WO2024176112A1

WO2024176112A1 - Large scale production of recombinant arylsulfatase a and compositions thereof

Info

Publication number: WO2024176112A1
Application number: PCT/IB2024/051618
Authority: WO
Inventors: Anna TCHOUDAKOVA; Andrew Keefe; Christopher Barton; Evan GRAESSLE; Wei Lu; Lan Yang; Brian Flaherty; Gang Yan
Original assignee: Takeda Pharmaceutical Co Ltd
Current assignee: Takeda Pharmaceutical Co Ltd
Priority date: 2023-02-21
Filing date: 2024-02-20
Publication date: 2024-08-29
Anticipated expiration: 2025-08-21

Abstract

The present invention provides, among other things, improved large-scale methods and compositions comprising recombinant arylsulfatase A (ASA) protein for enzyme replacement therapy, for example in treating Metachromatic Leukodystrophy Disease (MLD). In one aspect, the method of the present invention is for large-scale production of highly pure recombinant ASA with high cellular uptake, lysosomal targeting and bioactivity.

Description

LARGE SCALE PRODUCTION OF RECOMBINANT ARYLSULFATASE A AND

COMPOSITIONS THEREOF

BACKGROUND

[1] Metachromatic Leukodystrophy Disease (MLD) is an inherited, autosomal recessive disorder resulting from a deficiency of a lysosomal enzyme, Arylsulfatase A (ASA). ASA, which is encoded by the ARSA gene in humans, is an enzyme that breaks down ASA substrates including galactosylceramide-3-0 sulfate (cerebroside sulfate or sulfatide) into cerebroside and sulfate. In the absence of the enzyme, cells of the central and peripheral nervous system are unable to metabolize ASA substrates, and as a result, sulfatides accumulate in the nervous system (e.g., myelin sheaths, neurons and glial cells) and to a lesser extent in visceral organs. The consequence of these molecular and cellular events is progressive demyelination and axonal loss within the CNS and PNS, which is accompanied clinically by severe motor and cognitive dysfunction.

[2] Central nervous system (CNS) degeneration, which results in cognitive impairment (e.g., mental retardation, nervous disorders, and blindness, among others) is a clinical feature of MLD. MLD can manifest itself in young children (late-infantile form), where affected children typically begin showing symptoms just after the first year of life (e.g., at about 15-24 months), and generally do not survive past the age of 5 years. MLD can manifest itself in children (juvenile form), where affected children typically show cognitive impairment by about the age of 3-10 years, and life-span can vary (e.g., in the range of 10-15 years after onset of symptoms). MLD can manifest itself in adults (adult-onset form) and can appear in individuals of any age (e.g., typically at age 16 and later) and the progression of the disease can vary greatly.

[3] Enzyme replacement therapy (ERT) is a potential therapy for treating MLD, which involves administering exogenous replacement ASA enzyme, particularly recombinant Arylsulfatase A (rASA) (e.g., recombinant human Arylsulfatase A (rhASA)) to patients with MLD. SUMMARY OF THE INVENTION

[4] The present invention provides, among other things, compositions comprising purified recombinant ASA protein and large-scale methods of making recombinant ASA in high yields and with high purity and using recombinant ASA protein, for example, in enzyme replacement therapy, for treating diseases e.g., MLD.

[5] Due to increased clinical dose and frequency of enzyme replacement therapy, there is a need for a process of producing highly pure recombinant ASA at a high-yield for therapeutic use. The present invention provides, among other things, an improved process for producing recombinant ASA that results in higher yield and better product quality, for example, in a large-scale manufacturing process, while maintaining a stable product for longterm storage at or below -65°C and accelerated storage at 5±3° C or stress storage (25±2 °C), for example, storage in the presence of a surfactant or buffering agent.

[6] In some aspects, the present invention provides a large-scale production method producing a high yield of a highly pure recombinant ASA product. In some embodiments, the process utilizes CHO host cell lines (e.g. CHO cell line lacking glutamate synthase) and affinity resin purification. In some embodiments, the purified ASA utilizes human cell lines (e.g. human fibroblast cell line (e.g., HT-1080)). In some embodiments, the purified recombinant ASA features distinct glycosylation characteristics (e.g., that facilitate bioavailability, improved uptake, and/or improved efficacy of the recombinant ASA protein). In some embodiments, the recombinant ASA is produced from CHO cells and has unexpectedly high di-Mannose-6-phosphate (di-M6P) leading to particularly effective in vivo cellular uptake.

[7] Further, the recombinant ASA of the present invention shows increased bioactivity. Without wishing to be bound by any particular theory, it is contemplated that an increase in formylglycine levels (%FG) of the recombinant ASA protein results in increased enzyme activity.

[8] In some embodiments, addition of a reducing agent, e.g., DTT after the viral inactivation step in the process of the present invention leads to production of a dimeric rhASA form, which is desirable for purification.

[9] As described in the Examples section, exemplary recombinant ASA proteins purified using processes described herein conform to the marketing purity requirements in the US and many other countries. Potential product impurities, for example, host cell DNA, host cell proteins (HCP) and product-related low-molecular weight (LMW) species were reduced in the present invention to a level comparable or below acceptable limits for other processes and/or industry standard. Without wishing to be bound by any particular theory, it is contemplated that impurities (e.g., HCP levels) are controlled and minimized by the improved capture and downstream chromatographic steps of the present invention. In some embodiments, the improved downstream steps include, for example, a washing step with greater than 400 mM arginine (e.g., 650 mM arginine). In some embodiments, a washing step includes a wash with greater than 400 mM arginine hydrochloride buffer (e.g., 650 mM arginine hydrochloride). In some embodiments, a washing step includes a wash with between about 400-1000 mM arginine. In some embodiments, the improved downstream steps include, for example, cleaning the affinity column with between about 4-8M guanidium hydrochloride (e.g., 6M guanidium hydrochloride), for example, at every 2 or more cycles (e.g., every 3 cycles).

[10] In some embodiments, provided herein is an affinity elution buffer with a delayed pH shift. In some embodiments, an affinity elution buffer is 50 mM glycine hydrochloride, 50 mM NaCl, pH 3.1. Without wishing to be bound by any particular theory, it is contemplated that this elution buffer results in a higher pH of the elution pool, increasing ASA product stability.

[11] In summary, large-scale methods of the present invention for purifying recombinant ASA protein provide advantages such as cost and time reductions by improving yield, reducing host cell derived impurities, e.g., in large-scale manufacturing processes, and providing purified ASA compositions with beneficial attributes, e.g., In some embodiments, a dimeric rhASA with an improved glycan pattern including di-M6P increases cellular uptake. In some embodiments, increased % formylglycine improves bioactivity, thereby improving efficacy and dosing of enzyme replacement therapy for treatment of diseases, e.g., MLD.

[12] In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein having the amino acid sequence of SEQ ID NO: 1, at least 70% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[13] In some embodiments, between about 75% to greater than 95% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises 100% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, substantially all of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[14] In some embodiments, between about 85% to about 99% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the at least 85% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[15] In some embodiments, between about 77% to about 89% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, between about 78% to about 86% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 77%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 87%, at least 88% or at least 89% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[16] In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 1% of total glycans in N-linked glycosylation sites of the recombinant ASA protein comprises are di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or greater of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein between about 6.4-9.6% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 6.9%, at least 7.9% or at least 8.9% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 6.4% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 6.9% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein between about 7-21% of total glycans in N-linked glycosylation sites is di- mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein between about 7.8-20.9% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein between about 10-20% of total glycans in N-linked glycosylation sites is di- mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20% or at least 21% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 7% of total glycans in N-linked glycosylation sites is di-mannose-6- phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 7.8% of total glycans in N- linked glycosylation sites is di-mannose-6-phosphate (di-M6P).

[17] In some embodiments, the N-linked glycosylation sites comprise one or more ofN140, N166, and/or N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise N140 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise N166 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise one or more of N140, N166 or N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise one or more of N140, N166 and N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise N140, N166 and N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites are N140, N166 and N332 of SEQ ID NO: 1.

[18] In some embodiments, the recombinant ASA protein has a specific activity of between about 58-176 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of at least 100 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 50 U/mg to about 130 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 50 U/mg to about 70 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 70 U/mg to about 100 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 71 U/mg to about 96 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 101 U/mg to about 134 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 90 U/mg to about 150 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 60-110 U/mg. In some embodiments, the recombinant ASA protein has a specific activity of between about 71-96 U/mg. In some embodiments, the recombinant ASA protein has a specific activity of between about 80-150 U/mg. In some embodiments, the recombinant ASA protein has a specific activity of between about 100-150 U/mg. In some embodiments, the recombinant ASA protein has a specific activity of between about 100-140 U/mg. In some embodiments, the recombinant ASA protein has a specific activity of between about 100-130 U/mg. In some embodiments, the recombinant ASA protein has a specific activity of at least about 100 U/mg.

