant ASA protein purified according to embodiments of the invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 1. In some embodiments, recombinant ASA proteins purified according to embodiments of the invention are substantially identical to the mature human ASA protein (SEQ ID NO: 1). In some embodiments, recombinant ASA proteins purified according to embodiments of the invention have an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1. In some embodiments, recombinant ASA proteins purified according to embodiments of the invention include fragments and portions of mature human ASA protein.

[0083] Alternatively, the recombinant ASA protein purified according to embodiments of the present invention is a full-length ASA protein. In some embodiments, the recombinant ASA protein may be a homolog or analog of a full-length human ASA protein. For example, a homolog or analog of a full-length human ASA protein may be a modified full-length human ASA protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to a wild-type or naturally occurring ASA protein (e.g., SEQ ID NO: 2), but retains substantial ASA protein activity. Thus, in some embodiments, the recombinant ASA protein purified according to embodiments of the present invention is substantially homologous to a full-length human ASA protein (SEQ ID NO: 2). In some embodiments, the recombinant ASA protein purified according to embodiments of the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 2. In some embodiments, the recombinant ASA protein purified according to embodiments of the invention is substantially identical to SEQ ID NO: 2. In some embodiments, the recombinant ASA protein purified according to embodiments of the invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2. In some embodiments, the recombinant ASA protein purified according to embodiments of the invention includes fragments and portions of full-length human ASA protein. As used herein, a full-length ASA protein typically includes a signal peptide sequence.

[0084] Homologs or analogs of human ASA protein can be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those found in references that summarize such methods. In some embodiments, conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N, and (g) E, D. In some embodiments, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.

[0085] In some embodiments, the recombinant ASA protein may contain a portion that binds to a receptor on the surface of a target cell to facilitate cellular uptake and / or lysosomal targeting. For example, such a receptor may be the cation-independent mannose-6-phosphate receptor (CI-MPR) that binds to mannose-6-phosphate (M6P) residues. In addition, the CI-MPR also binds to other proteins, including IGF-II. In some embodiments, the recombinant ASA protein contains M6P residues on the surface of the protein. In particular, the recombinant ASA protein may contain bis-phosphorylated oligosaccharides that have a higher binding affinity to the CI-MPR. In some embodiments, a suitable enzyme contains up to about an average of about at least 20% bis-phosphorylated oligosaccharides per enzyme. In other embodiments, a suitable enzyme may contain about 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% bis-phosphorylated oligosaccharides per enzyme.

[0086] In some embodiments, recombinant ASA enzyme can be fused with lysosomal targeting that can bind to receptors on the surface of target cells.Suitable lysosomal targeting moieties can be IGF-I, IGF-II, RAP, p97, and variants, homologs, or fragments thereof (e.g., including those peptides that have at least 70%, 75%, 80%, 85%, 90%, or 95% identical sequence to wild-type mature human IGF-I, IGF-II, RAP, p97 peptide sequence).Lysosomal targeting moieties can be conjugated or fused to ASA protein or enzyme at the N-terminus, C-terminus, or internally.

[0087] Generation of recombinant ASA protein The present invention can be used to purify recombinant ASA protein produced by various means. For example, ASA protein can be produced recombinantly by expressing nucleic acid encoding ASA using an engineered host cell system. Alternatively, ASA protein can be produced by activating endogenous ASA gene.

[0088] It is contemplated that the present invention may be used to purify recombinant ASA proteins produced using a variety of expression systems. Suitable expression systems include, for example, egg cells, baculovirus, plant, yeast, or mammalian cells.

[0089] In some embodiments, the ASA enzyme is produced in mammalian cells. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include BALB / c mouse myeloma line (NSO / 1, ECACC number 85110503), human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands), monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney lines (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977), human fibrosarcoma cell lines (e.g., HT1080), baby hamster kidney cells (BHK21, ATCC CCL 10), Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980), mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1) 587), human cervical cancer cells (HeLa, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary cancer (MMT 060562, ATCC) CCL51), TRI cells (Mather et al.,Annals NYAcad. Sci., 383:44-68, 1982), MRC 5 cells, FS4 cells, and a human hepatocellular carcinoma line (Hep G2).

[0090] In some embodiments, the inventive methods according to the invention are used to purify recombinant ASA enzyme produced from human cells (e.g., HT1080).In some embodiments, the inventive methods according to the invention are used to purify recombinant ASA enzyme produced from CHO cells.

[0091] Typically, the cell that is engineered to express recombinant ASA can contain a transgene that encodes the recombinant ASA protein described herein.It should be understood that the nucleic acid that encodes recombinant ASA can contain regulatory sequences, gene control sequences, promoters, non-coding sequences, and / or other suitable sequences for expressing recombinant ASA.Typically, the coding region is operably linked to one or more of these nucleic acid components.

[0092] "Regulatory sequences" typically refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences. Sometimes "regulatory sequences" are also referred to as "gene control sequences."

[0093] A "promoter" typically refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. A promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Thus, an "enhancer" is a nucleotide sequence capable of stimulating promoter activity, and may be an intrinsic element of the promoter or a heterologous element inserted to increase the level or tissue specificity of the promoter. Promoters may be derived in their entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or may contain synthetic nucleotide portions. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.

[0094] "3' non-coding sequences" typically refer to nucleotide sequences located downstream of a coding sequence, and include polyadenylation recognition sequences and other sequences that encode regulatory signals capable of affecting mRNA processing or gene expression. Polyadenylation signals are usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of a pre-mRNA.

[0095] "Translation leader sequence" or "5' non-coding sequence" refers to a nucleotide sequence that is typically located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect the processing of the primary transcript into mRNA, the stability of the mRNA, or the efficiency of translation.

[0096] Typically, the term "operably linked" or "operably linked" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.

[0097] The coding region of the transgene may contain one or more silent mutations to optimize codon usage for a particular cell type.For example, the codons of the ASA transgene may be optimized for expression in vertebrate cells.In some embodiments, the codons of the ASA transgene may be optimized for expression in mammalian cells.In some embodiments, the codons of the ASA transgene may be optimized for expression in human cells.

[0098] Optionally, the construct may contain additional components such as one or more of splice sites, enhancer sequences, selectable marker genes under the control of a suitable promoter, amplifiable markers under the control of a suitable promoter, and matrix attachment regions (MARs) or other elements known in the art that enhance expression of the region into which it is inserted.

[0099] Once transfected or transduced into a host cell, suitable vectors can be expressed extrachromosomally (episomal) or can be integrated into the host cell genome.

[0100] Cell culture media and conditions A variety of cell culture media and conditions can be used to produce recombinant ASA protein. For example, recombinant ASA protein can be produced in serum-containing or serum-free media. In some embodiments, recombinant ASA protein is produced in serum-free media. In some embodiments, recombinant ASA protein is produced in animal-free media, i.e., media lacking animal-derived components. In some embodiments, recombinant ASA protein is produced in chemically defined media. As used herein, the term "chemically defined nutrient medium" refers to a medium in which substantially all of the chemical components are known. In some embodiments, chemically defined nutrient media are free of animal-derived components, such as serum, serum-derived proteins (e.g., albumin or fetuin), and other components. In some cases, chemically defined media contain one or more proteins (e.g., protein growth factors or cytokines). In some cases, chemically defined nutrient media contain one or more protein hydrolysates. In other cases, chemically defined nutrient media are protein-free media, i.e., serum-free media that do not contain proteins, hydrolysates, or components of unknown composition.

[0101] In some embodiments, the chemically defined medium may be supplemented with one or more animal-derived components. Such animal-derived components include, but are not limited to, fetal bovine serum, horse serum, goat serum, donkey serum, human serum, and serum-derived proteins such as albumin (e.g., bovine serum albumin or human serum albumin). The addition of serum is desirable because it contains components such as vitamins, amino acids, growth factors, and hormones, but it also constitutes a concentrated source of exogenous proteins that may interfere with the purification of recombinant proteins. For example, fetuin protein is a family of related serum proteins secreted by cells, primarily hepatocytes, and has three characteristic domains (Brown WM, et al., Eur J Biochem. 1992 Apr 1; ​​205(1): 321-31). Fetuin is abundant during fetal life and therefore is often abundant in commercially available fetal serum, constituting a major protein contaminant during purification. Therefore, in some embodiments, a suitable medium is a xeno-free medium, e.g., a medium that does not contain any bovine serum or bovine serum-derived components. For example, a xeno-free medium may contain one or more of human serum albumin, human transferrin, human insulin, and human lipids. In some embodiments, a suitable medium contains fetuin-depleted serum. Fetuin can be depleted from serum using various methods known in the art. For example, fetuin can be depleted from serum by antibody affinity chromatography. (See, e.g., Toroian D and Price PA, Calcif Tissue Int (2008) 82:116-126). In some embodiments, a suitable medium is fetuin-free.

[0102] A variety of cell culture conditions can be used to produce recombinant ASA protein on a large scale, including, but not limited to, roller bottle culture, bioreactor culture, and bioreactor batch culture, and bioreactor fed-batch culture. In some embodiments, recombinant ASA protein is produced by cells cultured in suspension. In some embodiments, recombinant ASA protein is produced by adherent cells.

[0103] Purification of recombinant arylsulfatase A Embodiments of the invention include purification processes for the production of arylsulfatase A ("ASA"), specifically recombinant human ASA ("rhASA"), drug substance. A variety of techniques may be used to produce purified ASA drug substance, in whole or in part, optionally with the modifications described herein. For example, FIG. 1 shows a generalized flow diagram of an embodiment of the invention. This exemplary purification process begins with thawing and pooling of rhASA unpurified bulk (UPB), which is then captured and filtered by ultrafiltration and diafiltration ("UFDF"). After filtration, the captured material may be virally inactivated (not shown) prior to chromatographic purification. In a particular illustrated embodiment, the following four purification process steps use a series of four chromatographic columns: an anion exchange column (e.g., a Q Sepharose Fast Flow ("Q-FF") column), a ceramic hydroxyapatite type I (HA) column, a phenyl column, and a cation exchange (e.g., SP) column. After the fourth and final chromatographic step, the SP eluate is concentrated and diafiltered using tangential flow ultrafiltration. The resulting filtrate is then filtered (e.g., through a Planova® 20N virus reduction filter), followed by a second concentration and ultrafiltration step to achieve the target protein concentration. Note that FIG. 2 shows another exemplary embodiment of the invention. The process optionally begins as in FIG. 1, using one or more different chromatography resins (e.g., TMAE anion exchange resin). After the fourth and final chromatography step, the SP eluate is pooled and the pH of the pool is adjusted. In some embodiments, the pH is adjusted to 5.5-6.5 (e.g., about 6.0). The resulting pH-adjusted cation exchange is then virus filtered, followed by a single concentration and diafiltration step to achieve the final target protein concentration. Optional improvements and further embodiments of these processes are described herein.

[0104] As used herein, a "contaminant" is a substance that is different from the desired polypeptide product, e.g., arylsulfatase A (ASA). A contaminant can be a variant of the desired polypeptide (e.g., a deamidated or amino-aspartate variant of the desired polypeptide) or another molecule, e.g., a polypeptide, a nucleic acid, an endotoxin, and the like.

[0105] As used herein, "purifying" a polypeptide from a composition or sample that contains the polypeptide and one or more contaminants means increasing the purity of the polypeptide in the composition or sample by removing (completely or partially) at least one contaminant from the composition or sample. A "purification step" can be part of an overall purification process that results in a composition that contains at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight of the polypeptide of interest, based on the total weight of the composition. The purity of arylsulfatase A can be measured, for example, by one or more of host cell protein (HCP) Western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse phase HPLC, and size exclusion HPLC. In some embodiments, the specific activity of the purified arylsulfatase A is at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg, e.g., as measured by a method described herein.In some embodiments, the purified recombinant ASA has a potency of about 50-200 U / mg (e.g., about 50-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-140 U / mg, 60-130 U / mg, 60-120 U / mg, 60-110 U / mg, 60-100 U / mg, 70-140 U / mg, 70-130 U / mg, 70-120 U / mg, 70-140 U / mg, 70-150 U / mg, 70-160 U / mg, 70-170 U / mg, 70-180 U / mg, 70-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-140 U / mg, 60-130 U / mg, 60-120 U / mg, 60-110 U / mg, 60-100 U / mg, 70-140 U / mg, 70-130 U / mg, 70-120 U / mg, 70-140 U / mg, 70-150 U / mg, 70-16 U / mg, 70-110U / mg, 70-100U / mg, 80-140U / mg, 80-130U / mg, 80-120U / mg, 80-110U / mg, 80-100U / mg, 90-140U / mg, 90-130U / mg, 90-120U / mg, 90-110U / mg, 90-100U / mg, 100-140U / mg, 100-130U / mg, 100-120U / mg, 100-110U / mg, 110-140U / mg, 110-130U / mg, 110-120U / mg, 120-140U / mg, 120-130U / mg, or 130-140U / mg).

[0106] The starting material for the purification process is any impure preparation. For example, the impure preparation can be unprocessed cell culture medium containing recombinant ASA protein secreted from cells (e.g., mammalian cells) that produce ASA protein or raw cell lysate containing ASA protein. In some embodiments, the impure preparation can be a partially processed cell culture medium or cell lysate. For example, the cell culture medium or cell lysate can be concentrated, diluted, processed by virus inactivation, virus treatment, or virus removal. In some embodiments, virus removal can use nanofiltration and / or chromatography techniques, among others. In some embodiments, virus inactivation can use solvent inactivation, detergent inactivation, pasteurization, acidic pH inactivation, and / or ultraviolet inactivation. The cell culture medium or cell lysate can also be treated with proteases, DNases, and / or RNases to reduce the levels of host cell proteins and / or nucleic acids (e.g., DNA or RNA). In some embodiments, unprocessed or partially processed biomaterials (e.g., cell culture media or cell lysates) can be frozen for a period of time and stored at a desired temperature (e.g., 2-8°C, -4°C, -25°C, -75°C) and thawed for purification. As used herein, impure preparations are also referred to as starting materials or loading materials.

[0107] The purification methods described herein may include, but are not limited to, one or more of the following steps: depth filtration, viral inactivation, ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography), mixed mode chromatography, hydrophobic interaction chromatography, ultrafiltration / diafiltration, and viral removal filtration. In some embodiments, the purification methods described herein further include affinity chromatography.

[0108] In the chromatography step, the appropriate amount of resin used when packed into the chromatography column is reflected by the dimensions of the column, i.e., the diameter of the column and the height of the resin, and varies depending on, for example, the amount of protein in the applied solution and the binding capacity of the resin used. However, it is within the scope of the present disclosure to increase the scale of the production and purification process to obtain the production and purification of ASA on an industrial scale. Thus, parameters such as the size, diameter, and flow rate of the column may be increased to match the speed and efficiency of such large-scale production. In some embodiments, the diameter of the column is in the range of about 50-100 mm, the volume is in the range of about 100-300 ml, and the flow rate is in the range of about 40-400 cm / hr (e.g., about 100 cm / hr-150 cm / hr) or about 5-100 ml.

[0109] Ultrafiltration - Capture In some embodiments of the invention, the purification methods disclosed herein include one or more steps of upstream ultrafiltration to capture ASA (e.g., human recombinant ASA) produced from the perfusion bioreactor. Ultrafiltration, as used herein, refers to membrane filtration using filter pore sizes as small as 0.001-0.1 μm that can be used to concentrate and desalt dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), exchange buffers, and for gross fractionation. Ultrafiltration methods used in embodiments of the invention include tangential flow ultrafiltration or cross-flow filtration.

[0110] Tangential flow filtration and ultrafiltration, as used herein, refer to an arrangement in which the feed stream passes parallel to the membrane plane, while a portion passes through the membrane (permeate) while the remainder (retenate) is recycled back to the feed vessel. In some embodiments, the pore size of the tangential flow ultrafiltration filter is selected to allow recombinant ASA to permeate through the filter. In other embodiments, the pore size is selected to retain substantially all of the ASA in the feed that passes over the filter. As described elsewhere, ASA exists as a 480 kDa octamer under acidic conditions (pH≦about 5.0), but dissociates into dimers at neutral pH levels. Thus, the pH of the feed can be adjusted in combination with the selection of an appropriate pore size to either retain ASA on the filter membrane or allow it to pass as a permeate.

[0111] Pore ​​sizes with molecular weight cutoffs of at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 300 kDa, at least 400 kDa, or at least 500 kDa can be selected. For example, a filter with a pore size of at least 10 kDa will retain most of the proteins in the feed with a molecular weight of about 11 kDa or greater. As another example, a filter with a pore size of at least 400 kDa will retain most of the proteins with a molecular weight greater than 400 kDa. In some embodiments, the feed retention is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more. Similarly, pore sizes can be selected for isolation of a particular size permeate. For example, a filter having a pore size of at least 500 kDa allows the passage of most proteins having a molecular weight less than 500 kDa, in some embodiments, the passage is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more.

[0112] The filtration area or capacity may also be optimized for use in the processes disclosed herein. Considerations influencing the selection of the filtration area include robustness, cost, feed flow rate (i.e., cross flow rate), transmembrane pressure, permeate flux rate, equipment compatibility, and throughput. In some embodiments, the permeate flux is about 50-100 liters / m 2 per hour ("LMH"). In some embodiments, the feed streams total about 250-600 LMH. In some embodiments, the feed streams total about 250-350 LMH. In some embodiments, the feed streams total about 175-245 LMH. In some embodiments, the feed streams total about 170-230 LMH. In some embodiments, the feed streams total about 120-160 LMH. In some embodiments, the feed streams total about 15-30 LMH. In some embodiments, the feed streams total about 11-21 LMH. In some embodiments, the filtration area is about 0.02 m 2 , approx. 0.14m 2 , about 0.7, or about 3.5 m 2 In certain embodiments, the transmembrane pressure is about 55-60 psi total. In some embodiments, the transmembrane pressure is about 15-25 psi total. In some embodiments, the transmembrane pressure is about 10-20 psi total. In some embodiments, the transmembrane pressure is about 5-15 psi.

[0113] Ultrafiltration filters for use in embodiments of the present invention may include membrane materials known to those skilled in the art, including, but not limited to, polyethersulfone and stabilized cellulose. One exemplary filter cassette for use in embodiments of the present invention is Sartorius' ECO®. Another exemplary filter cassette for use in embodiments of the present invention is Sartorius' HYDROSART® 30 kD standard membrane.

[0114] deep filtration The purification method described herein may include one or more steps of depth filtration. Depth filters are a variety of filters that use porous filtration media to retain particles through the media, rather than just on the surface of the media. Depth filters include filtration media with graded density, allowing larger particles to be trapped near the surface of the filter, while smaller particles penetrate the larger open areas at the surface of the filter, and only be trapped by smaller openings closer to the center of the filter. Some embodiments use a depth filtration step only during the upstream recovery stage (i.e., before the subsequent chromatographic purification step), while other embodiments use a depth filter during one or more additional stages of purification. In some embodiments of the present invention, Cuno Zeta Plus depth filters are used.

[0115] Depth filtration may optionally be followed by 0.45 micron (±2 μm) filtration to remove particulates and reduce bioburden in preparation for downstream treatment processes.

[0116] Viral inactivation The purification methods described herein may include one or more steps of viral inactivation. In some embodiments, viral inactivation includes a solvent and / or detergent. The solvent or detergent may include, for example, polysorbate 80, tri-n-butyl-phosphate (TnBP), or both. Viral inactivation may include incubation in the solvent or detergent for 3 to 24 hours. In another embodiment, viral inactivation includes viral filtration, for example, by using a Planova™ filter.

[0117] It is understood that these methods are directed to producing preparations of the enzyme that are substantially free of infectious virus and may be designated a "virus-safe product."In addition, it is contemplated that the various methods may be used independently or in combination.

[0118] Viral inactivation can be achieved by the addition of one or more "viral inactivating agents" to the solution containing the enzyme. In some embodiments, the viral inactivation step is performed before the chromatographic purification step (i.e., before loading the impure preparation onto the first chromatographic column) to ensure that the viral inactivation agent is not present in the final product in any amount or concentration that would compromise the safety of the product when the agent is used as a pharmaceutical product or when the product is used for the preparation of a pharmaceutical product, while other embodiments use depth filters during one or more further stages of purification. For example, in some embodiments, the inventive method according to the present invention further comprises a step of virus removal after the last chromatographic column.

[0119] The term "virus inactivating agent" is intended to indicate an agent (e.g., a detergent) or method that can be used to inactivate lipid enveloped viruses as well as non-lipid enveloped viruses. The term "virus inactivating agent" should be understood to encompass both combinations of such agents and / or methods, and only one type of such agent or method, whenever appropriate.

[0120] Typical virus inactivating agents are detergents and / or solvents, most typically detergent-solvent mixtures. It should be understood that the virus inactivating agent is optionally a mixture of one or more detergents and one or more solvents. A variety of detergents and solvents can be used for virus inactivation. The detergent can be selected from the group consisting of non-ionic and ionic detergents, and is selected to be substantially non-denaturing. Typically, non-ionic detergents are used to facilitate the subsequent elimination of the detergent from the rASA preparation in the subsequent purification step. Suitable detergents are described, for example, in U.S. Pat. No. 4,314,997 and U.S. Pat. No. 4,315,919 to Shanbrom et al. Typical detergents are those sold under the trademarks Triton X-100 and Tween 20 or Tween 80. Preferred solvents for use with the virus inactivating agent are di- or tri-alkyl phosphates, for example, as described in U.S. Pat. No. 4,764,369 by Neurath and Horowitz. A typical solvent is tri(n-butyl) phosphate (TnBP). A particularly preferred viral inactivating agent for the practice of the invention is Tween 80, although other agents or combinations of agents may alternatively be used. Typical agents were added in volumes such that the concentration of Tween-80 in the solution containing ASA is in the range of about 0.5-4.0% by weight, preferably at a concentration of about 1% by weight. TnBP may then be added to a final concentration of 0.3%, calculated based on the new volume of the sample containing ASA.

[0121] The virus inactivation step is carried out under conditions that inactivate enveloped viruses resulting in a substantially virus-safe rhASA-containing solution. Generally, such conditions include a temperature of 4-37°C, e.g., 19-28°C, 23-27°C, typically about 25°C, and an incubation time found to be effective by validation studies. Generally, an incubation time of 1-24 hours, preferably 10-18 hours, e.g., about 14 hours, is sufficient to ensure sufficient virus inactivation. However, the appropriate conditions (temperature and incubation time) will vary depending on the virus inactivation agent used, the pH, and the protein concentration and lipid content of the solution.

[0122] Other methods for viral removal or inactivation can also be used to produce virus-safe products, such as the addition of methylene blue with subsequent inactivation by irradiation with ultraviolet light.

[0123] The purification methods described herein may include one or more steps of virus removal filtration. Typically, virus filtration is performed after purification of the enzyme by one or more steps of chromatography. In some embodiments, the virus filtration step is performed by passing the ASA containing the solution resulting from the purification step through a sterile filter, followed by passing the sterile filtered solution through a nanofilter. By "sterile filter" is meant a filter that removes substantially all microorganisms capable of transmitting and / or causing an infection. While it is typical for the filter to have a pore size of about 0.1 microns, this pore size may range from about 0.05 to 0.3 microns. It may be feasible to replace or combine virus filtration of the sample as is performed in the purification process in which the sample is contacted with a detergent.

[0124] Some embodiments of the present invention include at least two steps of virus inactivation and / or filtration. For example, virus inactivation before column chromatography can be combined with virus removal after all of the chromatography steps are completed. Post-chromatography virus removal can be performed before or after one or more steps of ultrafiltration / diafiltration (UFDF) (e.g., tangential flow ultrafiltration). In certain examples, the eluate is obtained from the final step of chromatographic purification (e.g., cation exchange (SP) chromatography), and the pH of the elution pool is adjusted to about 5.5, about 6.0, about 6.5, or about 7.0, followed by virus filtration. Thus, in certain embodiments, a single step of UFDF begins with virus filtration (e.g., using a Planova™ filter). In other examples, the eluate obtained from the final step of chromatographic purification (e.g., cation exchange (SP) chromatography) is subjected to a first step of UFDF without pH adjustment, followed by virus filtration and a second step of UFDF.

[0125] In certain embodiments, the pH adjusted cation exchange eluate pool is virus filtered on a Planova 20N filter. In some embodiments, the yield of the pH adjusted cation exchange eluate compared to the input after virus filtration, as assessed by A280 absorbance, is about 90-100%, i.e., about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more. Thus, in some embodiments, essentially no recombinant ASA is lost during virus filtration. The yield for virus filtration is significant, as a pH adjusted to about 6.0 demonstrates that the octamer of ASA (which is about 20 nm in diameter) can dissociate into a dimeric form. Thus, the pore size of the virus filter can be selected to ensure that only the dimeric form is filtered (i.e., the octameric form can be retained by the filter or cause blockage of the virus filter). For example, a viral filter with a pore size of 20 nm retains the octameric but not dimeric form of ASA.

[0126] Affinity chromatography The purification methods described herein may include one or more steps of affinity chromatography (eg, immunoaffinity chromatography, immobilized metal ion affinity chromatography, and / or immobilized ligand affinity chromatography).

[0127] Briefly, affinity chromatography is a chromatographic technique that relies on highly specific interactions, such as between receptors and ligands, antigens and antibodies, or enzymes and substrates. As known by those skilled in the art, the selective molecules used in the affinity chromatography step in the purification methods described herein may be based on various properties of recombinantly produced ASA that can be exploited by the selective molecules (e.g., three-dimensional structure, glycosylation, etc.). Exemplary selective molecules (or capture reagents) that can be used in the affinity chromatography step include protein A, protein G, antibodies, metal ions (e.g., nickel), specific substrates, ligands, or antigens. In some embodiments, a selective molecule suitable for the affinity chromatography step of the present invention uses an anti-arylsulfatase A antibody (e.g., an anti-human arylsulfatase A antibody). A suitable anti-arylsulfatase A antibody may be obtained commercially or through immunization of a non-human animal (e.g., mouse, rat, rabbit, chicken, goat, sheep, horse, or other suitable animal for generating antibodies against human proteins).

[0128] Generally, the molecule of interest (e.g., recombinant ASA) is captured on a solid or stationary phase or medium through interaction with the selective molecule, while other undesired molecules are not captured if they are not bound by the selective molecule(s). The solid medium can then be removed from the mixture and optionally washed, and the molecule of interest can be released from capture by elution. In some embodiments, the affinity column can be eluted by changing the ionic strength through a gradient. For example, salt concentration, pH, pI, and ionic strength can be used to separate or form a gradient to separate.

[0129] In some embodiments, recombinant ASA proteins can be produced with tags to facilitate purification by affinity chromatography. As known by those skilled in the art, protein tags can include, for example, glutathione-S-transferase (GST), hexahistidine (His), maltose binding protein (MBP), among others. In some embodiments, lectins are used in affinity chromatography to separate components within a sample. For example, certain lectins specifically bind to certain carbohydrate molecules and can be used to separate glycoproteins from non-glycosylated proteins or one glycoform from another.

[0130] Ion exchange chromatography The purification methods described herein may include one or more steps of ion exchange chromatography (eg, anion exchange chromatography and / or cation exchange chromatography).

[0131] As known to those skilled in the art, ion exchangers (e.g., anion exchangers and / or cation exchangers) can be based on various materials for the matrix and the attached charged groups. For example, agarose-based (such as SEPHAROSE™ CL-6B, SEPHAROSE™ Fast Flow and SEPHAROSE™ High Performance), cellulose-based (such as DEAE SEPHACEL™), dextran-based (such as SEPHADEX™), silica-based and synthetic polymer-based matrices can be used, and the materials mentioned can be cross-linked to some extent.

[0132] Ion exchange resin can be prepared according to known methods. Typically, an equilibration buffer that allows the resin to bind its counterion can be passed through the ion exchange resin before loading the sample or composition containing the polypeptide and one or more contaminants onto the resin. For convenience, the equilibration buffer can be the same as the loading buffer, but this is not necessary.

[0133] In any embodiment of the present invention, the ion exchange resin can be regenerated with a regeneration buffer after elution of the polypeptide, so that the column can be reused.Generally, the salt concentration and / or pH of the regeneration buffer can be such that substantially all of the contaminants and the polypeptide of interest are eluted from the ion exchange resin.Generally, the regeneration buffer has a very high salt concentration to elute the contaminants and the polypeptide from the ion exchange resin.

[0134] Anion exchange chromatography An embodiment of the present invention includes, for example, providing a sample of arylsulfatase A (e.g., recombinant arylsulfatase A) and subjecting the sample to anion exchange chromatography, for example, anion exchange chromatography as described herein. For anion exchange resins, the charge groups covalently attached to the matrix can be diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and / or quaternary ammonium (Q). In some embodiments, the anion exchange resin used is a Q Sepharose column. Anion exchange chromatography can be performed using, for example, Q SEPHAROSE™ Fast Flow, Q SEPHAROSE™ High Performance, Q SEPHAROSE™ XL, CAPTO™ Q, DEAE, TOYOPEARL GIGACAP™ Q, FRACTOGEL™ TMAE (trimethylaminoethyl, quaternary ammonia resin), ESHMUNO™ Q, NUVIA™ Q, or UNOSPHERE™ Q. Other anion exchangers may be used within the scope of the present invention, including quaternary amine resins or "Q-resins" (e.g., CAPTO™-Q, Q-SEPHAROSE®, QAE SEPHADEX®); diethylaminoethane (DEAE) resins (e.g., DEAE-TRISACRYL®, DEAE These include, but are not limited to, SEPHAROSE®, benzoylated naphthoylated DEAE, diethylaminoethyl SEPHACEL®; AMBERJET® resin; AMBERLYST® resin; AMBERLITE® resin (e.g., AMBERLITE® IRA-67, AMBERLITE® strong base, AMBERLITE® weak base), cholestyramine resin, ProPac® resin (e.g., PROPAC® SAX-10, PROPAC® WAX-10, PROPAC® WCX-10); TSK-GEL® resin (e.g., TSKgel DEAE-NPR; TSKgel DEAE-5PW); and ACCLAIM® resin.In some embodiments, subjecting the sample of arylsulfatase A to anion exchange chromatography is performed at a temperature of about 23°C or less, about 18°C ​​or less, about 16°C or less, e.g., about 23°C, about 20°C, about 18°C, or about 16°C.