[19] In some embodiments, the recombinant ASA of the present invention is characterized by a proteoglycan map. In some embodiments, the proteoglycan map is determined by High Performance Anion Exchange Chromatography with Fluorescence Detection (HPAEC-FLD). In some embodiments, the HPAEC-FLD uses a 2- aminobenzamide (2 -AB) labeling method. In some embodiments, the proteoglycan map is determined using liquid chromatography with UV and mass spectrometry detection (LC-UV- MS). In some embodiments, provided herein is a composition comprising recombinant ASA protein, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 1% to about 12% capped M6P glycan, about 20% to about 40% total M6P glycan, about 5% to about 21% of di-M6P glycanM6P glycan, about 3% to about 18% hybrid glycan, about 3% to about 26% neutral glycan, and about 28% to about 43% sialic acid moieties per molecule of ASA protein. In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of about 7% to about 11% capped M6P glycanM6P glycan, about 21% to about 40% total M6P protein, about 7% to about 21% of di- M6P ASA protein, about 10% to about 18% hybrid glycan, about 3% to about 21% neutral glycan, and about 28% to about 42% sialic acid moieties per molecule of ASA protein. In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of about 7.3% to about 10.6% capped M6P glycan, about 21.6% to about 39.4% total M6P protein, about 7.8% to about 20.9% of di-M6P ASA protein, about 10.6% to about 17.1% hybrid glycan, and about 28.6% to about 41.3% sialic acid moieties per molecule of ASA protein. In some embodiments, about 1% to about 10% capped M6P glycan, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of about 24% to about 32% total M6P glycan, about 3% to about 11% hybrid glycan, about 16% to about 26% neutral glycan, and about 34% to about 43% sialic acid moieties per molecule of ASA protein. In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising about 21.6% to about 39.4% total M6P protein, and about 7.8% to about 20.9% of di-M6P ASA protein per molecule of ASA protein.

[20] In some embodiments, the composition comprises at least about 5% di-M6P glycan. In some embodiments, the composition comprises at least about 10% di-M6P glycan. In some embodiments, the composition comprises at least about 15% di-M6P glycan. In some embodiments, the composition comprises at least about 6.4% di-M6P glycan. In some embodiments, the composition comprises at least about 6.9% di-M6P glycan. In some embodiments, the composition comprises at least about 7% di-M6P glycan. In some embodiments, the composition comprises at least about 7.8% di-M6P glycan.

[21] In some embodiments, the composition comprises a ratio of mono-M6P to di- M6P of between about 5: 1 to 1 : 1. In some embodiments, the composition comprises a ratio of mono-M6P to di-M6P of between about 2: 1 to 1 :1. [22] In some embodiments, the recombinant ASA protein contains less than 100 ng/mg Host Cell Protein (HCP). In some embodiments, the recombinant ASA protein contains less than 70 ng/mg Host Cell Protein (HCP).

[23] In some embodiments, the recombinant ASA protein is present in the composition at a concentration of at least 20 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of at least 25 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of at least 30 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of at least 40 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of at least 45 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of at least 50 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of about 30 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of between about 10-50 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of between about 20-40 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of between about 25-35 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of between about 35-45 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of about 30 mg/ml. In some embodiments, the recombinant ASA protein is present in the composition at a concentration of about 40 mg/ml.

[24] In some embodiments, the recombinant ASA protein is purified from CHO cells. In some embodiments, the recombinant ASA protein is purified from human cells.

[25] In some embodiments, provided herein is a method of treating metachromatic leukodystrophy (MLD), the method comprising administering the composition provided herein to a subject in need of treatment.

[26] In some embodiments, the composition is administered at a dose of at least 150 mg. In some embodiments, the composition is administered at a dose of 150 mg.

[27] In some embodiments, the pharmaceutical composition is administered to the CSF. [28] In some embodiments, the pharmaceutical composition is administered by intrathecal or intraventricular injection. In some embodiments, the pharmaceutical composition is administered by intrathecal injection.

[29] In some embodiments, the pharmaceutical composition is administered at least once weekly. In some embodiments, the pharmaceutical composition is administered once weekly. In some embodiments, the pharmaceutical composition is administered at a dose of 150 mg and once weekly.

[30] In some embodiments, administration of the pharmaceutical composition results in a reduction in the amount of glycosaminoglycans within the CSF of the patient.

[31] In some embodiments, provided herein is a method of purifying recombinant aryl sulfatase A (ASA) protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 70 ng/mg HCP. In some embodiments, provided herein is a method of purifying recombinant aryl sulfatase A (ASA) protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 7 pg/mg Host Cell DNA. In some embodiments, provided herein is a method of purifying recombinant arylsulfatase A (ASA) protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 2.8% of HMW impurities.

[32] In some embodiments, the method comprises 5 chromatography steps or less. In some embodiments, the method comprises 4 chromatography steps or less. In some embodiments, the method comprises 3 or 4 chromatography steps. In some embodiments, the method comprises 4 chromatography steps.

[33] In some embodiments, the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 70 ng/mg HCP. In some embodiments, the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 7 pg/mg Host Cell DNA. In some embodiments, the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 2.8% of HMW impurities.

[34] In some embodiments, the affinity chromatography is carried out using a single column.

[35] In some embodiments, the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography in sequential order.

[36] In some embodiments, the method further comprises a step of viral inactivation. In some embodiments, the viral inactivation step comprises addition of a reducing agent. In some embodiments, the reducing agent is dithiothreitol (DTT). In some embodiments, 10-1000 mol of DTT is added per mol of recombinant ASA protein. In some embodiments, 10 mol of DTT is added per mol of recombinant ASA protein. In some embodiments, the method comprises purification from CHO cells. In some embodiments, the CHO cells lack glutamine synthetase. In some embodiments, the CHO cells are grown in a medium comprising L-glutamine and copper.

[37] In some embodiments, the recombinant ASA protein contains less than 70 ng/mg HCP. In some embodiments, at least 70% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 97% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[38] In some embodiments, at least 1% of total glycans in N-linked glycosylation sites of the recombinant ASA protein are di-mannose-6-phosphate (di-M6P). In some embodiments, the N-linked glycosylation sites comprise one or more of N140, N166, and/or N332 of SEQ ID NO: 1.

[39] In some embodiments, the recombinant ASA protein has a specific activity of at least 100 U/mg as determined by an in vitro assay. [40] In some embodiments, the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 7% to about 11% capped M6P glycan, about 21% to about 40% total M6P glycan, about 10% to about 25% mono-M6P glycan, about 7% to about 21% of di-M6P glycan, about 7% to about 21% of mono-M6P glycan, about 10% to about 18% hybrid glycan, about 3% to about 21% neutral glycan, or about 28% to about 42% sialic acid moieties per molecule of ASA protein.

[41] In some embodiments, the amount of di-M6P glycan is at least 5%. In some embodiments, the amount of di-M6P glycan is at least 10%. In some embodiments, the amount of di-M6P glycan is at least 15%.

[42] In some embodiments, the ratio of a mono-M6P to di-M6P is between about 2: 1 to 1 :1. In some embodiments, the recombinant ASA protein contains less than 70 ng/mg HCP.

[43] In some embodiments, the recombinant ASA protein has an amino acid sequence of SEQ ID NO: 1. In some embodiments, CHO cells comprise one or more exogenous nucleic acids encoding the recombinant ASA protein and/or the FGE. In some embodiments, the one or more exogenous nucleic acids are integrated in the genome of the cells. In some embodiments, the one or more exogenous nucleic acids are present on one or more extra-chromosomal constructs. In some embodiments, the one or more exogenous nucleic acids are present on a single extra-chromosomal construct. In some embodiments, the cells overexpress the recombinant ASA protein. In some embodiments, the cells overexpress FGE.

[44] In some aspects, provided herein is a method for large-scale production of recombinant aryl sulfatase (ASA) protein in CHO cells, comprising culturing CHO cells coexpressing a recombinant ASA protein and a formylglycine generating enzyme (FGE) in suspension in a large-scale culture vessel in medium containing copper.

[45] In some embodiments, the method of purifying recombinant arylsulfatase A protein from an impure preparation comprises affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography.

[46] In some embodiments, the method comprises purifying recombinant arylsulfatase A protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography in sequential order.

[47] In some embodiments, elution from affinity chromatography column(s) is carried out using an elution buffer comprising 20-80 mM glycine-HCl and 20-80 mM NaCl at about pH 2-5. In some embodiments, elution from affinity chromatography column(s) is carried out using an elution buffer comprising 50 mM glycine-HCl and 50 mM NaCl at pH 3.1. In some embodiments, elution is carried out at about pH 2-5.

[48] In some embodiments, the method comprises addition of a reducing agent during or after a viral inactivation step. In some embodiments, the reducing agent is dithiothreitol (DTT). In some embodiments, 10-1000 mol of DTT is added per mol of recombinant ASA protein. In some embodiments, 10 mol of DTT is added per mol of recombinant ASA protein.

[49] In some embodiments, at least 70% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[50] In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[51] In some embodiments, provided herein is a method, wherein at least 97% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[52] In some embodiments, provided herein is a method, wherein the scale is between about 200 liters to about 2000 liters. In some embodiments, the scale is about 200 liters. In some embodiments, the scale is about 300 liters. In some embodiments, the scale is about 400 liters. In some embodiments, the scale is about 500 liters. In some embodiments, the scale is about 600 liters. In some embodiments, the scale is about 700 liters. In some embodiments, the scale is about 800 liters. In some embodiments, the scale is about 900 liters. In some embodiments, the scale is about 1000 liters. In some embodiments, the scale is about 1100 liters. In some embodiments, the scale is about 1200 liters. In some embodiments, the scale is about 1300 liters. In some embodiments, the scale is about 1400 liters. In some embodiments, the scale is about 1500 liters. In some embodiments, the scale is about 1600 liters. In some embodiments, the scale is about 1700 liters. In some embodiments, the scale is about 1800 liters. In some embodiments, the scale is about 1900 liters. In some embodiments, the scale is about 2000 liters.