[0135] Typical mobile phases for anion exchange chromatography include relatively polar solutions, such as water, acetonitrile, organic alcohols such as methanol, ethanol, and isopropanol, or solutions containing 2-(N-morpholino)-ethanesulfonic acid (MES). Thus, in certain embodiments, the mobile phase includes about 0%, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% of a polar solution. In certain embodiments, the mobile phase comprises from about 1% to about 100%, from about 5% to about 95%, from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% of the polar solution at any given time during the course of the separation.

[0136] In certain embodiments, the rASA is loaded at a binding capacity of about 23 AU / L resin or less, e.g., about 19 AU / L resin or less, about 15 AU / L resin or less, or about 12 AU / L resin or less, e.g., about 12 AU / L resin to about 15 AU / L resin, or about 15 AU / L resin to about 19 AU / L resin. In some embodiments, the arylsulfatase A sample is loaded onto an anion exchange chromatography column at a binding capacity of at least about 4.5 g / L resin (e.g., at least about 5 g / L resin, 6 g / L resin, 7 g / L resin, 8 g / L resin, 9 g / L resin, 10 g / L resin, 11 g / L resin, 12 g / L resin, 13 g / L resin, 14 g / L resin, or 15 g / L resin). In some embodiments, the sample of arylsulfatase A is loaded onto an anion exchange chromatography column at a binding capacity in the range of about 4.5-20 g / L resin (e.g., in the range of about 5-20 g / L resin; 5-19 g / L resin, 5-18 g / L resin, 5-17 g / L resin, 5-16 g / L resin, 5-15 g / L resin, 7.5-20 g / L resin, 7.5-19 g / L resin, 7.5-18 g / L resin, 7.5-17 g / L resin, 7.5-16 g / L resin, 7.5-15 g / L resin, 10-20 g / L resin, 10-19 g / L resin, 10-18 g / L resin, 10-17 g / L resin, 10-16 g / L resin, or 10-15 g / L resin).

[0137] The aqueous solution containing ASA and contaminant(s) can be loaded onto the anion exchange resin as a mobile phase using a loading buffer having a salt concentration and / or pH such that the polypeptide and contaminants bind to the anion exchange resin. The resin can then be washed with one or more column volumes of loading buffer, followed by one or more column volumes of wash buffer, increasing the salt concentration. Finally, ASA can be eluted with an elution buffer having an increasing salt concentration. Optionally, elution of the enzyme can also be mediated by gradually or stepwise decreasing the pH. Fractions containing ASA activity can be collected and combined for further purification.

[0138] In some embodiments, the loading of the sample of arylsulfatase A onto the anion exchange chromatography column is performed using a loading buffer. In one embodiment, the loading buffer does not contain sodium chloride. In another embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration of the loading buffer is about 1 mM to about 25 mM, for example, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or about 5 mM to about 10 mM. In some embodiments, the salt concentration in the mobile phase is a gradient (for example, a linear or non-linear gradient). In some embodiments, the salt concentration in the mobile phase is constant. In some embodiments, the salt concentration in the mobile phase may be increased or decreased stepwise. In some embodiments, the loading of the sample of arylsulfatase A onto the anion exchange chromatography column is performed at a pH of about 5 to about 9, for example, about 6 to about 8, for example, about 7.

[0139] In some embodiments, washing of the anion exchange chromatography column is performed with one or more wash buffers. For example, washing of the anion exchange column can include two or more (e.g., first and second) wash steps, each using a different wash buffer. In one embodiment, the wash buffer does not contain sodium chloride. In another embodiment, the wash buffer contains sodium chloride. For example, the sodium chloride concentration of the wash buffer is about 50 mM to about 200 mM, for example, about 50 mM to about 150 mM, about 100 mM to about 200 mM, or about 100 mM to about 150 mM, for example, about 80 mM, about 100 mM, about 120 mM, or about 140 mM. In some embodiments, washing of the anion exchange chromatography column is performed at a pH of about 5 to about 9, for example, about 6 to about 8, for example, about 7.

[0140] In one embodiment, the elution buffer contains sodium phosphate. For example, the sodium phosphate concentration of the elution buffer is about 20 mM to about 50 mM, for example, about 25 mM to about 45 mM, for example, about 30 mM, about 35 mM, or about 40 mM. In another embodiment, the elution buffer does not contain sodium chloride. In yet another embodiment, the elution buffer contains sodium chloride. For example, the sodium chloride concentration of the elution buffer is about 200 mM to about 300 mM, for example, about 240 mM to about 280 mM. In some embodiments, elution of arylsulfatase A from the anion exchange chromatography column is performed at a pH of about 5 to about 9, for example, about 6 to about 8, for example, about 7.

[0141] In some embodiments, elution of arylsulfatase A from an anion exchange chromatography column includes one or more steps of elution peak collection, e.g., starting at about 50 mAU on the ascending surface and down to about 50 mAU on the descending surface, e.g., from about 100 mAU on the ascending surface to about 50 mAU on the descending surface, from about 200 mAU on the ascending surface to about 50 mAU on the descending surface, from about 50 mAU on the ascending surface to about 100 mAU on the descending surface, from about 50 mAU on the ascending surface to about 200 mAU on the descending surface, or from about 100 mAU on the ascending surface to about 100 mAU on the descending surface, as measured, e.g., by spectrophotometry, e.g., at 280 nM.

[0142] It will be apparent to one skilled in the art that many different buffers may be used in the loading, washing, and elution steps. Typically, however, the column may be equilibrated with 1-10 column washes of a buffer comprising 0.05 M MES-Tris, pH 7.0. For convenience, the sample may be loaded in a buffer from a previous step of the purification process, or the sample may be loaded using the loading buffer. The column may be washed with 1-10 column volumes of the buffer used for equilibration, followed by washing with a washing buffer comprising 0.02 MES-Tris, 0.12 M NaCl, pH 7.0. Alternatively, the column may be equilibrated, loaded, and washed using any other equilibration, loading, and washing buffers described herein for anion exchange chromatography. The sample may be eluted in a buffer comprising 0.02 MES-Tris, 0.26 M NaCl, pH 7.0. Alternatively, the sample may be eluted in any other elution buffer described herein for anion exchange chromatography.

[0143] The loading buffer, washing buffer, and elution buffer described herein may include one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is about 1 mM to about 500 mM, for example, about 10 mM to about 250 mM, about 20 mM to about 100 mM, about 1 mM to 5 mM, about 5 mM to 10 mM, about 10 mM to 50 mM, or about 50 mM to about 100 mM, for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0144] Yield, activity, and purity after anion exchange chromatography may vary. In some embodiments, the activity yield of arylsulfatase A is at least about 75%, e.g., at least about 85%, e.g., about 85% to about 99%, or about 90% to about 99%. In some embodiments, the protein yield (AU or absorbance units) is about 10% to 50%, e.g., about 20% to about 35%, or about 25% to about 30%, e.g., as measured by spectrophotometry, e.g., at 280 nm. In some embodiments (e.g., using a TMAE column as described below), the activity yield (AU or absorbance units) of the elution pool protein is at least greater than 95%, e.g., about 70% to 400%, e.g., about 80% to about 390%, or about 90% to about 350%, or about 100% to 150%, e.g., as measured by spectrophotometry, e.g., at 280 nm. In some embodiments (e.g., using a TMAE column as described below), the host cell protein (HCP) log reduction value (LRV) is about 0.5 to about 1.1, e.g., about 0.6 to 0.9, or about 0.7 to 0.8. In some embodiments (e.g., using a TMAE column as described below), the purity is at least 75%, e.g., at least 80%, at least 85%, at least 90% or more, e.g., as measured by capillary electrophoresis-SDS PAGE. In preferred embodiments, the activity yield after anion exchange chromatography, the HCP LRV, and the purity (as measured by capillary electrophoresis-SDS PAGE) are at least about 90%, at least about 0.6, and at least about 80%, respectively.

[0145] In a preferred embodiment of the invention, an anion exchange column with a high loading capacity is used. In an embodiment of the invention, the column is characterized by a loading range of about 3-20 g / L (i.e., about 5-15 g / L, about 10-15 g / L, about 10-20 g / L). In some embodiments, the loading capacity is significantly more than 4.3 g / L (e.g., about 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, or 20 g / L or more). In some embodiments, the binding capacity of the resin is about 75-100 AU / L (e.g., about 75 AU / L, about 80 AU / L, about 85 AU / L, about 90 AU / L, about 95 AU / L). In some embodiments, the loading capacity is greater than about 80 AU / L. In some embodiments, the high loading capacity column is a TMAE column. In certain embodiments, the column is selected from the group consisting of a Fractogel® TMAE column, a Nuvia Q column, a Q Sepharose Fast Flow column, a Capto Q column, a Q Sepharose XL column, an Eshmuno Q column, a UNOsphere Q column, or a GigaCap Q column.

[0146] In certain embodiments of the invention, the TMAE column is pre-equilibrated with a buffer containing about 20 mM MES-Tris and 1000 mM NaCl at pH 7.0. In certain embodiments, the column is equilibrated with a buffer containing 50 mM MES-Tris at pH 7.0. In some embodiments, the loading flow rate of the TMAE column is about 75-125 cm / hr (i.e., about 75-115 cm / hr, about 75-110 cm / hr, about 75-105 cm / hr, about 75-100 cm / hr, about 85-115 cm / hr, about 85-110 cm / hr, about 85-105 cm / hr, about 85-100 cm / hr, about 95-115 cm / hr, about 95-110 cm / hr, about 95-105 cm / hr, about 95-100 cm / hr, about 100-120 cm / hr, about 100-115 cm / hr, about 100-110 cm / hr, about 100 cm / hr). Loading conditions can be optimized and evaluated by absorbance at A280 as described herein.

[0147] In certain embodiments using a TMAE column (e.g., a Fractogel TMAE column), very little product is lost to the flow-through during loading, even at load capacities of over 15 g / L. The ability to increase the loading capacity while minimizing flow-through losses is a significant improvement in purification methodologies. In certain embodiments of the invention, the amount of flow-through product loss is less than 30% of the load (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%).

[0148] In some embodiments, after loading, the TMAE column is washed at least once. In certain embodiments, the column is washed twice. The first or second wash buffer may contain an optimized level of sodium chloride. In some embodiments, the amount of sodium chloride in the first or second wash buffer is about 50-150 mM (e.g., about 50-140 mM, about 50-130 mM, about 50-120 mM, about 50-110 mM, about 50-100 mM, about 50-90 mM, about 50-80 mM, about 80-150 mM, about 80-140 mM, about 80-130 mM, about 80-120 mM, about 80-110 mM, about 80-100 mM, about 80-90 mM, about 80 mM, or about 120 mM). In some embodiments, the first wash buffer comprises 50 mM MES-Tris at pH 7.0. In some embodiments, the second wash buffer comprises 20 mM MES-Tris, 100 mM NaCl at pH 7.0. Further optimization of the wash conditions, particularly the second wash conditions, is encompassed within the embodiments of the present invention. For example, increasing the salt concentration of the second wash may improve the log reduction value (LRV) and overall purity of the host cell protein (HCP), but decrease both the activity and A280 yield. As described herein, certain wash conditions may be balanced with the elution conditions described below to provide an optimal combination of purity, activity, and yield.

[0149] In an embodiment of the present invention, the recombinant ASA that binds to the TMAE column is eluted with an elution buffer. In some embodiments, the amount of sodium chloride in the elution buffer is optimized. In certain embodiments, the amount of sodium chloride in the elution buffer is about 150 to 300 mM (e.g., about 150 to 290 mM, about 150 to 280 mM, about 150 to 270 mM, about 150 to 260 mM, about 150 to 250 mM, about 150 to 240 mM, about 150 to 230 mM, about 150 to 220 mM, about 150 to 210 mM, about 170 to 290 mM, about 170 to 280 mM, about 170 to 270 mM, about 170 to 260 mM, about 170 to 250 mM, about 170 to 240 mM, about 170 to 230 mM, about 170 to 220 mM, about 170 to 210 mM). The elution buffer may be about 180-290 mM, about 180-280 mM, about 180-270 mM, about 180-260 mM, about 180-250 mM, about 180-240 mM, about 180-230 mM, about 180-220 mM, about 180-210 mM, about 180, about 220, or about 260). In certain examples, the elution buffer comprises 50 mM MES-Tris and 1 M NaCl at pH 7.0. In some embodiments, the A280 yield after elution is greater than 60% of the load (e.g., about 60%, about 70%, about 80% or more). Further optimization of elution conditions is encompassed within embodiments of the invention. For example, increasing the elution salt concentration (i.e., conductivity) provides better yields but results in poorer purity and HCP removal. As noted above, the specific wash conditions must be balanced with the elution conditions to provide the optimal combination of purity, activity, and yield.

[0150] Cation exchange chromatography In some embodiments, the method further comprises subjecting the sample of arylsulfatase A to cation exchange chromatography, e.g., sulfopropyl (SP) cation exchange chromatography, e.g., as described herein. In some embodiments, the sample of arylsulfatase A is subjected to anion exchange chromatography prior to cation exchange chromatography. In an exemplary embodiment, the cation exchange chromatography comprises sulfopropyl (SP) cation exchange chromatography, although other cation chromatographic membranes or resins can be used, e.g., MUSTANG™ S membrane, S-SEPHAROSE™ resin, or Blue SEPHAROSE™ resin. In some embodiments, the method further comprises concentrating and / or filtering the sample of arylsulfatase A, e.g., by ultrafiltration and / or diafiltration, e.g., by tangential flow ultrafiltration. The cation exchange chromatography can be performed at an optimal temperature, e.g., as described herein, to enhance target binding and / or reduce impurity binding. For example, cation exchange chromatography can be carried out at a temperature of about 23°C, 18°C, 16°C, or lower.

[0151] In one embodiment, the cation exchange chromatography comprises sulfopropyl (SP) cation exchange chromatography. In another embodiment, the cation exchange chromatography is a polishing step. The cation exchange chromatography (e.g., sulfopropyl (SP) cation exchange chromatography) can be performed using, for example, one or more of TOYOPEARL® SP-650, TOYOPEARL® SP-550, TSKGEL® SP-3PW, TSKGEL® SP-5PW, SP SEPHAROSE™ Fast Flow, SP SEPHAROSE™ High Performance, SP SEPHAROSE™ XL, SARTOBIND® S membrane, POROS® HS50, UNOSPHERE™ S, and MACROCAP™ S.

[0152] The aqueous solution containing arylsulfatase A and contaminant(s) can be loaded onto the cation exchange resin using a loading buffer having a salt concentration and / or pH such that the polypeptide and contaminants bind to the cation exchange resin. The resin can then be washed with one or more column volumes of equilibrated butter or loading buffer, followed by one or more column volumes of wash buffer to increase the salt concentration. Finally, arylsulfatase A can be eluted in an elution buffer. Fractions containing arylsulfatase A activity can be collected and combined for further purification.

[0153] In typical embodiments, the NaCl concentration and / or pH of the loading buffer, washing buffer, and / or elution buffer can be optimized, for example, as described herein, to enhance target binding and / or reduce impurity binding. In some embodiments, the NaCl concentration in the loading buffer is about 20 mM, 15 mM, 10 mM, or less. In some embodiments, the loading buffer has a pH of about 4.5, 4.3, 4.0, or less. In some embodiments, the NaCl concentration in the washing buffer is about 20 mM, 15 mM, 10 mM, or less. In some embodiments, the NaCl concentration in the elution buffer is about 55 mM, 50 mM, 45 mM, 40 mM, or less.

[0154] In some embodiments, subjecting the sample of arylsulfatase A to cation exchange chromatography includes loading the sample of arylsulfatase A onto a cation chromatography column (e.g., a sulfopropyl (SP) cation exchange column), washing the cation exchange chromatography column, and eluting the arylsulfatase A from the column. In some embodiments, the column may be equilibrated with more than 3, e.g., 5-10 column volumes of 0.01 M NaAc, 0.01 M NaCl, 0.03 M acetic acid, pH 4.2.

[0155] In some embodiments, the sample may be loaded in a buffer from a previous step of the purification process, or the sample may be loaded using a loading buffer. In one embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration of the loading buffer is about 1 mM to about 25 mM, such as about 5 mM to about 20 mM, such as about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In another embodiment, the loading buffer contains sodium acetate. For example, the sodium acetate concentration of the loading buffer is about 10 mM to about 100 mM, such as about 20 mM, about 40 mM, or about 60 mM. In some embodiments, the loading of the sample of arylsulfatase A onto the cation exchange chromatography column is performed at a pH of about 3.0 to about 6.0, such as about 4.0 to about 5.0, such as about 4.0, about 4.3, or about 4.5. In some embodiments, the arylsulfatase A sample is loaded onto a cation exchange chromatography column at a binding capacity of about 15 AU / L resin or less, e.g., about 14 AU / L resin or less, or about 12 AU / L resin or less, e.g., from about 10 AU / L resin to about 14 AU / L resin, or from about 10 AU / L resin to about 12 AU / L resin.

[0156] In some embodiments, washing of the cation exchange chromatography column is performed with one or more wash buffers. For example, washing of the cation exchange column can include two or more (e.g., first and second) wash steps, each with a different wash buffer. The column can be washed with 1 to 10 column volumes of the buffer used for equilibration. Alternatively, the column can be equilibrated, loaded, and washed with any other equilibration, loading, and washing buffer described herein for cation exchange chromatography. In one embodiment, the wash buffer contains sodium chloride. For example, the sodium chloride concentration of the wash buffer is about 1 mM to about 25 mM, e.g., about 5 mM to about 20 mM, or about 10 mM to about 15 mM, e.g., about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In another embodiment, the wash buffer contains sodium acetate. For example, the sodium acetate concentration of the loading buffer is about 10 mM to about 100 mM, e.g., about 20 mM, about 40 mM, or about 60 mM. In some embodiments, washing of the cation exchange chromatography column is performed at a pH of about 3.0 to about 6.0, e.g., about 4.0 to about 5.0, e.g., about 4.0, about 4.3, or about 4.5.

[0157] In some embodiments, elution of arylsulfatase A from the cation exchange chromatography column is performed with an elution buffer. In one embodiment, the elution buffer contains sodium chloride. For example, the sodium chloride concentration of the elution buffer is about 25 mM to about 75 mM, such as about 45 mM to about 60 mM, such as about 45 mM, about 50 mM, about 55 mM, or about 55 mM. In some embodiments, elution of arylsulfatase A from the cation exchange chromatography column is performed at a pH of about 3.0 to about 6.0, such as about 4.0 to about 5.0, such as about 4.0, about 4.3, or about 4.5. Thus, as one particular example, the sample may be eluted in a buffer comprising 0.02 M NaAc, 0.05 M NaCl, pH 4.5. Alternatively, the sample may be eluted in any other elution buffer described herein for cation exchange chromatography.

[0158] In some embodiments, the elution of arylsulfatase A from a cation exchange chromatography column includes one or more steps of elution peak collection. For example, the elution peak collection starts at about 50mAU on the ascending surface and to about 50mAU on the descending surface, for example, from about 100mAU on the ascending surface to about 50mAU on the descending surface, from about 200mAU on the ascending surface to about 50mAU on the descending surface, from about 50mAU on the ascending surface to about 100mAU on the descending surface, from about 50mAU on the ascending surface to about 200mAU on the descending surface, or from about 100mAU on the ascending surface to about 100mAU on the descending surface, as measured, for example, by spectrophotometry at 280nM. The collected elution peaks can be pooled.

[0159] The loading buffer, washing buffer, and elution buffer described herein may include one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is about 1 mM to about 500 mM, for example, about 10 mM to about 250 mM, about 20 mM to about 100 mM, about 1 mM to 5 mM, about 5 mM to 10 mM, about 10 mM to 50 mM, or about 50 mM to about 100 mM, for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0160] In some embodiments, subjecting the sample of arylsulfatase A to cation exchange chromatography is performed at a temperature of about 23° C. or less, about 18° C. or less, about 16° C. or less, e.g., about 23° C., about 20° C., about 18° C., or about 16° C. In some embodiments, subjecting the sample of arylsulfatase A to cation exchange chromatography is performed at about 23° C. to about 16° C., e.g., about 23° C., about 20° C., about 18° C., or about 16° C., and loading the sample of arylsulfatase A onto the cation exchange chromatography column is performed at a pH of about 4.5 to about 4.3, e.g., about 4.5, about 4.4, or about 4.3. In some embodiments, subjecting the sample of arylsulfatase A to cation exchange chromatography is performed at about 23° C., and loading the sample of arylsulfatase A onto the cation exchange chromatography column is performed at a pH of about 4.5. In some embodiments, subjecting the sample of arylsulfatase A to cation exchange chromatography is performed at about 23° C. and loading the sample of arylsulfatase A onto the cation exchange chromatography column is performed at a pH of about 4.3. In some embodiments, subjecting the sample of arylsulfatase A to cation exchange chromatography is performed at about 18° C. and loading the sample of arylsulfatase A onto the cation exchange chromatography column is performed at a pH of about 4.5. In some embodiments, subjecting the sample of arylsulfatase A to cation exchange chromatography is performed at about 18° C. and loading the sample of arylsulfatase A onto the cation exchange chromatography column is performed at a pH of about 4.3.

[0161] The yield after cation exchange chromatography may vary. In some embodiments, the activity yield of arylsulfatase A is at least about 75%, e.g., at least about 80%, e.g., about 80% to about 105%. In some embodiments, the protein yield (AU or absorbance units) is about 65% to 100%, e.g., about 70% to about 95%, e.g., as measured by spectrophotometry, e.g., at 280 nm.

[0162] The purity and activity after cation exchange chromatography are greatly improved. In some embodiments, the log reduction value (LRV) of host cell protein (HCP) is about 1.0 to about 2.5, e.g., about 1.5 to about 2.0, or about 1.7 to about 1.9. The specific activity of the purified arylsulfatase A can be at least about 50 U / mg to about 140 U / mg, e.g., at least about 70 U / mg, at least about 90 U / mg, at least about 100 U / mg, or at least about 120 U / mg, e.g., as measured by the methods described herein. In some embodiments, the arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. The purity of arylsulfatase A can be measured, for example, by one or more of host cell protein (HCP) Western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reversed-phase HPLC, and size-exclusion HPLC.In some embodiments, reducing the salt concentration of the loading buffer and lowering its pH enhances the binding of ASA to the cation exchange column, but does not affect the binding of impurities.In other words, as described above, the optimal balance of salt concentration and pH can increase the yield after cation exchange chromatography without adversely affecting purity.

[0163] In some embodiments, the pH of the cation exchange elution pool may be adjusted. In some embodiments, the pH is adjusted immediately prior to viral filtration. The cation eluate (e.g., SP eluate) may be adjusted to a pH of about 5.5, about 6.0, about 6.5, or about 7.0 using a pH adjustment buffer containing 0.25 M sodium phosphate, 1.33 M sodium chloride, 0.34 M sodium citrate, pH 7.0. In some embodiments, the pH adjusted SP eluate pool is viral filtered on a Planova 20N filter. In some embodiments, the yield of the pH adjusted cation exchange eluate relative to the input after viral filtration is about 90-100%, i.e., about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more, as assessed by A280 absorbance. The yield for virus filtration is significant, as it demonstrates that adjusting the pH to about 6.0 can dissociate the octamer of ASA (which is about 20 nm in diameter) into a dimeric form. Therefore, the pore size of the virus filter can be selected to ensure that only the dimeric form is filtered (i.e., the octamer form can be retained by the filter or cause blockage of the virus filter). For example, a virus filter with a pore size of 20 nm retains the octameric form of ASA, but not the dimeric form.

[0164] Mixed-mode chromatography The purification method described herein may include one or more steps of mixed-mode chromatography.Mixed-mode chromatography is a type of chromatography, typically in liquid chromatography, that applies several separation modes to reduce a mixture of different molecules.For example, mixed-mode separation may include the features of combination phase and reverse phase with ion exchange at the same time.These stationary phases with more than one interaction type are available from several column manufacturers.

[0165] In one aspect, the disclosure features a method of purifying arylsulfatase A from a sample, the method including, for example, providing a sample of arylsulfatase A (e.g., recombinant arylsulfatase A) and subjecting the sample of arylsulfatase A to mixed-mode chromatography, e.g., ceramic hydroxyapatite (HA) chromatography, e.g., mixed-mode chromatography as described herein, such as a method including hydroxyapatite type I or type II chromatography. In some embodiments, the mixed-mode chromatography is performed using one or more of CHT™ Ceramic Hydroxyapatite Type I Medium, CHT™ Ceramic Hydroxyapatite Type II Medium, BIO-GEL® HT Hydroxyapatite, and BIO-GEL® HTP Hydroxyapatite.

[0166] In some embodiments, subjecting the sample of arylsulfatase A to mixed mode chromatography includes loading the sample of arylsulfatase A onto a mixed mode chromatography column (e.g., HA chromatography), washing the mixed mode chromatography column, and eluting the arylsulfatase A from the column. In some embodiments, subjecting the sample of arylsulfatase A to mixed mode exchange chromatography is performed at a temperature of about 23° C. or less, about 18° C. or less, about 16° C. or less, e.g., about 23° C., about 20° C., about 18° C., or about 16° C.

[0167] In some embodiments, loading the sample of arylsulfatase A onto the mixed-mode chromatography column is performed using a loading buffer. In one embodiment, the loading buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is about 1 mM to about 10 mM, for example, about 1 mM to about 5 mM, about 5 mM to about 10 mM, for example, about 1 mM, about 2 mM, or about 5 mM. In another embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration of the loading buffer is about 100 mM to about 400 mM, for example, about 200 mM to about 300 mM, for example, about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0168] In some embodiments, the loading of the arylsulfatase A sample onto the mixed mode chromatography column is carried out at a pH of about 5 to about 9, such as about 6 to about 8, eg, about 7.

[0169] In some embodiments, the mixed mode chromatography comprises ceramic hydroxyapatite (HA) chromatography. Hydroxyapatite (HAP) usually refers to a crystalline form of calcium phosphate. The mechanism of HAP involves non-specific interactions between negatively charged protein carboxyl groups on the resin and positively charged calcium ions, and positively charged protein amino groups on the resin and negatively charged phosphate ions. Basic or acidic proteins can be selectively adsorbed onto the column by adjusting the pH of the buffer, and elution can be achieved by varying the salt concentration of the buffer. Furthermore, it is clear that many buffer compositions as well as combinations of buffers can be used. Typically, however, the column can be equilibrated with 1-10 column washes of a buffer comprising 0.001 M NaPO4, 0.02 M MES-Tris, 0.26 M NaCl, pH 7.0. For convenience, the sample can be loaded in a buffer from a previous step of the purification process, or the sample can be loaded using a loading buffer. The column may be washed with 1-10 column volumes of the buffer used for equilibration, followed by washing with a wash buffer comprising 0.005 M NaPO4, 0.02 M MES-Tris, 0.26 M NaCl, pH 7.0. Alternatively, the column may be equilibrated, loaded, and washed with any other equilibration, loading, and washing buffer described herein for mixed-mode chromatography. The sample may be eluted in a buffer comprising 0.04 M NaPO4, pH 7.0. Optionally, the column may be cleared by washing with 1-10 column volumes of 0.4 M NaPO4, pH 12. Alternatively, the sample may be eluted in any other elution buffer described herein for mixed-mode chromatography.

[0170] In some embodiments, washing of the mixed mode chromatography column is performed with one or more wash buffers. For example, washing of the mixed mode chromatography column can include two or more (e.g., a first and a second) wash steps, each with a different wash buffer.

[0171] In one embodiment, the washing buffer contains sodium phosphate. For example, the sodium phosphate concentration of the washing buffer is about 1 mM to about 10 mM, for example, about 1 mM to about 5 mM, about 5 mM to about 10 mM, for example, about 1 mM, about 5 mM, or about 10 mM. In another embodiment, the washing buffer contains sodium chloride. For example, the sodium chloride concentration of the washing buffer is about 50 mM to about 600 mM, for example, about 100 mM to about 500 mM, or about 200 to about 400 mM, for example, about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0172] In some embodiments, washing of the mixed mode chromatography column is carried out at a pH of about 5 to about 9, such as about 6 to about 8, for example about 7.

[0173] In some embodiments, elution of arylsulfatase A from the mixed mode chromatography column is performed at a pH of about 5 to about 9, e.g., about 6 to about 8, e.g., about 7. In some embodiments, elution of arylsulfatase A from the mixed mode chromatography column includes one or more steps of elution peak collection. For example, elution peak collection begins at about 50 mAU on the ascending surface to about 50 mAU on the descending surface, e.g., from about 100 mAU on the ascending surface to about 50 mAU on the descending surface, from about 200 mAU on the ascending surface to about 50 mAU on the descending surface, from about 50 mAU on the ascending surface to about 100 mAU on the descending surface, from about 50 mAU on the ascending surface to about 200 mAU on the descending surface, or from about 100 mAU on the ascending surface to about 100 mAU on the descending surface, as measured, e.g., by spectrophotometry, e.g., at 280 nM.

[0174] The loading buffer, washing buffer, and elution buffer described herein may include one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is about 1 mM to about 500 mM, for example, about 10 mM to about 250 mM, about 20 mM to about 100 mM, about 1 mM to 5 mM, about 5 mM to 10 mM, about 10 mM to 50 mM, or about 50 mM to about 100 mM, for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0175] In some embodiments, purification of ASA by mixed-mode chromatography is followed by purification by ion exchange chromatography (e.g., anion exchange chromatography), however, these steps can be performed in the reverse order.

[0176] Yields after mixed-mode chromatography can vary. In some embodiments, the activity yield of arylsulfatase A is at least about 80%, e.g., at least about 90%, e.g., about 80% to about 115%. In some embodiments, the protein yield (AU or absorbance units) is about 30% to 80%, e.g., about 35% to about 75%, or about 50% to about 70%, e.g., as measured by spectrophotometry, e.g., at 280 nm.

[0177] The purity after mixed-mode chromatography is greatly improved. In some embodiments, the specific activity of the purified arylsulfatase A is at least about 50 U / mg to about 140 U / mg, e.g., at least about 70 U / mg, at least about 90 U / mg, at least about 100 U / mg, or at least about 120 U / mg, as measured, for example, by the methods described herein. In some embodiments, the arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. The purity of the arylsulfatase A can be measured, for example, by one or more of host cell protein (HCP) Western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse phase HPLC, and size exclusion HPLC. In some embodiments, the log reduction value (LRV) of the host cell protein (HCP) is about 0.3 to about 0.6, for example, about 0.4 to 0.5.