[53] In some embodiments, the scale is greater than about 2000 liters. In some embodiments, the scale is about 2500 liters. In some embodiments, the scale is about 3000 liters. In some embodiments, the scale is about 3500 liters. In some embodiments, the scale is about 4000 liters. In some embodiments, the scale is about 4500 liters. In some embodiments, the scale is about 5000 liters.

[54] As used herein, the terms “ASA protein,” “ASA,” “ASA enzyme,” or grammatical equivalents, refer to a preparation of recombinant ASA protein molecules unless otherwise specifically indicated. “Purified ASA” refers to a purified recombinant ASA protein or enzyme.

[55] As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

[56] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWING

[57] The Figures described below, which together make up the Drawings, are for illustration purposes only, not for limitation.

[58] FIG. 1 is a schematic diagram of rhASA depicting protein modification sites, which shows 3 N-linked glycosylation sites (depicted by hexagons) and active site post- translational modifications of Cys51.

[59] FIG. 2A is a schematic flowchart of the upstream purification process of rhASA, wherein recombinant ASA is produced in a large scale from CHO cells starting from thawing a vial of CHO cells and expanding cells in a shake flask, followed by wave bag, then in a 500 liters seed reactor to a 1500 liters production reactor, where media exchange is carried out by perfusion for 25 days at 1.0 VVD (volume of media per bioreactor volume per day), followed by clarification and a single column affinity chromatography step daily. Following this, Sartopore filtration is carried out, followed by optional storage of intermediate purified product. FIG 2B shows the steps of the downstream polishing and purification process, after affinity chromatography, which includes viral inactivation, DTT reduction, and sequential chromatography. For example, in some embodiments, a ultra-high binding capacity anion exchange resin with wide pH and flow rate working range is used (e.g. Nuvia Q), followed by a weak cation exchange or mixed mode resin (e.g. Capto MMC ImpRes) and subsequently a hydrophobic interaction chromatography resin for high- resolution intermediate and polishing steps (e.g. Capto Phenyl ImpRes).

[60] FIG. 3 A is a graph of stability of protein concentration following long-term storage (<-65°C). FIG. 3B is a graph of stability of pH following long-term storage (<-65°C). FIG. 3C is a graph of size exclusion chromatography (main peak) following long-term storage. FIG. 3D is a graph of size exclusion chromatography (high molecular weight peak) following long-term storage. (<-65°C). FIG. 3E is a graph showing specific activity of rhASA following long-term storage (<-65°C).

[61] FIG. 4A is a graph of stability of protein concentration following storage under accelerated conditions (5±3°C). FIG. 4B is a graph of stability of pH following storage under accelerated conditions (5±3°C). FIG. 4C is a graph of size exclusion chromatography (main peak) under accelerated conditions (5±3°C). FIG. 4D is a graph of size exclusion chromatography (high molecular weight peak) under accelerated conditions (5±3°C). FIG. 4E is a graph showing specific activity of rhASA under accelerated conditions (5±3°C).

[62] FIG. 5A is a graph of stability of protein concentration following storage under stress conditions (25±2°C). FIG. 5B is a graph of stability of pH following storage under stress conditions (25±2°C). FIG. 5C is a graph of size exclusion chromatography (main peak) under stress conditions (25±2°C). FIG. 5D is a graph of size exclusion chromatography (high molecular weight peak) under stress conditions (25±2°C). FIG. 5E is a graph showing specific activity of rhASA under accelerated conditions (25±2°C).

[63] FIG. 6A-FIG. 6C are graphs of impurities in purified rhASA (peak percentage) relative to the amount of a reducing agent, for example, DTT added (redox equivalence) into the viral inactivation pool. The low molecular weight (LMW) species were reduced by the addition of DTT. However, higher levels of DTT also increased high molecular weight species. DTT addition at 10 mol/mol DTT/rhASA redox units decreased LMW without increasing the HMW species. [64] FIG. 7A-FIG. 7D are graphs that show reduction in sulfatides in ASA treated animals. FIG. 7A is a graph that shows the reduction in short-chain sulfatides with 16 carbon non-hydroxylated fatty acids in ASA treated animals. FIG. 7B is a graph that shows the reduction in short-chain sulfatides with 18 carbon non-hydroxylated fatty acids in ASA treated animals. FIG. 7C is a graph that shows the reduction in total short-chain sulfatide fatty acids in ASA treated animals. FIG. 7D is a graph that shows C18:00-OH levels which represent short-chain sulfatides with 18 carbon hydroxylated fatty acids. FIG. 7E is a graph that shows C20:0 levels which represents short-chain sulfatides with 20 carbon non- hydroxylated fatty acids.

DEFINITIONS

[65] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[66] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 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 than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

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

[68] 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 mannose-6-phosphate (M6P) tags on acid hydrolase precursors in the Golgi apparatus that are destined for transport to the lysosome. In addition to mannose-6- phosphates, the CI-MPR also binds other proteins including IGF-II. The CI-MPR is also known as “M6P/IGF-II receptor,” “CI-MPR/IGF-II receptor,” “IGF-II receptor” or “IGF2 Receptor.” These terms and abbreviations thereof are used interchangeably herein.

[69] Chromatography: As used herein, the term “chromatography” refers to a technique for separation of mixtures. Typically, the mixture is dissolved in a fluid called the “mobile phase,” which carries it through a structure holding another material called the “stationary phase.” Column chromatography is a separation technique in which the stationary bed is within a tube, i.e., column.

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

[71] 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 a process to wash loaded resins to remove captured ions.

[72] Eluate: As used herein, the term “eluate” refers to a combination of mobile phase “carrier” and the analyte material that emerge from the chromatography, typically as a result of eluting.

[73] 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, enzyme is taken up by cells and transported to the lysosome, where the enzyme acts to eliminate material that has accumulated in the lysosomes due to the enzyme deficiency. Typically, for lysosomal enzyme replacement therapy to be effective, the therapeutic enzyme is delivered to lysosomes in the appropriate cells in target tissues where the storage defect is manifest. The purification processes described herein may be used to purify and formulate recombinant Arylsulfatase A as a drug substance for ERT of MLD.

[74] Equilibrate or Equilibration'. As used herein, the terms “equilibrate” or “equilibration” in relation to chromatography refer to the process of bringing a first liquid (e.g., buffer) into balance with another, generally to achieve a stable and equal distribution of components of the liquid (e.g., buffer). For example, in some embodiments, a chromatographic column may be equilibrated by passing one or more column volumes of a desired liquid (e.g., buffer) through the column.

[75] Improve, increase, or reduce'. As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A “control individual” is an individual afflicted with the same form of lysosomal storage disease as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).

[76] Impurities'. As used herein, the term “impurities” refers to substances inside a confined amount of liquid, gas, or solid, which differ from the chemical composition of the target material or compound. Impurities are also referred to as contaminants.

[77] Load'. As used herein, the term “load” refers to, in chromatography, adding a sample-containing liquid or solid to a column. In some embodiments, particular components of the sample loaded onto the column are then captured as the loaded sample passes through the column. In some embodiments, particular components of the sample loaded onto the column are not captured by, or “flow through”, the column as the loaded sample passes through the column.

[78] Polypeptide'. As used herein, a “polypeptide”, generally speaking, is a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides sometimes include “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain, optionally.

[79] Pool. As used herein, the term “pool” in relation to chromatography refers to combining one or more fractions of fluid that has passed through a column together. For example, in some embodiments, one or more fractions which contain a desired component of a sample that has been separated by chromatography (e.g., “peak fractions”) can be “pooled” together generate a single “pooled” fraction.

[80] Replacement enzyme'. As used herein, the term “replacement enzyme” refers to any enzyme that can act to replace at least in part the deficient or missing enzyme in a disease to be treated. In some embodiments, the term “replacement enzyme” refers to any enzyme that can act to replace at least in part the deficient or missing lysosomal enzyme in a lysosomal storage disease to be treated. In some embodiments, a replacement enzyme (e.g., rASA) is capable of reducing accumulated materials in mammalian lysosomes or that can rescue or ameliorate one or more lysosomal storage disease (e.g., MLD) symptoms. Replacement enzymes suitable for the invention include both wild-type or modified lysosomal enzymes and can be produced using recombinant and synthetic methods or purified from nature sources. A replacement enzyme can be a recombinant, synthetic, gene- activated or natural enzyme.

[81] Soluble-. As used herein, the term “soluble” refers to the ability of a therapeutic agent to form a homogenous solution. In some embodiments, the solubility of the therapeutic agent in the solution into which it is administered and by which it is transported to the target site of action is sufficient to permit the delivery of a therapeutically effective amount of the therapeutic agent to the targeted site of action. Several factors can impact the solubility of the therapeutic agents. For example, relevant factors which may impact protein solubility include ionic strength, amino acid sequence and the presence of other cosolubilizing agents or salts (e.g., calcium salts). In some embodiments, therapeutic agents in accordance with the present invention are soluble in its corresponding pharmaceutical composition.