[0178] Hydrophobic Interaction Chromatography (HIC) The purification method described herein may include subjecting a sample of arylsulfatase A to hydrophobic interaction chromatography (HIC). In one embodiment, the hydrophobic interaction chromatography comprises phenyl chromatography. In other embodiments, the hydrophobic interaction chromatography comprises butyl chromatography or octyl chromatography. In some embodiments, subjecting a sample of arylsulfatase A to HIC is performed at a temperature of about 23° C. or less, about 18° C. or less, or about 16° C. or less, for example, about 23° C., about 20° C., about 18° C., or about 16° C. In some embodiments, the sample of arylsulfatase A is subjected to mixed-mode chromatography prior to HIC.

[0179] Hydrophobic interaction chromatography exploits the attraction of a given molecule in a polar or non-polar environment; for proteins, this tendency is controlled by the hydrophobic or hydrophilic nature of the residues on the exposed outer surface of the protein. Hence, proteins are fractionated based on varying degrees of attraction to hydrophobic matrices, typically inert supports with alkyl linker arms of chain length between 2 and 18 carbons. The stationary phase consists of small non-polar groups (butyl, octyl, or phenyl) attached to a hydrophilic polymer backbone (e.g., cross-linked Sepharose™, dextran, or agarose). Hence, HIC columns are typically butyl SEPHAROSE™ columns or phenyl SEPHAROSE™ columns, most typically phenyl SEPHAROSE™ columns. In some embodiments, the hydrophobic interaction chromatography comprises phenyl chromatography using one or more of Phenyl SEPHAROSE™ High Performance, Phenyl SEPHAROSE™ 6 Fast Flow (low sub), or Phenyl SEPHAROSE™ 6 Fast Flow (high sub).

[0180] In some embodiments, subjecting a sample of arylsulfatase A to hydrophobic interaction chromatography includes loading the sample of arylsulfatase A onto a HIC column, washing the HIC column, and eluting arylsulfatase A from the column. Loading, washing, and elution in HIC essentially follow the same principles as those described above for ion exchange chromatography, but often apply conditions that are almost the opposite of those used in ion exchange chromatography. Thus, the process of HIC involves the use of a high salt loading buffer that unfolds the protein and exposes hydrophobic sites. The protein is retained by the hydrophobic ligands of the column and is exposed to a buffer gradient with a decreasing salt concentration. When the salt concentration is decreased, the protein returns to its native structure and is eventually eluted from the column. Alternatively, the protein can be eluted with PEG.

[0181] In some embodiments, the loading of the sample of arylsulfatase A onto the HIC column is performed using a loading buffer. In one embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration of the loading buffer is about 0.5 M to about 2.5 M, for example, about 1 M or about 1.5 M. In another embodiment, the loading buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is about 10 mM to about 100 mM, for example, about 25 mM, about 50 mM, or about 75 mM. In some embodiments, the loading of the sample of arylsulfatase A onto the HIC column is performed at a pH of about 5 to about 7, for example, about 5.5 to about 6.5, for example, about 5.5, about 6.0, or about 6.5. In some embodiments, the arylsulfatase A sample is loaded at a binding capacity of about 12 AU / L resin or less, e.g., about 10 AU / L resin or less, about 9 AU / L resin or less, about 7 AU / L resin or less, or about 5 AU / L resin or less, e.g., from about 5 AU / L resin to about 9 AU / L resin, or from about 5 AU / L resin to about 7 AU / L resin.

[0182] The use of phenyl SEPHAROSE™ as a solid phase in HIC is common in this disclosure. Furthermore, it is easily understood that there are many different possibilities when it comes to the exact conditions and buffers and buffer combinations used in the loading, washing, and elution processes. In a typical embodiment, the column can be equilibrated in a buffer containing 0.05 M NaPO4, 1 M NaCl, pH 5.5. For convenience, the sample can be loaded in a buffer from a previous step of the purification process, or the sample can be loaded using a loading buffer.

[0183] In some embodiments, washing of the HIC column is performed with one or more wash buffers. For example, washing of the HIC column can include two or more (e.g., first and second) wash steps, each using a different wash buffer. In some embodiments, the wash buffer contains sodium chloride. For example, the sodium chloride concentration of the wash buffer is about 100 mM to about 1.5 M, for example, about 250 mM to about 1 M, for example, about 250 mM, about 500 mM, about 750 mM, or about 1 M. In another embodiment, the wash buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is about 10 mM to about 100 mM, for example, about 25 mM, about 50 mM, or about 75 mM. In some embodiments, washing of the HIC column is performed at a pH of about 5 to about 7, for example, about 5.5 to about 6.5, for example, about 5.5, about 6.0, or about 6.5. For example, washing may be performed with 1-2 column washes of equilibration buffer, followed by 1-5 column volumes of 0.02 M MES, 0.05 M NaPO4, 0.5 M NaCl, pH 5.5. Alternatively, the column may be equilibrated, loaded, and washed with any other equilibration, loading, and washing buffers described herein for HIC.

[0184] In some embodiments, elution of arylsulfatase A from the HIC column is performed using an elution buffer. In some embodiments, the elution buffer contains sodium chloride. For example, the sodium chloride concentration of the elution buffer is about 30 mM to about 100 mM, for example, about 45 mM to about 85 mM, for example, about 50 mM, about 60 mM, about 70 mM, or about 80 mM. In some embodiments, elution of arylsulfatase A from the HIC column is performed at a pH of about 5 to about 9, for example, about 6 to about 8, for example, about 7. For example, arylsulfatase A can be eluted using 0.02 M MES-Tris, 0.06 M NaCl, pH 7.0. Alternatively, the sample can be eluted in any other elution buffer described herein for HIC.

[0185] In some embodiments, elution of arylsulfatase A from the HIC column includes one or more steps of elution peak collection, e.g., starting at about 50 mAU on the ascending surface and down to about 50 mAU on the descending surface, e.g., from about 100 mAU on the ascending surface to about 50 mAU on the descending surface, from about 200 mAU on the ascending surface to about 50 mAU on the descending surface, from about 50 mAU on the ascending surface to about 100 mAU on the descending surface, from about 50 mAU on the ascending surface to about 200 mAU on the descending surface, or from about 100 mAU on the ascending surface to about 100 mAU on the descending surface, as measured, e.g., by spectrophotometry, e.g., at 280 nM.

[0186] In some embodiments, purification of arylsulfatase A by HIC is followed by purification by ion exchange chromatography (e.g., anion exchange chromatography) and / or mixed-mode chromatography, however, these steps can be performed in the reverse order.

[0187] The loading buffer, washing buffer, and elution buffer described herein may include one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is about 1 mM to about 500 mM, for example, about 10 mM to about 250 mM, about 20 mM to about 100 mM, about 1 mM to 5 mM, about 5 mM to 10 mM, about 10 mM to 50 mM, or about 50 mM to about 100 mM, for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0188] The yield after HIC can vary. In some embodiments, the activity yield of arylsulfatase A is at least about 60%, e.g., at least about 70%, e.g., about 70% to about 100%. In some embodiments, the protein yield (AU or absorbance units) is about 45% to 100%, e.g., about 50% to about 95%, or about 55% to about 90%, e.g., as measured by spectrophotometry, e.g., at 280 nm.

[0189] The purity after HIC is greatly improved, in some embodiments, the specific activity of the purified arylsulfatase A is at least about 50 U / mg to about 140 U / mg, e.g., at least about 70 U / mg, at least about 90 U / mg, at least about 100 U / mg, or at least about 120 U / mg, e.g., as measured by the methods described herein.

[0190] In some embodiments, the arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. The purity of the arylsulfatase A can be measured, for example, by one or more of host cell protein (HCP) Western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse phase HPLC, and size exclusion HPLC. In some embodiments, the log reduction value (LRV) of the host cell protein (HCP) is about 0.6 to about 1.2, for example, about 0.7 to 0.95.

[0191] Ultrafiltration / Diafiltration The purification method described herein may include one or more steps of downstream ultrafiltration and / or diafiltration. In some embodiments, the method further includes concentrating and / or filtering the sample of arylsulfatase A, for example, by ultrafiltration and / or diafiltration, for example, by tangential flow ultrafiltration.

[0192] Ultrafiltration refers to a membrane separation process in which membranes, driven by a pressure gradient, fractionate components of a liquid as a function of their solvated size and structure. Diafiltration is a specialized type of ultrafiltration process in which the retentate is diluted with water and re-ultrafiltered to reduce the concentration of soluble permeant components and further increase the concentration of retained components. Ultrafiltration is often combined with diafiltration and the purification steps of ultrafiltration / diafiltration (UFDF).

[0193] Embodiments of the invention employ at least one, at least two, at least three or more downstream UFDF purification steps. One or more diafiltrations can occur within a UFDF step (e.g., UFDF). In some embodiments, the protein yield (AU or absorbance units) after downstream UFDF is about 90% to 105%, e.g., about 95% to about 100%, or about 97% to about 99%, as measured, e.g., by spectrophotometry, e.g., at 280 nm, compared to the amount from the preceding purification step. In some embodiments, essentially no protein is lost during UFDF.

[0194] In some embodiments of the present invention, downstream UFDF results in rASA that is at least about 95%, at least about 97%, at least about 98%, at least about 99% or more pure as measured by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and / or reverse phase-high performance liquid chromatography (RP-HPLC). In some embodiments, arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. The purity of arylsulfatase A can be measured, for example, by one or more of host cell protein (HCP) Western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse phase HPLC, and size exclusion HPLC. As described below, the specific activity of rASA is at least about 60 U / mg to about 100 U / mg, e.g., at least about 65 U / mg, at least about 90 U / mg, at least about 70 U / mg, or at least about 90 U / mg, as measured, for example, by a sulfatase release assay.

[0195] In some embodiments, arylsulfatase A is purified by tangential flow ultrafiltration in an acidic environment, by separation from contaminants according to their size. At low pH, arylsulfatase A forms an octamer with a theoretical molecular weight of 480 kDa, and therefore most of the contaminants will pass through a relatively wide membrane, while being retained by the membrane (Sommerlade et al., (1994) Biochem. J., 297; 123-130; Schmidt et al., (1995) Cell, 82 271-278; Lukatela et al., (1998) Biochemistry, 37, 3654-3664).

[0196] In an exemplary embodiment, the diafiltration buffer comprises 0.01 M sodium phosphate-citrate, 0.137 M NaCl, pH 6.0.

[0197] In some embodiments, the starting material for this process is a suspension of arylsulfatase A, as eluted from the chromatography column in the preceding step of the process, in which the pH is adjusted to 4-5 by the addition of 0.2-1 M sodium acetate, pH 4.5. Diafiltration is then carried out against 1-10 buffer volumes of sodium acetate, pH 4.0-5.5, in a manner well known to those skilled in the art. Filtration can be carried out by application of several different filter types with nominal weight cut-off values ​​in the range of 20-450 kDa, but typically filters with cut-off values ​​in the range of 100-300 kDa are used. For further processing of the solution containing arylsulfatase A, the pH is adjusted to a value in the range of 7-8 by the addition of a Tris system to a final concentration of about 20-50 mM.

[0198] As an alternative to acidic tangential flow ultrafiltration as described above, separation of ASA from contaminants can be obtained by acidic gel filtration using essentially the same conditions and composition of buffers. Filtration is performed at low pH through a gel filtration column equilibrated with a solution at low pH, for example a 0.2-0.9 M sodium acetate solution at pH 4-5. Alternatively, the solution of arylsulfatase A can be concentrated by tangential flow ultrafiltration through a 20-50 kDa filter prior to gel filtration. The concentration range can vary widely such that arylsulfatase A can be concentrated from about 0.1 mg / ml to about 50 mg / ml, preferably up to about 5 mg / ml.

[0199] In some embodiments, the sample pool is concentrated against a Biomax A-screen, 30 kDa. Diafiltration is performed against 3-5 column washes of 20 mM sodium acetate at pH 5.4-5.7.

[0200] Characterization of purified ASA protein The purified recombinant ASA protein can be characterized using a variety of methods.

[0201] purity The purity of the purified recombinant ASA protein is typically measured by the levels of various impurities (e.g., host cell proteins or host cell DNA) present in the final product. For example, the levels of host cell proteins (HCPs) can be measured by ELISA or SDS-PAGE. In some embodiments, the purified recombinant ASA protein contains less than 150 ng of HCPs / mg ASA protein (e.g., less than 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 30, 20, 10 ng of HCPs / mg ASA protein). In some embodiments, the purified recombinant ASA protein contains less than about 150 pg / mg, 140 pg / mg, 130 pg / mg, 120 pg / mg, 110 pg / mg, 100 pg / mg, 90 pg / mg, 80 pg / mg, 70 pg / mg, 60 pg / mg, 50 pg / mg, 40 pg / mg, 30 pg / mg, 20 pg / mg, or 10 pg / mg of host cell DNA.

[0202] In some embodiments, the purified recombinant ASA protein, when subjected to SDS-PAGE using Coomassie Brilliant Blue staining, has no new bands with intensities greater than 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% of the assay control. In some embodiments, the purified recombinant ASA protein, when subjected to SDS-PAGE using Western blot for HCP, has no bands with intensities greater than the assay control for the 15 kDa HCP band, and has no new bands with intensities greater than 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or 1.0% of the assay control. In some embodiments, the purified recombinant ASA protein has no new bands with intensities greater than 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% of the assay controls when subjected to SDS-PAGE with silver staining. In some embodiments, the log reduction value (LRV) of the host cell protein (HCP) is about 0.3 to about 0.6, e.g., about 0.4 to 0.5. A variety of assay controls can be used, particularly those acceptable to regulatory agencies such as the FDA.

[0203] The purity of the purified recombinant ASA protein can also be measured by one or more of size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), capillary electrophoresis-SDS PAGE (CE-SDS PAGE), and / or reversed phase-high performance liquid chromatography (RP-HPLC) (e.g., using a column of octadecyl (C18) bonded silica at acidic pH with TFA as counterion). In some embodiments of the invention, the main peak in the chromatogram is ASA. Parameters that can be varied or optimized to increase resolution include gradient conditions, organic modifiers, counterions, temperature, column pore size and particle size, solvent composition, and flow rate. Purity levels can be identified by the main peak percentage, as known to those skilled in the art. For example, purity can be measured by integrating the observed main and minor peaks and calculating the percentage of the main peak of the total area. In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein, as measured by the percentage of the main peak by SEC-HPLC, is 95% or greater (e.g., about 96%, about 97%, about 98%, about 99% or greater). In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein, as measured by the percentage of the main peak by RP-HPLC, is 98% or greater (i.e., about 98%, about 99% or greater).