[82] Stability. As used herein, the term “stable” refers to the ability of the therapeutic agent (e.g., a recombinant enzyme) to maintain its therapeutic efficacy (e.g., all or the majority of its intended biological activity and/or physiochemical integrity) over extended periods of time. The stability of a therapeutic agent, and the capability of the pharmaceutical composition to maintain stability of such therapeutic agent, may be assessed over extended periods of time (e.g., for 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 upon storage and during processes (such as freeze/thaw, mechanical mixing and lyophilization). For protein stability, it can be measure by formation of high molecular weight (BMW) aggregates, loss of enzyme activity, generation of peptide fragments and shift of charge profiles.

[83] Viral Processing'. As used herein, the term “viral processing” refers to “viral removal,” in which viruses are simply removed from the sample (e.g. viral filtration), or “viral inactivation,” in which the viruses remain in a sample but in a non-infective form. In some embodiments, viral removal may utilize nanofiltration and/or chromatographic techniques, among others. In some embodiments, viral inactivation may utilize solvent inactivation, detergent inactivation, pasteurization, acidic pH inactivation, and/or ultraviolet inactivation, among others.

DETAILED DESCRIPTION

[84] The present invention provides, among other things, compositions comprising purified recombinant ASA protein and large-scale methods of making and using recombinant ASA protein for enzyme replacement therapy, for use in treating diseases e.g. MLD.

[85] The present invention provides, among other things, a large-scale process for producing a high yield of recombinant ASA of high product quality. In some aspects, the present invention provides a high yield of a highly pure recombinant ASA product. In some embodiments, the process utilizes CHO host cell lines (e.g. CHO cell line lacking glutamate synthase) and affinity resin purification. In some embodiments, the process utilizes human cell lines. The purified recombinant ASA of the present invention features distinct glycosylation characteristics from the host cell line (e.g., that facilitate bioavailability, improved uptake, and/or improved efficacy of the recombinant ASA protein). For example, in some embodiments, an increase in di-Mannose-6-phosphate (di-M6P) in the glycan map of the ASA leads to improved in vivo cellular uptake as measured by cellular uptake bioassays as well as pharmacokinetic and pharmacodynamic studies.

[86] In some embodiments, an increase in formylglycine levels (%FG) of the recombinant ASA protein results in increased enzyme activity, thereby improving efficacy.

[87] Potential product impurities, for example, host cell DNA, host cell proteins (HCP) and product-related low-molecular weight (LMW) species were reduced to a level comparable or below acceptable limits for other processes and/or industry standard. In some embodiments, impurities are controlled and minimized by the improved capture and downstream chromatographic steps of the present invention (e.g., affinity purification process).

[88] In summary, large-scale processes of the present invention for purifying recombinant ASA protein provide advantages such as cost and time reductions by improving yield, reducing host cell derived impurities, and providing purified ASA compositions with beneficial attributes, e.g. in some embodiments a glycan pattern including di-M6P, increases cellular uptake, and increased % formylglycine improves bioactivity, thereby improving efficacy of enzyme replacement therapy for treatment of diseases, e.g., MLD.

[89] In some embodiments, characteristic features of purified recombinant ASA protein (e.g., a characteristic glycan map such as purified recombinant ASA protein having a threshold amount of mannose-6-phosphated recombinant ASA protein) as well as other properties such as a low level of impurities of such as Host Cell Protein (HCP), Host Cell DNA (HCD), and/or a particular specific activity) can result in desirable properties of compositions comprising a purified recombinant ASA protein (e.g., improved stability to storage, improved therapeutic properties).

[90] Various aspects of the invention are described in further detail in the following subsections. The use of 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 stated otherwise.

Arylsulfatase A (ASA, ARSA, cerebroside sulfatase)

[91] Arylsulfatase A (ASA, ARSA, or cerebroside-sulfatase) is an enzyme that breaks down cerebroside 3-sulfate (or sulfatide) into cerebroside and sulfate. Specifically, galactosyl sulfatide is normally metabolized by the hydrolysis of 3-O-sulphate linkage to form galactocerebroside through the combined action of the lysosomal enzyme Arylsulfatase A (EC 3.1.6.8) (Austin et al. Biochem J. 1964, 93, 15C-17C) and a sphingolipid activator protein called saposin B. A deficiency of Arylsulfatase A occurs in all tissues from patients with the late infantile, juvenile, and adult forms of Metachromatic Leukodystrophy (MLD). As used herein, the Arylsulfatase A protein will be termed “ASA” or “ARSA”.

[92] 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. ASA exists as a 480 kDa octamer in acidic conditions (pH < about 5.0), but dissociates into dimers at neutral pH levels. In human urine, the enzyme consists of two non-identical 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 consist of two subunits of slightly different sizes varying 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 in the case of 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 phosphorylated and sulfated oligosaccharide of the complex type (Waheed A et al. Biochim Biophys Acta. 1985, 847, 53-61, Braulke T et al. Biochem Biophys Res Commun. 1987, 143, 178-185). In normal cultured fibroblasts, a precursor polypeptide of 62 kDa is produced, which translocates via mannose-6-phosphate receptor binding (Braulke T et al. J Biol Chem. 1990, 265, 6650-6655) to an acidic prelysosomal endosome (Kelly BM et al. Eur J Cell Biol. 1989, 48, 71-78).

[93] The methods described herein can be used to purify arylsulfatase A from any source, e.g., from tissues, or cultured cells (e.g., CHO cells that recombinantly produce arylsulfatase A, human cells that recombinantly produce ASA), and purify Arylsulfatase A of any origin. In some embodiments, the recombinant ASA is recombinant human ASA.

[94] The length (18 amino acids) of the human Arylsulfatase A signal peptide is based on the consensus sequence and a specific processing site for a signal sequence. Hence, from the human ASA cDNA (EMBL GenBank accession numbers J04593 and X521151), the cleavage of the signal peptide occurs in all cells after residue number 18 (Ala), resulting in the mature form of the human aryl sulfatase A. As used herein, recombinant aryl sulfatase A will be abbreviated “rASA”. Recombinant human ASA is designated “rhASA” and is recombinantly produced for enzyme replacement therapy. The mature form of arylsulfatase A including the mature form of human arylsulfatase A will be termed “mASA” and the mature recombinant human ASA will be termed “mrhASA”.

[95] Multiple forms of arylsulfatase A have been demonstrated on electrophoresis and isoelectric focusing of enzyme preparations from human urine (Luijten JAFM et 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. Neurology. 1979, 29, 16-20) and liver (Stevens RL et al. Biochim Biophys Acta. 1976, 445, 661-671, 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 reduces the molecular size and complexity of the electrophoretic pattern, which suggests that much of the charge heterogeneity of arylsulfatase A is due to variations in the carbohydrate content of the enzyme.

[96] 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 two or more arginine residues (James GT, Arch Biochem Biophys. 1979, 97, 57-62). Many anions are inhibitors of the enzyme at concentrations in the millimolar range or lower.

[97] The human arylsulfatase A gene structure has been described. As used herein, this gene will be termed “ARSA.” However, “ARSA” may also refer to arylsulfatase A protein in some cases. The ARSA gene is located near the end of the long arm of chromosome 22 (22ql3.31-qter), it spans 3.2 kb (Kreysing et al. Eur J Biochem. 1990, 191, 627-631) and consists of eight exons specifying the 507 amino acid enzyme unit (Stein et al. J Biol Chem. 1989, 264, 1252-1259). For example, Messenger RNAs of 2.1, 3.7, and 4.8 kb have been detected in fibroblast cells, with the 2.1-kb message apparently responsible for the bulk of the active arylsulfatase A generated by the cell (Kreysing et al. Eur J Biochem. 1990, 191, 627-631). The ARSA sequence has been deposited at the EMBL GenBank with the accession number X521150. Differences between the published cDNA and the coding part of the 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 the cDNA sequence described by Kreysing et al. (Eur J Biochem. 1990, 191, 627-631) have been deposited at the EMBL GenBank with the following accession numbers J04593 and X521151, respectively.

[98] Several polymorphisms and more than 40 disease-related 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). The disease- related mutations in the ARSA gene can be categorized in two broad groups that correlate fairly well with the clinical phenotype of MLD. One group (I) produces no active enzyme, no immunoreactive protein, and expresses no ASA activity when introduced into cultured animal cell lines. The other group (A) generates small amounts of cross-reactive material and low levels of functional enzyme in cultured cells. Individuals homozygous for a group (I) mutation, or having two different mutations from this group, express late infantile MLD. Most individuals with one group (I)-type and one group (A)-type mutation develop the juvenile-onset form, whereas those with two group (A)-type mutations generally manifest adult MLD. Some of the mutations have been found relatively frequently, whereas others have been detected only in single families. It is possible to trace specific mutations through members of many families, however general carrier screening is not yet feasible.