[0204] specific activity The purified recombinant ASA protein may also be characterized by assessing its functional and / or biological activity. The enzymatic activity of the recombinant ASA composition may be measured using methods known in the art. Typically, the method involves detecting the removal of sulfate from a synthetic substrate, known as a sulfate release assay. One example of an enzymatic activity assay involves the use of ion chromatography. This method quantifies the amount of sulfate ions enzymatically released by the recombinant ASA from the substrate. The substrate may be a natural or synthetic substrate. In some cases, the substrate is a heparin substrate, a dermatan substrate, or a functional equivalent thereof. Typically, the released sulfate ions are analyzed by ion chromatography using a conductivity detector. In this example, the results may be expressed as U / mg protein, with one unit being defined as the amount of enzyme required to release one μmole of sulfate ions from the substrate per hour. In some embodiments, the purified recombinant ASA has a specific activity of at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg. In some embodiments, the purified recombinant ASA is about 50-200 U / mg (e.g., about 50-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-200 U / mg, 60-190 U / mg, 60-180 U / mg, 60-170 U / mg, 60-160 U / mg, 60-150 U / mg, 60-140 U / mg, 50-150 U / mg, 60-160 U / mg, 60-170 U / mg, 60-180 U / mg, 60-190 U / mg, 60-180 U / mg, 60-170 U / mg, 60-160 U / mg, 60-150 U / mg, 60-140 U / mg, 60-150 U / mg, 60-160 U / mg, 60-170 U / mg, 60-180 U / mg, 60-190 U / mg, 60-190 U / mg, 60-180 U / mg, 60-170 U / mg, 60-160 U / mg, 60-150 U / mg, 60-140 U / mg, 60-150 U / mg, 60-160 U / mg, 60-170 U / mg, 60-180 U / mg, 60-180 U / mg, 60-190 U / mg 0U / mg, 60~130U / mg, 60~120U / mg, 60~110U / mg, 60~100U / mg, 70~200U / mg, 70~190U / mg, 70~180U / mg, 70~170U / mg, 70~160U / mg, 70~150U / mg mg, 70~140U / mg, 70~130U / mg, 70~120U / mg, 70~110U / mg, 70~100U / mg, 80~200U / mg, 80~190U / mg, 80~180U / mg, 80~170U / mg, 80~160U / mg,80~150U / mg, 80~140U / mg, 80~130U / mg, 80~120U / mg, 80~110U / mg, 80~100U / mg, 90~200U / mg , 90~190U / mg, 90~180U / mg, 90~170U / mg, 90~160U / mg, 90~150U / mg, 90~140U / mg, 90~130U / m g, 90~120U / mg, 90~110U / mg, 90~100U / mg, 100~200U / mg, 100~190U / mg, 100~180U / mg, 100~1 70U / mg, 100~160U / mg, 100~150U / mg, 100~140U / mg, 100~130U / mg, 100~120U / mg, 100~110U / m g, 110~200U / mg, 110~190U / mg, 110~180U / mg, 110~170U / mg, 110~160U / mg, 110~150U / mg, 11 0~140U / mg, 110~130U / mg, 110~120U / mg, 120~200U / mg, 120~190U / mg, 120~180U / mg, 120~17 0 U / mg, 120-160 U / mg, 120-150 U / mg, 120-140 U / mg, 120-130 U / mg, 130-200 U / mg, 130-190 U / mg, 130-180 U / mg, 130-170 U / mg, 130-160 U / mg, 130-150 U / mg, or 130-140 U / mg).

[0205] In another example, the enzyme activity of the recombinant ASA composition can be measured by measuring the removal of sulfate from 4-methylumbelliferyl-sulfate (4-MUF-sulfate) substrate to form fluorescent methylumbelliferone. In this example, the fluorescent signal generated by the test sample can be used to calculate the enzyme activity (mU / mL) using a standard of 4-MUF. One milliunit of activity is defined as the amount of enzyme required to convert one nanomole of 4-MUF-sulfate to 4-MUF in one minute at 37° C. The specific activity can then be calculated by dividing the enzyme activity by the protein concentration.

[0206] In some embodiments, activity is measured by hydrolysis of paranitrocatechol sulfate (pNCS), a synthetic chromogenic substrate with the end product paranitrocatechol (pNC) absorbing light at 515 nm. The following equation can be used to calculate the enzyme activity in μmol pNCS hydrolyzed / min×ml (=Units / ml): Vtot(ml) ×ΔA=Units / ml(1) εM / 1000 × Vsample (ml) × incubation time (min) where: ΔA = absorbance of sample - absorbance of blank Vtot(ml) = total reaction volume (ml) (in this case 0.15 ml) V sample (ml) = added sample volume (ml) (in this case 0.05 ml) εM = molar extinction coefficient for the product pNC, in this case 12 400 M-1 cm-1. Equation 1 is: ΔA×(0.15 / (12 400 / 1000×0.05×30))= It can be simplified as X μmol / (min × ml) (= Units / ml) (1). To calculate the specific activity in μmol pNC consumed / (min×mg) (=Units / mg), Equation 1 is divided by the protein concentration of the sample: Equation 1 / Protein concentration (mg / ml) = Yμmol / (min x mg) = Units / mg(2)

[0207] In any example, the protein concentration of the recombinant ASA composition can be measured by any suitable method known in the art for measuring protein concentration. In some cases, the protein concentration is measured by ultraviolet absorption assay. Such absorption assays are typically performed at a wavelength of about 280 nm (A280).

[0208] In some embodiments, the purified recombinant ASA is about 1.0×10 3 mU / mg~100.0×10 3 mU / mg, approximately 1.0×10 3mU / mg~50.0×10 3 mU / mg, approximately 1.0×10 3 mU / mg~40.0×10 3 mU / mg, approximately 1.0×10 3 mU / mg~30.0×10 3 mU / mg, approximately 1.0×10 3 mU / mg~20.0×10 3 mU / mg, approximately 1.0×10 3 mU / mg~10.0×10 3 mU / mg, approximately 4.0 × 10 3 mU / mg~8.0×10 3 mU / mg, approximately 4.0 × 10 3 mU / mg~10.0×10 3 mU / mg, approximately 4.5 × 10 3 mU / mg~10.0×10 3 mU / mg, approximately 5.0 × 10 3 mU / mg~10.0×10 3 mU / mg, approximately 5.5 × 10 3 mU / mg~15.0×10 3 mU / mg, or approximately 4.0 × 10 3 mU / mg~20.0×10 3 In some embodiments, the purified recombinant ASA has a specific activity on the 4-methylumbelliferone substrate in the range of about 1.0×10 3 mU / mg, approximately 2.0×10 3 mU / mg, approximately 3.0 × 10 3 mU / mg, approximately 4.0 × 10 3 mU / mg, approximately 5.0 × 10 3 mU / mg, approximately 10.0 × 10 3 mU / mg, approximately 15.0 × 10 3 mU / mg, approximately 20.0 × 10 3 mU / mg, approximately 25.0 × 10 3 mU / mg, approximately 30.0 × 10 3 mU / mg, approximately 35.0 × 10 3 mU / mg, approximately 40.0 × 10 3 mU / mg, approximately 45.0 × 10 3 mU / mg, approximately 50.0 × 10 3It has a specific activity of mU / mg or more of 4-methylumbelliferone substrate.

[0209] Charge Profile The purified recombinant ASA can be characterized by the charge profile associated with the protein. Typically, the charge profile of a protein typically reflects the side chain charge pattern of residues present on the surface of the protein. The charge profile can be determined by performing an ion exchange (IEX) chromatography (e.g., HPLC) assay on the protein. In some embodiments, "charge profile" refers to a set of values ​​that indicate the amount of protein that elutes from an ion exchange column at a time point after loading the column with a mobile phase containing the exchange ions.

[0210] Typically, a suitable ion exchange column is an anion exchange column. For example, the charge profile can be determined by strong anion exchange (SAX) chromatography using a high performance liquid chromatography (HPLC) system. In general, recombinant ASA is adsorbed to an immobilized positively charged strong anion exchange column, and a gradient of increasing ionic strength using a mobile phase at a defined flow rate elutes the recombinant ASA species from the column in proportion to the strength of their ionic interaction with the positively charged column. More negatively charged (more acidic) ASA species elute later than less negatively charged (less acidic) ASA species. Protein concentration in the eluate is detected by ultraviolet absorption (at 280 nm).

[0211] In some embodiments, recombinant ASA is adsorbed onto an immobilized positively charged Mini Q PE column in 20 mM TRIS-HCl at about pH 8.0, and a gradient of increasing ionic strength using a mobile phase consisting of 20 mM TRIS-HCL, 1 M sodium chloride, pH 8.0 at a flow rate of 0.8 ml / min elutes recombinant ASA species from the column in proportion to the strength of their ionic interaction with the positively charged column.

[0212] In some embodiments, the charge profile may be represented by a chromatogram of absorbance units versus time following elution from an HPLC column. The chromatogram may include a series of one or more peaks, each peak in the set identifying a subpopulation of recombinant ASA of the composition having a similar surface charge.

[0213] Glycan mapping In some embodiments, purified recombinant ASA protein can be characterized by its proteoglycan composition, typically referred to as glycan mapping. Without wishing to be bound by any theory, it is believed that the shape and complexity of the branched structure can affect in vivo clearance, lysosomal targeting, bioavailability, and / or efficacy.

[0214] Typically, the glycan map can be determined by enzymatic digestion and subsequent chromatographic analysis. A variety of enzymes can be used for enzymatic digestion, including, but not limited to, suitable glycosylases, peptidases (e.g., endopeptidases, exopeptidases), proteases, and phosphatases. In some embodiments, a suitable enzyme is alkaline phosphatase. In some embodiments, a suitable enzyme is neuraminidase. Glycans (e.g., phosphoglycans) can be detected by chromatographic analysis. For example, phosphoglycans can be detected by high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) or size exclusion high performance liquid chromatography (HPLC). The amount of glycan (e.g., phosphoglycan) represented by each peak on the glycan map can be calculated using a standard curve of glycan (e.g., phosphoglycan) according to methods known in the art and disclosed herein.

[0215] In some embodiments, purified recombinant ASA proteins according to the invention are characterized using a glycan map comprising at least seven peak groups, each representing neutral (Peak Group 1), monosialylated (Peak Group 2), capped mannose-6-phosphorylated (Peak Group 3), disialylated (Peak Group 4), mono-mannose-6-phosphorylated (Peak Group 5), hybrid (Peak Group 6), and di-mannose-6-phosphorylated (Peak Group 7) ASA proteins.

[0216] Peptide mapping In some embodiments, peptide mapping can be used to characterize amino acid composition, post-translational modifications, and / or cellular processing, such as signal peptide cleavage and / or glycosylation. Typically, recombinant proteins can be decomposed into individual peptide fragments through controlled or random cleavage to generate a pattern or peptide map. In some cases, purified ASA protein can be first subjected to enzymatic digestion before analytical analysis. Digestion can be performed using peptidases, glycoside hydrolases, phosphatases, lipases, or proteases, and / or combinations thereof, before analytical analysis. The structural composition of peptides can be determined using methods well known in the art. Exemplary methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR), or HPLC.

[0217] metal analysis In some embodiments, the purified recombinant ASA protein may be characterized by metal analysis. A variety of methods for analyzing trace metals in purified drug substances are known in the art and can be used to practice the present invention.

[0218] In some embodiments, the residual phosphoric acid is measured and compared to a reference sample. Without wishing to be bound by any theory, it is hypothesized that the residual phosphoric acid contributes to maintaining the pH of the drug substance. In some embodiments of the present invention, the residual phosphoric acid is about 10-50 ppm (i.e., about 10-45 ppm, about 10-40 ppm, about 10-30 ppm, about 20-50 ppm, about 20-45 ppm, about 20-40 ppm, about 20-30 ppm, about 30-50 ppm, about 30-40 ppm). In some embodiments, the pH range of recombinant ASA purified according to the methods disclosed herein is about 5 to 7 (i.e., about 5.5 to 7.0, about 5.5 to 6.5, about 5.5 to 6.0, about 6.0 to 7.0, about 6.0 to 6.5, about 6.0 to 6.4, about 6.0 to 6.3, about 6.0 to 6.2, about 6.0 to 6.1, about 6.1 to 6.2).

[0219] In some embodiments, the recombinant ASA purified according to the methods disclosed herein contains calcium. Without wishing to be bound by any theory, it is hypothesized that calcium ions present in the active site of ASA may be essential for enzymatic activity. In some embodiments of the present invention, calcium is present at a level of about 1-20 ppm (i.e., about 1-15 ppm, about 1-10 ppm, about 5-15 ppm, about 5-10 ppm, about 10-20 ppm, about 10-15 ppm, about 10-14 ppm, about 10-13 ppm, about 10-12 ppm).

[0220] Pharmaceutical Compositions and Administration The purified recombinant ASA protein can be administered to a patient with MLD according to known methods. For example, the purified recombinant ASA protein can be delivered intravenously, subcutaneously, intramuscularly, parenterally, transdermally, or mucosally (e.g., orally or nasally).

[0221] In some embodiments, the recombinant ASA or a pharmaceutical composition comprising same is administered to the subject by intravenous administration.

[0222] In some embodiments, recombinant ASA or a pharmaceutical composition comprising it is administered to a subject by intrathecal administration. As used herein, the term "intracereal administration" or "thecal injection" refers to injection into the spinal canal (the intrathecal cavity surrounding the spinal cord). Various techniques can be used, including but not limited to lateral ventricular injection through a burr hole or intracistern or lumbar puncture, etc. In some embodiments, "intracereal administration" or "intracereal delivery" according to the present invention refers to IT administration or delivery via the lumbar region or area, i.e., lumbar IT administration or delivery. As used herein, the term "lumbar region" or "lumbar region" refers to the region between the third and fourth lumbar (lower back) vertebrae, as well as more generally, the L2-S1 region of the spine. In some embodiments, recombinant ASA or a pharmaceutical composition comprising same is administered to a subject by intrathecal administration, as described in PCT International Publication Nos. WO2011 / 163648 and WO2011 / 163650, the entireties of which are incorporated herein by reference.

[0223] In some embodiments, recombinant ASA or pharmaceutical compositions comprising it are administered to a subject by subcutaneous (i.e., under the skin) administration.For such purpose, the formulation can be injected using a syringe.However, other devices for administering the formulation are available, such as injection devices (e.g., Inject-ease and Genject devices), pen-type injectors (e.g., GenPen), needleless devices (e.g., MediJector and BioJector), and subcutaneous patch delivery systems.

[0224] In some embodiments, intrathecal administration can be used in conjunction with other routes of administration, such as intravenous, subcutaneous, intramuscular, parenteral, transdermal, or transmucosal (eg, oral or nasal).

[0225] The present invention contemplates single and multiple administration of therapeutically effective amount of recombinant ASA or pharmaceutical composition comprising it as described herein.Recombinant ASA or pharmaceutical composition comprising it can be administered at regular intervals depending on the nature, severity and extent of the condition (e.g., lysosomal storage disease) of the subject.In some embodiments, therapeutically effective amount of recombinant ASA or pharmaceutical composition comprising it can be administered periodically at regular intervals (e.g., once a year, once every 6 months, once every 5 months, once every 3 months, every other month (once every 2 months), monthly (once a month), every other week (once every 2 weeks), weekly, daily, or continuously).

[0226] Recombinant ASA or pharmaceutical compositions containing it can be formulated with physiologically active acceptable carriers or excipients to prepare pharmaceutical compositions.Carriers and therapeutic agents can be sterile.Formulations must be suitable for the method of administration.