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

Recombinant ASA Protein

[100] As used herein, the term “recombinant ASA protein” refers to any molecule or a portion of a molecule that can substitute for 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 a recombinant ASA protein that is a polypeptide having an amino acid sequence substantially similar or identical to mature human ASA protein.

[101] Recombinant human ASA (rhASA) is a multimeric glycoprotein produced in a cell line comprising a gene encoding human lysosomal enzyme, arylsulfatase A. In some embodiments, the cell line is a non-human cell line. In some embodiments, the cell line is a Chinese hamster ovary cells (CHO cells). In some embodiments, the cell line is a CHOZN® GS'^/_ cell line. In some embodiments, the CHO cells lack glutamine synthetase. In some embodiments, the CHO cells are grown in a medium comprising L-glutamine and copper. In some embodiments, e.g. the cell line is a human cell line. In some embodiments, the cell line is HT-1080. Recombinant human ASA is typically produced as a full-length precursor molecule or full-length ASA protein that is post-translationally processed to a mature form by cleavage of the 18 amino acid N-terminal signal peptide. The full-length precursor or full- length ASA protein contains 507 amino acids. The N-terminal 18 amino acids are cleaved, resulting in a mature form that is 489 amino acids in length. It is contemplated that the N- terminal 18 amino acids are generally 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. Human Arylsulfatase A

[102] Thus, in some embodiments, a recombinant ASA protein purified by embodiments of the present invention is mature human ASA protein (SEQ ID NO: 1). In some embodiments, a recombinant ASA protein purified by embodiments of the present invention may be a homologue or an analogue of mature human ASA protein. For example, a homologue or an analogue of mature human ASA protein may be a modified mature human ASA protein containing one or more amino acid substitutions, deletions, and/or insertions as compared to a wild-type or naturally-occurring ASA protein (e.g., SEQ ID NO:1), while retaining substantial ASA protein activity. Thus, in some embodiments, a recombinant ASA protein purified by embodiments of the present invention is substantially homologous to mature human ASA protein (SEQ ID NO: 1). In some embodiments, a recombinant ASA protein purified by embodiments of the present invention has an amino acid sequence 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, a recombinant ASA protein purified by embodiments of the present invention is substantially identical to mature human ASA protein (SEQ ID NO: 1). In some embodiments, a recombinant ASA protein purified by embodiments of the present invention has an amino acid sequence 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, a recombinant ASA protein purified by embodiments of the present invention contains a fragment or a portion of mature human ASA protein.

[103] Alternatively, a recombinant ASA protein purified by embodiments of the present invention is full-length ASA protein. In some embodiments, a recombinant ASA protein may be a homologue or an analogue of full-length human ASA protein. For example, a homologue or an analogue of full-length human ASA protein may be a modified full-length human ASA protein containing one or more amino acid substitutions, deletions, and/or insertions as compared to a wild-type or naturally-occurring full-length ASA protein (e.g., SEQ ID NO:2), while retaining substantial ASA protein activity. Thus, in some embodiments, a recombinant ASA protein purified by embodiments of the present invention is substantially homologous to full-length human ASA protein (SEQ ID NO:2). In some embodiments, a recombinant ASA protein purified by embodiments of the present invention has an amino acid sequence 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, a recombinant ASA protein purified by embodiments of the present invention is substantially identical to SEQ ID NO:2. In some embodiments, a recombinant ASA protein purified by embodiments of the present invention has an amino acid sequence 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, a recombinant ASA protein purified by embodiments of the present invention contains a fragment or a portion of full-length human ASA protein. As used herein, a full-length ASA protein typically contains signal peptide sequence.

[104] Homologues or analogues of human ASA proteins can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods. In some embodiments, conservative substitutions of amino acids include substitutions made among 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, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.

[105] In some embodiments, the mature ASA protein is post-translationally modified by glycosylation at three asparagine (Asn) amino acid residues (bold and underlined in SEQ ID NO: 1), and conversion of cysteine 51 (Cys51) to formylglycine (FGE) rhASA includes six disulfide bridges between 12 of the 15 cysteine (Cys) residues.

[106] The mature secreted form of ASA is a multimeric protein of 489 amino acids, with a molecular weight of about 57 kDa. In lysosomes, under acidic pH conditions (pH < 6), the ASA protein associates into an octamer which shows optimal catalytic activity. Under neutral and alkaline pH, ASA octamer dissociates into an inactive dimer. A schematic diagram of rhASA depicting protein modification sites shows 3 N-linked glycosylation sites (depicted by hexagons) and active site post-translational modifications of Cys51 (FIG. 1). In some embodiments, the glycans contain mannose-6-phosphate (M6P) and di-mannose-6- phosphate (di-M6P), which is required for target cell uptake and trafficking to lysosomes by binding to membrane-bound M6P receptors.

[107] In some embodiments, the glycan moiety binds to a mannose-6-phosphate (M6P) receptor on the surface of target cells to facilitate cellular uptake and/or lysosomal targeting. For example, such a receptor may be the cation-independent mannose-6-phosphate receptor (CI-MPR) which binds the mannose-6-phosphate (M6P) residues. In addition, the CI-MPR also binds other proteins including IGF-II. In some embodiments, a recombinant ASA protein contains M6P residues on the surface of the protein. In particular, a recombinant ASA protein may contain bis-phosphorylated oligosaccharides which have 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.

[108] In some embodiments, recombinant ASA enzymes may be fused to a lysosomal targeting moiety that is capable of binding to a receptor on the surface of target cells. A suitable lysosomal targeting moiety can be IGF-I, IGF-II, RAP, p97, and variants, homologues or fragments thereof (e.g., including those peptide having a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to a wild-type mature human IGF-I, IGF-II, RAP, p97 peptide sequence). The lysosomal targeting moiety may be conjugated or fused to an ASA protein or enzyme at the N-terminus, C-terminus or internally.

[109] In some embodiments, cellular uptake is between about 88-251%. In some embodiments, cellular uptake is about 140-200%. In some embodiments, cellular uptake is between about 140% and 150%. In some embodiments, cellular uptake is between about 150% and 160%. In some embodiments, cellular uptake is between about 160% and 170%. In some embodiments, cellular uptake is between about 170% and 180%. In some embodiments, cellular uptake is between about 180% and 190%. In some embodiments, cellular uptake is between about 190% and 200%. In some embodiments, cellular uptake is between about 200% and 210%. In some embodiments, cellular uptake is between about 210% and 220%. In some embodiments, cellular uptake is between about 220% and 230%. In some embodiments, cellular uptake is between about 230% and 240%. In some embodiments, cellular uptake is between about 240% and 250%. In some embodiments, cellular uptake is between about 250% and 260%. In some embodiments, cellular uptake is between about 260% and 270%.

Purification of Recombinant Arylsulfatase A

[110] Embodiments of the invention include purification processes for the production of Arylsulfatase A (“ASA”). In some embodiments, the ASA protein is recombinant human ASA (“rhASA”), drug substances.

[Hl] A variety of techniques, in whole or in part, optionally with modifications as described herein, may be used to produce purified ASA drug substance.

[112] In some aspects, the present invention provides a large-scale method to produce high yields of highly pure ASA for therapeutic use, thereby meeting large dosing requirements for treating metachromatic leukodystrophy. In some aspects, the drug product is a sterile solution of recombinant human arylsulfatase A, 30 mg/mL, in 154 mM sodium chloride with 0.005% (vol/vol) polysorbate 20 (P-20) at pH 6.0. In some aspects, the drug substance is 40 mg/mL rhASA in 154 mM NaCl with 0.005% (v/v) polysorbate 20, pH 6.0.

[113] In some aspects, human cell lines are used. In some aspects, CHO cell lines are used to achieve high ASA yield in the process of the present invention. In some embodiments, CHO cell lines lack glutamine synthetase and cells are grown in the presence of exogenous L-glutamine in the medium. In some embodiments, copper is added to the medium.

[114] An expression vector comprising rhASA (GenBank Identifier: 7262293) and formylglycine generating enzyme (FGE) (GenBank identifier: 30840148) expression cassettes is used, in order to increase formylglycine at position 51 of rhASA by FGE, which is a post-translational modification required for sulfatase (e.g. ASA) enzymatic activity.

[115] In some exemplary embodiments, the process involves purification from CHO cells (such as CHOZN® GS-/- ) at a high cell density, and includes steps for clarification, and purification and polishing processes that use different resins to limit CHO host cell protein and impurity species. In some embodiments, the process includes an affinity chromatography capture step, which improves process yield and removes impurities (FIG. 2A). The process employs an antibody or peptide that specifically binds to ASA as an affinity ligand e.g, a micro ASA antibody to affinity capture and purify ASA for e.g., on a Capture Select affinity column. The process further includes a DTT reduction step , for purification of dimeric rhASA and improvement of yield. Changes in downstream chromatography steps ensure that impurities are minimized to below acceptable levels and also increases yield since resins with a high loading density are used for large scale purification. In some embodiments, a resin is used in bind/elute mode or flowthrough mode. For example, in some embodiments, a ultra- high binding capacity anion exchange resin with wide pH and flow rate working range is used (e.g. Nuvia Q), followed by a weak cation exchange or mixed mode resin (e.g. Capto MMC ImpRes) and subsequently a hydrophobic interaction chromatography resin for high- resolution intermediate and polishing steps (e.g. Capto Phenyl ImpRes) (FIG. 2B).

[116] In some aspects, provided herein is a method of purifying recombinant aryl sulfatase A (ASA) protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, cation exchange chromatography (or the cation exchange function from the mixed mode resin), and/or hydrophobic interaction chromatography, which are described in detail below, and wherein the purified recombinant ASA protein contains less than 70 ng/mg HCP. For example, in some embodiments, the method comprises 5 chromatography steps or less. In some embodiments, the recombinant ASA protein produced by the method of the present invention contains less than 65 ng/mg HCP. In some embodiments, the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 60 ng/mg HCP. In some embodiments, the recombinant ASA protein produced by the method of the present invention contains less than 55 ng/mg HCP. In some embodiments, the affinity chromatography is carried out using a single column. In some embodiments, the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography in sequential order.

[117] In some embodiments, the recombinant ASA protein produced by the method of the present invention contains less than 50 ng/mg HCP. In some embodiments, the recombinant ASA protein produced by the method of the present invention contains less than 45 ng/mg HCP. In some embodiments, the recombinant ASA protein produced by the method of the present invention contains less than 40 ng/mg HCP. In some embodiments, the recombinant ASA protein produced by the method of the present invention contains less than 35 ng/mg HCP. In some embodiments, the recombinant ASA protein produced by the method of the present invention contains less than 30 ng/mg HCP.

[118] In some embodiments, the affinity column is cleaned using one or more chaotropic agents. In some embodiments, the chaotropic agent is guanidium (e.g., guanidium hydrochloride. In some embodiments, the affinity column is cleaned using 4M-8M guanidium hydrochloride. In some embodiments, the affinity column is cleaned using 4M guanidium hydrochloride. In some embodiments, the affinity column is cleaned using 5M guanidium hydrochloride. In some embodiments, the affinity column is cleaned using 6M guanidium hydrochloride. In some embodiments, the affinity column is cleaned using 7M guanidium hydrochloride. In some embodiments, the affinity column is cleaned using 8M guanidium hydrochloride. In some embodiments, the chaotropic agent is urea. In some embodiments, the affinity column is cleaned using 6M-10M urea. In some embodiments, the affinity column is cleaned using 6M urea. In some embodiments, the affinity column is cleaned using 7M urea. In some embodiments, the affinity column is cleaned using 8M urea. In some embodiments, the affinity column is cleaned using 9M urea. In some embodiments, the affinity column is cleaned using 10M urea. In some embodiments, the affinity column is cleaned every cycle. In some embodiments, the affinity column is cleaned every 2, 3 or more cycles. In some embodiments, the affinity column is cleaned every 2 cycles. In some embodiments, the affinity column is cleaned every 3 cycles. In some embodiments, the affinity column is cleaned every 4 cycles.

[119] In some embodiments, the affinity column has a washing step using greater than 400 mM arginine buffer (e.g., greater than 400 mM arginine hydrochloride buffer). In some embodiments, the affinity column has a washing step using between 400-1000 mM arginine buffer. In some embodiments, the affinity column has a washing step using between 400-800 mM arginine buffer. In some embodiments, the affinity column has a washing step using greater than 1000 mM arginine buffer. In some embodiments, the affinity column has a washing step using 400 mM arginine buffer. In some embodiments, the affinity column has a washing step using 450 mM arginine buffer. In some embodiments, the affinity column has a washing step using 500 mM arginine buffer. In some embodiments, the affinity column has a washing step using 550 mM arginine buffer. In some embodiments, the affinity column has a washing step using 600 mM arginine buffer. In some embodiments, the affinity column has a washing step using 650 mM arginine buffer. In some embodiments, the affinity column has a washing step using 700 mM arginine buffer. In some embodiments, the affinity column has a washing step using 750 mM arginine buffer. In some embodiments, the affinity column has a washing step using 800 mM arginine buffer. In some embodiments, the affinity column has a washing step using 850 mM arginine buffer. In some embodiments, the affinity column has a washing step using 900 mM arginine buffer. In some embodiments, the affinity column has a washing step using 950 mM arginine buffer. In some embodiments, the affinity column has a washing step using 1000 mM arginine buffer. In some embodiments, the affinity column has a washing step using greater than 1000 mM arginine buffer.

[120] In some embodiments, a wash buffer is 50 mM Tris, 400 mM arginine, pH 7. In some embodiments, a wash buffer is 50 mM Tris, 650 mM arginine, pH 7. In some embodiments, a wash buffer is 50 mM Tris, 800 mM arginine, pH 7. In some embodiments, a wash buffer is 50 mM Tris, 1000 mM arginine, pH 7. In some embodiments, a wash buffer is 50 mM Tris, greater than 1000 mM arginine, pH 7.

[121] In some embodiments, in the process of affinity purification of ASA a resin ligand density of greater than 5 g/L is used. In some embodiments, a resin ligand density is between 5 g/L and 20 g/L. In some embodiments, a resin ligand density is 10 g/L. In some embodiments, a resin ligand density is 12 g/L. In some embodiments, a resin ligand density is 16 g/L. In some embodiments, a resin ligand density is 18 g/L. In some embodiments, a resin ligand density is 20 g/L.

[122] In some embodiments, an elution buffer is a delayed pH transition buffer, i.e. wherein the reduction in pH after elution from an affinity column is delayed. In some embodiments, a 50 mM glycine, 50 mM NaCl elution buffer is used. In some embodiments, an elution buffer has a pH of between 2-5. In some embodiments, an elution buffer has pH 3.0, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 and 4.0. In some embodiments, an elution buffer has a pH 3.1. In some embodiments, an elution buffer has a pH 3.3. In some embodiments, an elution buffer has a pH 3.5. In some embodiments, an elution buffer has a pH 3.7. In some embodiments, an elution buffer is 50 mM glycine, 50 mM NaCl, pH 3.1.

[123] In some embodiments, the recombinant ASA protein produced by the method of the present invention comprises at least 70% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises at least 97% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 1% of total glycans in N-linked glycosylation sites of the recombinant ASA protein is di-mannose-6-phosphate (di-M6P). In some embodiments, the N-linked glycosylation sites comprise one or more of N140, N166, and/or N332 of SEQ ID NO: 1.

[124] In some embodiments, the recombinant ASA protein produced by the method of the present invention has a specific activity of at least 100 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein has a specific activity of between about 60-110 U/mg. In some embodiments, the recombinant ASA protein has a specific activity of between about 71-96 U/mg.

[125] In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of about 1% to about 12% capped M6P glycan, about 20% to about 40% total M6P glycan, about 5% to about 21% of di-M6P glycan, about 3% to about 18% hybrid glycan, about 3% to about 26% neutral glycan, and about 28% to about 43% sialic acid moieties per molecule of ASA protein. In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 7% to about 11% capped M6P glycan, about 21% to about 40% total M6P protein, about 7% to about 21% of di-M6P glycan, about 10% to about 18% hybrid glycan, about 3% to about 21% neutral glycan, and about 28% to about 42% sialic acid moi eties per molecule of ASA protein. In some embodiments, about 1% to about 10% capped M6P glycan, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 24% to about 32% total M6P glycan, about 3% to about 11% hybrid glycan, about 16% to about 26% neutral glycan, and about 34% to about 43% sialic acid moieties per molecule of ASA protein.

[126] In some embodiments, wherein the recombinant ASA protein has an amino acid sequence of SEQ ID NO: 1.

[127] In some embodiments, CHO cells comprise one or more exogenous nucleic acids encoding the recombinant ASA protein and/or the FGE. In some embodiments, the one or more exogenous nucleic acids are integrated in the genome of the cells. In some embodiments, the one or more exogenous nucleic acids are present on one or more extra- chromosomal constructs. In some embodiments, the one or more exogenous nucleic acids are present on a single extra-chromosomal construct. In some embodiments, provided herein is a method wherein the cells overexpress the recombinant ASA protein. In some embodiments, cells of the present method overexpress FGE.

[128] In some aspects, provided is a method for large-scale production of recombinant aryl sulfatase (ASA) protein in CHO cells, comprising culturing CHO cells coexpressing a recombinant ASA protein and a formylglycine generating enzyme (FGE) in suspension in a large-scale culture vessel in medium containing copper. In some embodiments, the method of purifying recombinant arylsulfatase A protein from an impure preparation comprises affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography.

[129] In some embodiments, the method comprises purifying recombinant arylsulfatase A protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography in sequential order. In some embodiments, elution from affinity chromatography column(s) is carried out using an elution buffer comprising 50 mM glycine-HCl and 50 mM NaCl at pH 3.1.

[130] In some embodiments, the recombinant ASA comprises at least 70% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

In some embodiments, the recombinant ASA protein comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises at least 97% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). As used herein, Ca-formylglycine is used interchangeably with formylglycine (FGly).

[131] As used herein, a “contaminant” is a material that is different from the desired polypeptide product, e.g., arylsulfatase A (ASA). The contaminant may be a variant of the desired polypeptide (e.g., a deamidated variant or an amino-aspartate variant of the desired polypeptide) or another molecule, for example, polypeptide, nucleic acid, and endotoxin.