[0227] Suitable pharma- ceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose, or starch, sugars such as mannitol, sucrose, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oils, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, and combinations thereof. Pharmaceutical preparations can be mixed, if desired, with auxiliary substances (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors, and / or aromatic substances, and the like, which do not adversely react with the active compositions or interfere with their activity. In some embodiments, water-soluble carriers suitable for intravenous administration are used.

[0228] The composition or medicament may also contain a small amount of wetting agent or emulsifier, or pH buffering agent if desired.The composition may be liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.The composition may also be formulated as suppository with conventional binder and carrier such as triglyceride.Oral formulation may contain standard carrier such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0229] The composition or medicament can be formulated according to normal procedures as a pharmaceutical composition adapted for administration to humans. For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer. If necessary, the composition can also include a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of active agent. If the composition is administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0230] In some embodiments, arylsulfatase A is formulated in an isotonic solution such as 154 mM NaCl, or 0.9% NaCl, and 10-50 mM sodium phosphate pH 6.5-8.0, or sodium phosphate, glycine, mannitol, or the corresponding potassium salts. a) Formulation Buffer I containing (in mM): Na2HPO4 (3.50-3.90), NaH2PO4 (0-0.5), glycine (25-30), mannitol (230-270), and water for injection; or b) Formulated in a physiological buffer such as Formulation Buffer II containing (in mM): Tris-HCl (10), glycine (25-30), mannitol (230-270), and water for injection.

[0231] The arylsulfatase A purified by the method herein can be used as a medicine to reduce the level of sphingolipid 3-O-sulfogalactosylceramide (galactosylsulfatide) in cells in the peripheral nervous system and / or in the central nervous system in subjects suffering from and / or diagnosed with metachromatic leukodystrophy. Administration of ASA leads to impaired motor learning and / or increased neuromotor conduction velocity and / or neuroconduction amplitude. As used herein, the term "therapeutically effective amount" is determined primarily based on the total amount of therapeutic agent contained in the pharmaceutical composition of the present invention. In general, a therapeutically effective amount is sufficient to achieve a significant benefit to a subject (e.g., treat, regulate, cure, prevent, and / or ameliorate an underlying disease or condition). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, such as an amount sufficient to modulate lysosomal enzyme receptors or their activity, thereby treating such lysosomal storage disease or its symptoms (e.g., reducing or eliminating the presence or occurrence of "zebra bodies" or cellular vacuolar degeneration after administering the composition of the present invention to a subject). In general, the amount of therapeutic agent (e.g., recombinant lysosomal enzyme) administered to a subject in need thereof will vary depending on the subject's characteristics. Such characteristics include the subject's condition, disease severity, general health, age, sex, and weight. Those skilled in the art can easily determine the appropriate dosage depending on these and other related factors. In addition, both objective and subjective assays can be used, as appropriate, to identify optimal dosage ranges.

[0232] The therapeutically effective amount is generally administered in a dosage regimen that may include multiple unit doses.For any particular therapeutic protein, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosage regimen) may vary depending on the route of administration in combination with other pharmaceutical agents.The specific therapeutically effective amount (and / or unit dose) for any particular patient may also vary depending on a variety of factors, including the disease to be treated, the severity of the disease, the activity of the specific pharmaceutical agent used, the specific composition used, the age, weight, general health, sex, and diet of the patient, the administration time, administration route, and / or excretion or metabolic rate of the specific fusion protein used, the duration of treatment, and factors as are well known in the medical field.

[0233] It is further understood that for any particular subject, specific dosage regimens will be adjusted over time according to the individual needs and the professional judgment of the person administering or directing the administration of enzyme replacement therapy, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the invention. EXAMPLES

[0234] Example 1. Tangential Flow Ultrafiltration - Capture This example demonstrates that tangential flow ultrafiltration (also known as cross-flow filtration) can be used to capture recombinant ASA protein directly from the production bioreactor.

[0235] Specifically, raw bulk material was captured from a production-rich bioreactor by tangential flow ultrafiltration, which was performed on a Sartorius Hydrosart® 30 kD standard membrane within the manufacturer's specifications.

[0236] Further experiments were performed in which tangential flow filtration was performed using Sartorius HYDROSART® ECO membranes while maintaining the same composition and molecular weight cutoff. The ECO membranes allowed the process to use lower cross flow rates.

[0237] The permeate flux values ​​(LMH) obtained from 12 excursion experimental conditions performed with ECO and standard membranes were analyzed using the contour profile function. This function showed the response contour to two factors simultaneously. In this study, the feed flow rate and transmembrane pressure ("TMP") were compared with the permeate flux for each membrane. Exemplary results are shown in Figure 3.

[0238] As seen in the contour plots of the ECO vs. standard membrane excursion experiments, the ECO membrane demonstrated a much more robust flux output over the given parameter range tested: the ECO membrane was able to achieve higher flux rates at lower feed flow rates within the operating range.

[0239] More specifically, based on data analysis from design experiments, titer ELISA recoveries range from about 92% to 132%. Host cell protein (HCP) log reduction values ​​range from about 0.11 to 0.38. Titer ELISA recoveries, host cell protein (HCP) log reduction values ​​(LRVs), and percent activity recoveries were determined as described elsewhere in this application.

[0240] Results from this experiment demonstrated the feasibility of implementing a UFDF capture step in a stage III purification process of arylsulfatase A. Based on comparative UFDF excursion experiments, the ECO membrane was particularly effective as it was able to achieve higher flux rates at lower feed flow rates within the operating range. Design experiments were also performed with the ECO membrane to evaluate the potential effects of the operating parameters of loading challenge, TMP, feed flow rate, initial concentration factor, and processing temperature on the performance of the rhASA capture process. The yields of host cell protein (HCP) ELISA and activity steps were not significantly affected by the operating parameters of the ECO membrane.

[0241] Example 2. Depth filtration and viral inactivation This experiment demonstrates the conditions for depth filtration and viral inactivation that can be implemented during the purification process.

[0242] For example, the captured raw bulk material was thawed at 2-8°C for no more than 120 hours. The temperature of the bulk material was adjusted to approximately 18±2°C and filtered through a Cuno Zeta Plus depth filter followed by polishing filtration. A Maxicap Sartopore 2, 0.45±0.2 μm polishing filtration capsule was connected downstream of the depth filter to remove particulates and reduce bioburden in preparation for downstream treatment processes. The filter is flushed with Water for Injection ("WFI") followed by a MES-Tris, pH 7.1 equilibration solution.

[0243] After filtration, a final rinse was performed and the hold-up material was collected from the filtration train. Once filtration was complete, the filter train was flushed with a wash of 0.05 MES-Tris, pH 7.0. The collected wash was filtered and added to the pooled bulk material.

[0244] The filtered but unpurified bulk was then processed through a viral inactivation step by the addition of Polysorbate 80 and tri-n-butyl-phosphate (TnBP) followed by incubation for approximately 3-24 hours as follows: Polysorbate 80 was added to the filtered and pooled unpurified bulk from a stock solution of 10% (v / v) in 0.05 M MES-Tris, pH 7.1 to achieve a final concentration of 1%. Tri-n-butyl-phosphate (TnBP) was then added to a final concentration of approximately 0.3% calculated based on the new volume of unpurified bulk containing Polysorbate 80. The resulting pool was mixed to obtain uniformity and incubated with continuous mixing for approximately 3-24 hours.

[0245] When viral inactivation was complete, the pool was processed through a polyvinylidine difluoride (PVDF) 0.2 μm filter before being loaded onto the first chromatography column. In this experiment, the filtered viral inactivation pool was applied to a Q column (as described below) within approximately 28 hours of the start of the viral inactivation incubation. The viral inactivated intermediate was filtered, and at the end of the filtration, the filter was flushed with 0.05 M MES-Tris, pH 7.0 buffer to maximize product recovery.

[0246] Example 3. Anion exchange chromatography This example shows exemplary conditions for anion exchange chromatography, specifically when anion exchange chromatography is used as the first chromatographic step. In this particular example, Q SEPHAROSE™ Fast Flow (Q FF) resin was used in a quaternary amine anion exchanger operated in bind and elute mode.

[0247] A Q FF column was prepared using standard methods. ASA was bound to the Q column at pH 7.0 and eluted with 0.02 M MES-Tris, 0.26 M NaCl at pH 7.1. Loading, washing, and elution were performed at a flow rate of approximately 100 nm. Operating temperatures ranged from 16-23 °C. Q elution was collected by absorbance at 280 nm (A280) with set points of 0.5 AU / cm at the peak of the ascending surface to 0.25 AU / cm at the peak of the descending surface. Exemplary conditions used for the anion exchange column are shown in Table 2. [Table 2]

[0248] Further experiments were performed to compare the exemplary anion exchange resins for purity, activity, and throughput (e.g., target loading capacity). In particular, to facilitate scale-up, several high capacity anion exchange resins were screened and evaluated in this study.

[0249] Once "loaded", the resin was equilibrated, loaded, washed, and eluted using the conditions shown in Table 3. In this study, the binding conditions were kept the same as for the Q FF process described above. Flow-through (FT), wash (W), and elution / strip (EL / strip) fractions were collected and analyzed using A280 absorbance, titer assay, and SDS-PAGE (Coomassie and silver staining). [Table 3]

[0250] Seven resins in addition to Q FF were evaluated to determine binding capacity, selectivity, and recovery. Overall yields were comparable for all resins, ranging from 80-83%, indicating similar recovery and consistent experimental behavior. Some resins had less binding capacity than Q FF, while others had higher binding capacity. In particular, Fractogel showed the highest binding capacity, best selectivity, and comparable recovery of all of the resins tested. Additionally, only slight product loss was observed in the flow-through for Fractogel at a loading of 15 g / L.

[0251] Exemplary results showing the yield and binding capacity of Q FF and Fractogel resins are shown in Table 4. [Table 4]

[0252] Comparative confirmation experiment To validate the Fractogel process and evaluate its impact on subsequent purification steps and the quality of the final drug substance ("DS"), a comparison of Fractogel TMAE vs. Q FF purification process was performed from unpurified bulk material to drug substance, which includes the chromatography steps described in the Examples of the present invention. Further purification steps after anion exchange chromatography were as follows (shown in order of performance): HA column mixed mode chromatography, phenyl column hydrophobic interaction chromatography, SP column cation exchange chromatography, and two steps of UF / DF with an intervening viral filtration step.

[0253] Both the A280 and titer yields were comparable. HCP reduction was also similar across columns.

[0254] Furthermore, SDS-PAGE (silver) gels (Figure 4) showed similar band profiles for both Fractogel and Q FF throughout the entire process. CE-SDS data showed better purity for Fractogel than Q FF as the first step, but did not show similar results after the phenyl column step. This was confirmed by the HCP / rhASA ratio.

[0255] Quality attributes were evaluated for the SP eluates from both columns, as well as the control run SP eluate. The Fractogel and Q FF processes provided similar pure and acceptable SP eluates, although certain attributes differed slightly from the control due to differences in the crude load starting material.

[0256] Overall, the use of Fractogel as the first column provided similar process performance (yield, reduction in HCP) and quality characteristics of SP as the control process with Q FF, as confirmed by the results above. However, the Fractogel column provided a higher loading capacity and increased throughput.

[0257] In conclusion, the comparative validation runs showed that the Fractogel TMAE process demonstrated comparable performance (yield, HCP removal) to the Q FF process, but offered the added benefit of a higher target loading capacity (e.g., at least 10 g rhASA / L). No significant yield loss was observed for the characterized loading range of 5-20 g / L. Quality attributes of the SP eluate from the Fractogel runs were also comparable to the control Q FF runs.

[0258] Example 4. Mixed-mode hydroxyapatite (HA) chromatography This example provides exemplary conditions for mixed-mode hydroxyapatite chromatography that can be used in the purification of recombinant ASA protein.

[0259] Specifically, the anion exchange chromatography eluate prepared as described above was then purified by hydroxyapatite (HA) chromatography. A commercially available ceramic hydroxyapatite type 1 resin column was prepared. In this example, the column was pre-equilibrated with 0.250 M sodium phosphate buffer, pH 7.0.

[0260] The HA column was operated in bind and elute mode. The anion exchange eluate was first warmed to ambient temperature and adjusted to about 0.001 M sodium phosphate before loading onto the HA column. rhASA was bound to the column at pH 7.0 and eluted from the column at about 0.04 M sodium phosphate, pH 7.1. Loading, washing, and elution were performed at a flow rate of about 150 cm / hr. HA elution was collected by absorbance at 280 nm (A280) set points of 0.5 AU / cm at the peak of the ascending surface to 0.25 AU / cm at the peak of the descending surface. Exemplary conditions used for the HA column are shown in Table 5. [Table 5]

[0261] Example 5. Hydrophobic interaction (phenyl) chromatography This example provides exemplary conditions for hydrophobic interaction (phenyl) chromatography that can be used in the purification of recombinant ASA protein.

[0262] Specifically, the eluate from the HA column chromatography prepared as described above was then purified by phenyl column chromatography. A commercially available phenyl Sepharose Fast Flow column was packed and prepared for the purification of rhASA.

[0263] The phenyl column was operated in bind and elute mode. After warming to ambient temperature, the HA eluate was adjusted to about 1.1 M NaCl and pH about 5.6 with 0.5 M MES, free acid before loading onto the phenyl column. rhASA was bound to the column at pH 5.6 and eluted from the column with 0.02 M MES-Tris, 0.06 M NaCl, pH 7.1. Loading, washing, and elution were performed at a flow rate of about 150 cm / hr. The phenyl elution was collected by absorbance at 280 nm (A280) set points of 0.5 AU / cm at the peak of the ascending surface to 0.25 AU / cm at the peak of the descending surface. Exemplary conditions for the phenyl column are shown in Table 6. [Table 6]

[0264] Example 6. Cation exchange chromatography This example provides exemplary conditions for cation exchange chromatography that can be used in the purification of recombinant ASA protein.

[0265] Specifically, the eluate of the phenyl column chromatography prepared as described above was then purified by cation exchange chromatography. In this example, a SP-650M column (TOYOPEARL) was packed and prepared.

[0266] The SP column was operated in bind and elute mode. The phenyl column eluate was warmed to 16-23°C and then diluted with WFI to reduce conductivity and adjusted to pH 4.2 with approximately 1 M acetic acid before loading onto the SP column. rhASA was bound to the column at pH 4.2 and eluted from the column with 0.01 M sodium acetate, 0.01 M acetic acid, 0.05 M NaCl, pH 4.5. Loading, washing, and elution were performed at a flow rate of approximately 150 cm / hr. The SP elution was collected by absorbance at 280 nm (A280) set points of 0.25 AU / cm at the peak of the ascending surface to 0.25 AU / cm at the peak of the descending surface. Exemplary conditions for the SP column are shown in Table 7. [Table 7]

[0267] Example 7. Ultrafiltration / Diafiltration and Virofiltration This example provides exemplary conditions for ultrafiltration / diafiltration and viral filtration that can be used in the purification of recombinant ASA protein.

[0268] Ultrafiltration / Diafiltration1 The SP elution pool was concentrated and diafiltered using a 10 kDa Hydrosart Sartocon membrane. The product pool was concentrated to approximately 15 mg / mL and diafiltered using 6-8 volumes of 0.01 M sodium phosphate-citrate, 0.137 M NaCl, pH 6.0. The concentrated and diafiltered product pool was then processed through a 0.2 μm filter.