[132] As used herein, by “purifying” a polypeptide from a composition or sample comprising the polypeptide and one or more contaminants is meant increasing the degree of purity of the polypeptide in the composition or sample by removing (completely or partially) at least one contaminant from the composition or sample.

[133] A “purification step” may be part of an overall purification process resulting in a composition comprising 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 total weight of the composition.

[134] The purity of arylsulfatase A can be measured by, e.g., 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.

[135] The arylsulfatase A used in, e.g., the compositions and methods described herein, may also be described by a characteristic glycan map (e.g., any of the exemplary glycan maps described herein).

[136] In some embodiments, the specific activity of the purified arylsulfatase A is at least about 100 U/mg, 110 U/mg, 120 U/mg, 130 U/mg, 140 U/mg, 150 U/mg e.g., as determined by a method described herein. In some embodiments, the purified recombinant ASA has a specific activity ranging from about 100-200 U/mg (e.g., about 100-190 U/mg, 100-180 U/mg, 100-170 U/mg, 100-160 U/mg, 100-150 U/mg, 100-140 U/mg, 100-130 U/mg, 100-120 U/mg, 100-110 U/mg, 100-100 U/mg, 100-140 U/mg, 100-130 U/mg, 100- 120 U/mg, 100-110 U/mg), e.g., as determined by an in vitro method described herein.

[137] A starting material for the purification process is any impure preparation. For example, an impure preparation may be unprocessed cell culture medium containing recombinant ASA protein secreted from the cells (e.g., human cells, CHO cells) producing ASA protein or raw cell lysates containing ASA protein. In some embodiments, the ASA protein is a recombinant human ASA protein. In some embodiments, the CHO cells lack glutamine synthetase. In some embodiments, the CHO cells are grown in a medium comprising L-glutamine and copper.

[138] In some embodiments, an impure preparation may be partially processed cell medium or cell lysates. For example, cell medium or cell lysates can be concentrated, diluted, treated with viral inactivation, viral processing or viral removal. In some embodiments, viral removal may utilize nanofiltration and/or chromatographic techniques, among others. In some embodiments, viral inactivation may utilize solvent inactivation, detergent inactivation, pasteurization, acidic pH inactivation, and/or ultraviolet inactivation, among others. Cell medium or cell lysates may also be treated with protease, DNases, and/or RNases to reduce the level of host cell protein and/or nucleic acids (e.g., DNA or RNA). In some embodiments, unprocessed or partially processed biological materials (e.g., cell medium or cell lysate) may be frozen and stored at a desired temperature (e.g., 2-8 °C, -4 °C, -25 °C, -75 °C) for a period time and then thawed for purification. As used herein, an impure preparation is also referred to as starting material or loading material.

[139] In some embodiments, CHO cells used for purification lack glutamine synthetase. In some embodiments, CHO cells are grown in a medium comprising L- glutamine and copper. In some embodiments, the method yields recombinant ASA protein containing less than 35 ng/mg HCP. In some embodiments, CHO cells of the present method comprise one or more exogenous nucleic acids encoding the recombinant ASA protein and/or the FGE. In some embodiments, the one or more exogenous nucleic acids are integrated in the genome of the cells. In some embodiments, the one or more exogenous nucleic acids are present on one or more extra-chromosomal constructs. In some embodiments, the one or more exogenous nucleic acids are present on a single extra-chromosomal construct. In some embodiments, the cells overexpress the recombinant ASA protein. In some embodiments, the cells overexpress FGE.

[140] As explained in greater detail below, the characteristics of ASA composition produced by the present invention include, but are not limited to several features, e.g. the method of the present invention, in some embodiments, yields recombinant ASA protein comprising at least 60% conversion e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%) of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, at least 1% of total glycans in N-linked glycosylation sites of the recombinant ASA protein is di-mannose-6-phosphate (di-M6P). In some embodiments, the N-linked glycosylation sites comprise one or more of N140, N166, and/or N332 of SEQ ID NO: 1. In some embodiments, the recombinant ASA protein has a specific activity of at least 100 U/mg as determined by an in vitro assay. In some embodiments, the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 1% to about 12% capped M6P glycan, about 20% to about 40% total M6P glycan, about 10% to about 25% mono-M6P glycan, about 0.5% to about 15% of di-M6P glycan, about 5% to about 18% hybrid glycan, about 3% to about 26% neutral glycan, or about 28% to about 42% sialic acid moieties per molecule of ASA protein. In some embodiments, the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 7% to about 11% capped M6P glycan, about 21% to about 40% total M6P glycan, about 10% to about 18% hybrid glycan, about 3% to about 21% neutral glycan, or about 28% to about 42% sialic acid moieties per molecule of ASA protein. In some embodiments, the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 1% to about 10% capped M6P glycan, about 24% to about 32% total M6P glycan, about 3% to about 11% hybrid glycan, about 16% to about 27% neutral glycan, or about 34% to about 43% sialic acid moieties per molecule of ASA protein.

[141] In some embodiments, the amount of di-M6P glycan is at least 5%. In some embodiments, the amount of di-M6P glycan is at least 10%. In some embodiments, the amount of di-M6P glycan is at least 15%. In some embodiments, the ratio of a mono-M6P to di-M6P is between about 2:1 to 1 : 1.

[142] In some embodiments, the recombinant ASA protein contains less than 80 ng/mg HCP. In some embodiments, the recombinant ASA protein has an amino acid sequence of SEQ ID NO: 1. [143] The purification methods of purifying ASA from an impure preparation described herein include, affinity chromatography, and one or more of the following chromatographic steps, including but not limited to ion exchange chromatography (e.g., anion exchange chromatography and/or cation exchange chromatography), mixed mode chromatography, and/or hydrophobic interaction chromatography, viral inactivation, including reducing agent, e.g., DTT.

[144] In the chromatography steps, the appropriate volume of resin used when packed into a 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 e.g., 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 process as well as the purification process in order to obtain production and purification of ASA on an industrial scale. Accordingly parameters such as column size, diameter, and flow rate can be increased in order to comply with the speed and efficiency of such large-scale production. In some embodiments, the diameter of the column ranges from about 50-100 mm, the volume ranges from about 100-300 ml, and flow rate is between about 40-400 cm/hour (e.g., between about 100 cm/hour and 150 cm/hour) or about 5 to 100 ml.

[145] Exemplary steps of the purification process are depicted in FIG. 2A and 2B. The purification process described herein relates to a method of large-scale production of ASA. In some exemplary embodiments, the process is by culturing CHO cells co-expressing recombinant ASA protein and a formylglycine generating enzyme (FGE) in suspension in a large-scale culture vessel in medium containing copper.

[146] In some embodiments, the method of purifying recombinant arylsulfatase A (ASA) protein from an impure preparation comprises affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 70 ng/mg HCP. In some embodiments, the method comprises 5 chromatography steps or less. In some embodiments, the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 70 ng/mg HCP. In some embodiments, the affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are carried out in sequential order. Affinity chromatography

[147] The purification methods described herein can include one or more steps of affinity chromatography (e.g., immuno-affinity chromatography, immobilized metal ion affinity chromatography, and/or immobilized ligand affinity chromatography). In some embodiments, the affinity chromatography is carried out using a single column. The “high- yield” process of the present invention comprises affinity column purification, and improved capture and downstream chromatographic steps, which reduce impurities and greatly improve yield.

[148] Briefly, affinity chromatography is a chromatographic technique which relies on highly specific interactions, such as, for example, between a receptor and ligand, an antigen and antibody, or an enzyme and substrate. As is known by a person skilled in the art, selective molecules employed in an affinity chromatography step in the purification methods described herein are based on properties of recombinantly produced ASA e.g., three dimensional structure, glycosylation, etc.) that can be exploited by the selective molecule. Exemplary selective molecules (or capture reagents) that can be utilized in an affinity chromatography step include protein A, protein G, an antibody, a metal ion (e.g., nickel), specific substrate, ligand or antigen. In some embodiments, a suitable selective molecule for an affinity chromatography step of the present invention utilizes an anti-Arylsulfatase A antibody (e.g., an anti-human Arylsulfatase A antibody). Suitable anti-Arylsulfatase A antibodies may be obtained commercially or through immunization of non-human animals (e.g., a mouse, rat, rabbit, chicken, goat, sheep, horse or other suitable animal for producing antibodies against a human protein).

[149] Generally, a molecule of interest (e.g., recombinant ASA) is trapped on a solid or stationary phase or medium through interaction with a selective molecule, while other, undesired molecules are not trapped as they are not bound by the selective molecule(s). The solid medium is then removed from the mixture, optionally washed, and the molecule of interest released from the entrapment by elution. In some embodiments, ASA is eluted from an affinity column by changing the ionic strength through a gradient. For example, salt concentrations, pH, pl, and ionic strength may be used to separate or to form the gradient to separate. In some embodiments, elution of ASA from the affinity chromatography column is carried out using an elution buffer comprising 50 mM glycine-HCl and 50 mM NaCl at pH

3.1.

[150] In some embodiments, a recombinant ASA protein may be produced with a tag in order to facilitate purification by affinity chromatography. As is known by a person skilled in the art, protein tags may 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 the sample. For example, certain lectins specifically bind a particular carbohydrate molecule and can be used to separate glycoproteins from non-glycosylated proteins, or one glycoform from another glycoform.