[0269] The concentrated / diafiltered pool was 0.2 μm filtered and typically stored at 2-8° C. for no more than approximately 24 hours.

[0270] Viral Filtration After UF / DF1, a viral filtration could be performed to remove any virus-like particles from the process stream. For example, a filtration train was set up with a pump, Planova filter, and pressure gauge. The filter had a flow rate of 11.0 lb / in2 At a target pressure of 1000 psi, the filter was flushed with 1 L of 0.154 M NaCl through the retentate side and then 3 L more of 0.154 M NaCl through the permeate side. Upon completion, the filter was flushed with 4 L of 0.154 M NaCl to increase product recovery. Gold particle testing was performed on the used filter to ensure that filter integrity was maintained.

[0271] Ultrafiltration / Diafiltration2 The virus filtered material was concentrated to 15-30 mg / ml and diafiltered using a 10 kDa Hydrosart Sartocon UFDF membrane with 6-8 diavolumes of 0.154 M NaCl in diafiltration buffer. Upon completion of diafiltration, the material was further concentrated to 48-50 mg / ml. The system was then flushed with diafiltration buffer. An appropriate amount of system flush was then added back to the product concentrate to achieve a target final concentration of approximately 40 mg / ml.

[0272] Example 8. Single UF / DF unit operation The process described in Example 7 involves ultrafiltration / diafiltration ("UFDF") of the SP column eluate (UFDF1), viral filtration of the UFDF1 concentrate, and final UFDF of the viral filtrate (UFDF2) to pre-produce the final drug substance (DS). An exemplary flow diagram of such a process is shown in FIG.

[0273] In this example, further experiments were performed to determine the feasibility of eliminating one of the UFDF unit operations to reduce overall process time, cost, and potentially increase process yield. Therefore, a study was performed to evaluate the feasibility of performing viral filtration of the pH adjusted SP eluate followed by one single UFDF unit operation to produce a pre-finally filtered drug substance ("DS").

[0274] As mentioned above, the study used a partially purified feedstock that was previously purified from unrefined bulk material ("UPB") through a SP eluate. As mentioned above, the SP eluate was used as the load for the three UFDF experiments conducted in the current study, including two feasibility experimental runs and one control run of a dual UFDF process. The two experimental (known as feasibility) runs consisted of one UFDF, followed by pH adjustment of the SP eluate pool, and one ultrafiltration / dual diafiltration ("UFDFDF"). The SP eluate for the feasibility run was pH adjusted to 6.0 and then filtered using a filtration system such as the Planova filtration system. Virus filtration was performed using a 20N virus filter. Experimental runs were compared to a control run of UFDF1-virus filtration-UFDF2 as described above.

[0275] The SP eluate was adjusted to pH 6.0, virus filtered, and allocated into two equal volumes, with each experimental feasibility run using one of the two virus filtrate pools. An exemplary SP eluate pool pH-adjusted buffer formulation was designed as described in Table 8. A volume / volume addition method of approximately 0.075 L of adjustment buffer / L of SP eluate was determined to achieve a target pH of approximately 6.0. [Table 8]

[0276] Upon receipt of the SP elution pool for an experimental run, the pH was adjusted to 6.0, which produced an SP eluate having a pH of 5.98. The pH-adjusted SP eluate was then virus filtered onto a Planova 20N filter.

[0277] Table 9 includes a summary of the yields of the individual unit operation steps. As observed, all UFDF runs showed similar recovery yields based on A280. The experimental virus filtration unit operation produced a step yield of 106.6%, indicating that no A280 absorbing proteins were lost during filtration. The yields for virus filtration were significant as pH adjustment to approximately 6.0 demonstrated that the 20 nm diameter rhASA octameric molecule could dissociate into a dimeric form. Under acidic conditions (pH 5.0), the rhASA molecule exists as an octamer, but at neutral pH, it was believed to dissociate into a dimer. Since the Planova 20N has a pore size of 20 nm, it was important that the molecular conformation of the rhASA protein was not in the octameric form, as octameric rhASA could potentially remain / be lost within the virus filter. The step yield data confirmed the feasibility of performing virus filtration prior to the UFDF run by adjusting the pH of the SP eluate. These yield data indicate that rhASA was not lost during viral filtration, and pH adjustment was a critical step in both the efficiency of purification and targeting the drug substance formulation pH. [Table 9]

[0278] Upon completion of viral filtration, the viral filtrate pool was split equally into two aliquots: one aliquot was fed to experimental run 1 (UFDFDF) and the other was fed to experimental run 2 (UFDF).

[0279] As evidenced below, both experimental runs produced similar average permeabilities during the UF and initial DF segments. Additionally, the second DF segment of experiment 1 showed similar average permeabilities for the UF and initial DF segments. Both control runs UFDF1 and UFDF2 produced consistently lower average permeabilities when compared to the feasibility runs, which may be due to variation in scale of the instruments used. [Table 10]

[0280] The product quality of the drug substance from the control and experimental runs was compared by a series of analytical assays including SEC-HPLC, RP-HPLC, activity, SDS-PAGE / HCP Western, SDS-PAGE (Coomassie), glycan mapping, peptide mapping, and metal analysis.

[0281] SEC-HPLC and RP-HPLC Table 11 provides the size exclusion (SEC-HPLC) and reverse phase (RP-HPLC) results. Both SEC and RP data showed little difference between the experimental and control run drug substance, indicating that both experimental runs produced drug substance with comparable SEC and RP major peak percentages to the drug substance from the control run. These data demonstrated the feasibility of using either experimental run in place of the dual UFDF control process, as no discernible difference in purity was identified. [Table 11]

[0282] Activity Table 12 details the activity (U / mL) and specific activity (U / mg), and the concentration (mg / mL) was determined by dividing the absorbance (AU) by the known extinction coefficient of 0.67. Experimental run 1 (UFDF) was observed to have produced the lowest specific activity value, but was well within the desired specification range of 50-140 U / mg. Experimental run 2 (UFDF) produced a specific activity comparable to the control run. All three runs, two experimental and one control, were comparable, indicating that the method for obtaining the formulated drug substance does not affect the drug substance activity. [Table 12]

[0283] Host Cell Protein (HCP) Western and SDS-PAGE (Coomassie) Figures 5 and 6 show gel images of SDS-PAGE / HCP Western blot and SDS-PAGE (Coomassie), respectively. As seen in Figure 5, the experimental (feasibility) and control run samples (lanes 5, 7, 9) showed similar band patterns as the reference standard (lane 3), indicating that drug substance from all runs showed relatively low levels of HCP. Figure 6 also showed the comparability between the experimental (feasibility) and control runs against the reference standard, indicating that no additional band patterns were detected by SDS-PAGE (Coomassie). These data indicated that the product quality of drug substance from the experimental run conditions was comparable to that from the control run based on SDS-PAGE / HCP Western blot and SDS-PAGE (Coomassie) analysis. No HCP bands higher than the intensity of the 15 kDa HCP band control were observed in the HCP Western blot, and no new bands were observed above the intensity of the 1% assay control.

[0284] Glycan mapping Table 13 shows an exemplary glycan map. All glycosylation forms were found to be present at similar levels across all samples tested. These results showed that the product quality of the drug substance from the experimental (feasibility) run was comparable to that of the drug substance from the control run. [Table 13]

[0285] Metal Analysis and pH Metal analysis in the resulting drug substance (DS) for the experimental and control was also performed to measure the level of residual phosphate. Table 14 shows the residual phosphate and final pH of each drug sample. Residual phosphate was found to be comparable across all run conditions. pH was also found to be similar regardless of run condition. Since residual phosphate was hypothesized to contribute to maintaining drug substance pH, the residual phosphate results were consistent with the observation that the pH of all three lots of drug substance was maintained. These data showed that both experimental (feasibility) runs produced comparable levels of residual phosphate in the drug substance as the control run, and that all three drug substance lots were able to maintain the target pH of approximately 6.0 (i.e., 5.5-6.5) in the final formulation.

[0286] Metal analysis also detected elevated levels of calcium, as shown in Table 14. Calcium levels were fairly constant across all samples. Previous studies have shown that calcium ions are present in the active site of rhASA, which is important for enzyme activity. [Table 14]

[0287] Peptide mapping Peptide mapping analysis was performed. Again, all samples yielded similar profiles, suggesting that the run conditions tested yielded comparable rhASA peptide maps as controls.

[0288] conclusion The above experiments evaluated the feasibility of virus filtration prior to UFDF operation by pH adjustment of the SP eluate, the feasibility of a single UFDF step instead of two UFDF steps, and the feasibility of a single diafiltration (vs. dual filtration) within the UFDF step after virus filtration.

[0289] The high step yields for the viral filtration unit operation in the experimental feasibility run demonstrated the feasibility of viral filtration prior to the UFDF run. Two UFDF feasibility runs were performed because the viral filtrate allowed for the transfer of rhASA molecules into the filtrate. A single UFDF step was determined to be feasible because all product quality and process performance metrics showed equivalence of both feasibility runs to the control. The results also demonstrated the feasibility of a single diafiltration (experimental feasibility run 2) directly into the final drug product solution (0.9% saline solution) because the process was able to produce drug substance of comparable quality to that produced by both the control and dual diafiltration experiments (experimental feasibility run 1).

[0290] Based on these data, it was found that pH adjustment to about 6.0 using, for example, the buffers contained in Table 4 produces a pH-adjusted SP eluate that is suitable for viral filtration prior to a final UFDF operation. This process, illustrated in Figure 2, is shown to be a viable replacement in processes that include a UFDF step, followed by viral filtration, followed by another UFDF step. The pH adjustment-viral filtration-UFDF process has been demonstrated to produce drug substance of equivalent quality and purity to that produced by more complex, expensive, and time-consuming processes.

[0291] Example 9. Drug Substance of Recombinant Human Arylsulfatase A (rhASA) rhASA purified according to the process described above was formulated at 35-45 mg / ml in 154 mM NaCl buffer, having a pH of approximately 5.5-6.5.

[0292] The peptide and glycan maps of the final drug substance were confirmed to be those described in Example 8. Residual host cell DNA was present in amounts less than or equal to 100 pg / mg as measured by the THRESHOLD® DNA assay. Purity as measured by reverse phase and size exclusion HPLC showed major peak areas greater than 98% and 95%, respectively. Western blot showed no host cell protein (HCP) bands with intensities greater than that of the approximately 15 kDa HCP band in the assay control. No more than three HCP bands were detected. SDS-PAGE (Coomassie, reduced) analysis confirmed the reference standard with no new bands with intensities greater than 1% of the assay control. Specific activity was measured to be 50-140 U / mg.

[0293] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above detailed description, but is as set forth in the following claims.

[0294] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, imply a priority, precedence, or order of one claim element over another, or a temporal order in which acts of a method are performed, but rather is merely used as a marker to distinguish one claim element having a particular name from another element having the same name (but for the use of ordinal terms) and to distinguish the claim elements.

[0295] In this application and claims, the articles "a" and "an" as used herein should be understood to include plural referents unless specifically and clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the members of the group are present, used, or otherwise relevant to a given product or process, unless clearly indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which only one member of a group is present, used, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one member of a group, or all members of a group are present, used, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the present invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims, which are introduced into another claim (or any other related claim) that relies on the same underlying claim, unless otherwise indicated to create a contradiction or inconsistency or is obvious to a person skilled in the art. When elements are presented in a list (e.g., in a Markush group or similar format), it is to be understood that each subgroup of those elements is also disclosed, and that any element(s) may be removed from the group. In general, when the invention or aspects of the invention are considered to include certain elements, features, etc., it should be understood that an embodiment of the invention or an aspect of the invention consists of, or consists essentially of, such elements, features, etc. For simplicity, those embodiments have not been specifically described in so many words in every instance herein. It should also be understood that any embodiment or aspect of the invention may be specifically excluded from the claims, regardless of whether a specific exclusion is recited herein.Publications, websites, and other references referenced herein to describe the background of the invention or to provide further details regarding the practice of the invention are hereby incorporated by reference.

Claims

1. 1. A method for purifying a recombinant arylsulfatase A (ASA) protein, comprising the following successive steps: (a) performing one or more viral inactivation steps; (b) purifying the recombinant arylsulfatase A (ASA) protein from impure preparations by performing one or more chromatography steps, including cation exchange chromatography; (c) pooling the eluates from one or more chromatography steps; (d) adjusting the pH of the pooled eluate to 5.8-6.4; and (e) subjecting the pH adjusted eluate to a single step of post-chromatographic ultrafiltration and / or diafiltration, thereby exchanging the purified recombinant ASA protein directly into a saline-based formulation.

2. 10. The method of claim 1, wherein the pH is adjusted to 6.

0.

3. 3. The method of claim 1 or 2, wherein the pH is adjusted with a buffer containing sodium phosphate, sodium chloride, and sodium citrate having a pH of 7.

0.

4. 4. The method of claim 3, wherein the buffer comprises 0.1-0.5 M sodium phosphate having a pH of 7.0, 0.5-2.5 M sodium chloride, and 0.1-0.6 M sodium citrate.

5. The method according to any one of claims 1 to 4, wherein the single step of ultrafiltration and / or diafiltration comprises only one diafiltration.

6. The method according to any one of claims 1 to 5, wherein the ultrafiltration is tangential flow ultrafiltration.

7. 7. The method of any one of claims 1 to 6, wherein the cation exchange chromatography is the last chromatography step and the eluates from the cation exchange chromatography are pooled before adjusting the pH.

8. 8. The method of claim 1, wherein one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed prior to performing the cation exchange chromatography.

9. 9. The method of claim 8, wherein the anion exchange chromatography uses a column selected from the group consisting of Q Sepharose™ Fast Flow, Q Sepharose™ High Performance, Q Sepharose™ XL, Capto™ Q, DEAE, TOYOPEARL GigaCap® Q, Fractogel® TMAE, Eshmuno™ Q, Nuvia™ Q, and UNOsphere™ Q.

10. 9. The method of claim 8, wherein the mixed mode chromatography is hydroxyapatite (HA) chromatography.

11. 9. The method of claim 8, wherein the hydrophobic interaction chromatography is phenyl chromatography.

12. 12. The method of any one of claims 1 to 11, wherein the ultrafiltration uses a membrane filter comprising a pore size with a molecular weight cut-off of at least 10 kDA.

13. 13. The method of claim 12, wherein the ultrafiltration comprises a polyethersulfone or cellulose membrane.

14. The method according to any one of claims 1 to 13, wherein the one or more viral inactivation steps are followed by one or more chromatography steps.

15. 15. The method of claim 14, wherein the step of viral inactivation comprises adding a detergent to the impure preparation.

16. The method of claim 15, wherein the impure preparation is prepared from serum-free medium containing recombinant ASA protein secreted from mammalian cells.

17. The method of any one of claims 1 to 16, wherein the recombinant ASA protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 (corresponding to the wild-type human ASA protein).

18. 18. The method of claim 17, wherein the recombinant ASA protein has an amino acid sequence identical to SEQ ID NO:

1.

19. The method of any one of claims 1 to 18, wherein the purified recombinant ASA protein contains less than 100 ng / mg HCP and / or less than 100 pg / mg host cell DNA.

20. 20. The method of claim 19, wherein the purified recombinant ASA protein contains less than 60 ng / mg HCP and / or less than 50 pg / mg host cell DNA.

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