[151] In some embodiments, affinity column purification is followed by a sterilizing filtration using Sartopore 2, a polyethersulfone (PES) liquid filtration, using a membrane of 0.2 uM pore size.

[152] In some embodiments, the eluates are pooled and subjected to viral inactivation.

Viral Inactivation

[153] In some embodiments, the purification methods described herein include one or more steps of viral inactivation. In some embodiments, the viral inactivation comprises a solvent and/or a detergent. The solvent or detergent can include, for example, polysorbate 20, polysorbate 80, Tri-n-Butyl-Phosphate (TnBP), or a combination thereof. Viral inactivation may involve 3-24 hours of incubation in the solvent or detergent. In another embodiment, the viral inactivation comprises virus filtration, e.g., by using a Planova™ filter.

[154] It is understood that these methods are intended to give rise to a preparation of an enzyme, which is substantially free of infectious viruses and which can be denoted a “virus-safe product”. In addition, it is contemplated that the various methods can be used independently or in combination.

[155] Virus-inactivation can be accomplished by the addition of one or more “virus-inactivating agents” to a solution comprising the enzyme. In some embodiments, a virus-inactivating step is performed prior to chromatographic purification steps (i.e., before loading the impure preparation onto the first chromatography column) in order to assure that the agent is not present in the final product in any amounts or concentrations that will compromise the safety of the product when used as a pharmaceutical or when the product is used for the preparation of a pharmaceutical; other embodiments employ depth filters during one or more additional phases of purification. For example, in some embodiments, an inventive method according to the invention further includes a step of viral removal after the last chromatography column.

[156] The term “virus-inactivating agent” is intended to denote an agent (e.g., detergent) or a method, which can be used in order to inactivate lipid-enveloped viruses as well as non-lipid enveloped viruses. The term “virus-inactivating agent” is to be understood as encompassing both a combination of such agents and/or methods, whenever that is appropriate, as well as only one type of such agent or method.

[157] Typical virus-inactivating agents are detergents and/or solvents, most typically detergent- solvent mixtures. It is to be understood that the virus inactivating agent is optionally a mixture of one or more detergents with one or more solvents. A wide variety of detergents and solvents can be used for virus inactivation. The detergent may be selected from the group consisting of non-ionic and ionic detergents and is selected to be substantially non-denaturating. Typically, a non-ionic detergent is used as it facilitates the subsequent elimination of the detergent from the rASA preparation in the subsequent purification steps. Suitable detergents are described, e.g. by Shanbrom et al., in US Patent 4,314,997, and US Patent 4,315,919. Typical detergents are those sold under the trademarks Triton X-100 and Tween 20 or Tween 80. Preferred solvents for use in virus-inactivating agents are di- or trialkylphosphates as described e.g. by Neurath and Horowitz in US Patent 4,764,369. A typical solvent is tri(n-butyl) phosphate (TnBP). An especially preferred virus-inactivating agent for the practice of the present invention is Tween 80, but, alternatively, other agents or combinations of agents can be used. The typical agent added in such a volume that the concentration of Tween-80 in the ASA-containing solution is within the range of about

0.5-4.0% by weight, preferably at a concentration of about 1% by weight. TnBP can then be added to a final concentration of 0.3% calculated based on the new volume of the sample containing ASA.

[158] The virus-inactivation step is conducted under conditions inactivating enveloped viruses resulting in a substantially virus-safe rhASA-containing solution. In general, such conditions include a temperature of 4-37°C, such as 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 is sufficient, preferably 10-18 hours, such as about 14 hours, to ensure sufficient virus inactivation. However, the appropriate conditions (temperature and incubation times) depend on the virus-inactivating agent employed, pH, and the protein concentration and lipid content of the solution.

[159] It is contemplated that other methods for removal of or inactivating virus can also be employed to produce a virus-safe product, such as the addition of methylene blue with subsequent inactivation by radiation with ultraviolet light.

[160] The purification methods described herein can include one or more steps of viral 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 passage of the ASA containing solution which is a result of a purification step through a sterile filter and subsequently passage of the sterile filtered solution through a nanofilter. By “sterile filter” is meant a filter, which will substantially remove all micro-organisms capable of propagating and/ or causing infection. Whereas it is typical that the filter has a pore size of about 0.1 micron, the pore size could range between about 0.05 and 0.3 micron. It may be feasible to replace or combine virus filtration of the sample as performed in the purification process with contacting the sample with a detergent.

[161] In some embodiments, the pore size of a viral filter may be selected to ensure that only the dimeric form is filtered (i.e., that the octameric form may be retained by the filter, or cause viral filter plugging). For examples, a viral filter with a pore size of 20 nm will retain the octameric form of ASA, but not the dimeric form.

Addition of reducing agent (e.g. DTT)

[162] In some embodiments of the present invention, the viral inactivation step is followed by addition of a reducing agent, for example, dithiothreitol (DTT), for example, 10- 1000 mol of DTT is added per mol of recombinant ASA protein. In some embodiments, 10 mol of DTT is added per mol of recombinant ASA protein. Addition of a reducing agent results in dimeric form of ASA, which facilitates purification process.

[163] In some embodiments of the invention, addition of DTT reduces the low molecular weight impurities, minimizing elevated impurity levels due to CHO cells as measured by size exclusion chromatography, especially of low molecular weight (LMW) species, which are present at levels below the limit of quantitation in ASA purified from human cell lines, but are elevated in the process of the present invention. The LMW species are typically misfolded species that are unable to form the predominant dimeric species, likely formed by oxidized cysteine residues leading to disulfide scrambling and disrupting the normal dimer formation interface. In order to minimize LMW species, in the process of the present invention, an exemplary reducing agent, for example, dithiothreitol (DTT) is added reduce disulfide bonds. Addition of DTT is carried out at different concentrations (redox equivalent of 10 mol/mol to 1000 mol/mol of DTT/rhASA). In some embodiments, 10 mol DTT/mol ASA is added leading to reduced LMW species without increasing HMW species.

[164] In some embodiments, DTT is added at different steps of the purification process (unclarified, clarified, viral inactivation pool, and Nuvia Q eluate) as well as at different concentrations (redox equivalent of 10 mol/mol to 1000 mol/mol of DTT/rhASA). In some embodiments, addition of DTT is into the viral inactivation pool. In some embodiments, addition of DTT is after viral inactivation.

[165] In some embodiments, following DTT addition, the recombinant ASA is filtered through a Durapore PVDF membrane, and then subjected to ion exchange chromatography (e.g. anion exchange chromatography).

Ion exchange chromatography

[166] The purification methods described herein can include one or more steps of ion exchange chromatography (e.g., anion exchange chromatography and/or cation exchange chromatography).

[167] As is known by a person skilled in the art, ion exchangers (e.g., anion exchangers and/or cation exchangers) may be based on various materials with respect to the matrix as well as to the attached charged groups. For example, the following matrices may be used, in which the materials mentioned may be more or less crosslinked: 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.

[168] The ion exchange resin can be prepared according to known methods. Typically, an equilibration buffer, which allows the resin to bind its counter ions, can be passed through the ion exchange resin prior to loading the sample or composition comprising the polypeptide and one or more contaminants onto the resin. Conveniently, the equilibration buffer can be the same as the loading buffer, but this is not required.

[169] In an optional embodiment of the invention, the ion exchange resin can be regenerated with a regeneration buffer after elution of the polypeptide, such that the column can be re-used. Generally, the salt concentration and/or pH of the regeneration buffer can be such that substantially all contaminants and the polypeptide of interest are eluted from the ion exchange resin. Generally, the regeneration buffer has a very high salt concentration for eluting contaminants and polypeptide from the ion exchange resin.

Anion Exchange Chromatography

[170] Embodiments of the invention include, for example, providing a sample of arylsulfatase A (e.g., recombinant arylsulfatase A), and subjecting the sample to anion exchange chromatography, e.g., anion exchange chromatography described herein. In some embodiments, the Nuvia Q™ anion exchange filter is used. For the anion exchange resin, the charged groups which are covalently attached to the matrix can be, for example, diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and/or quaternary ammonium (Q). In some embodiments, the anion exchange resin employed is a Q Sepharose column. The anion exchange chromatography can be performed using, e.g., Q SEPHAROSE™ Fast Flow, Q SEPHAROSE™ High Performance, Q SEPHAROSE™ XL, CAPTO™ Q, DEAE, TOYOPEARL GIGACAP® Q, FRACTOGEL® TMAE (trimethylaminoethyl, a quarternary ammonia resin), ESHMUNO™ Q, NUVIA™ Q, or UNOSPHERE™ Q. Other anion exchangers can be used within the scope of the invention, including but not limited to, but are not limited to, quaternary amine resins or “Q-resins” (e.g., CAPTOTM-Q, Q- SEPHAROSE®, QAE SEPHADEX®); diethylaminoethane (DEAE) resins (e.g., DEAE- TRISACRYL®, DEAE SEPHAROSE®, benzoylated naphthoylated DEAE, diethylaminoethyl SEPHACEL®); AMBERJET® resins; AMBERLYST® resins;

AMBERLITE® resins (e.g., AMBERLITE® IRA-67, AMBERLITE® strongly basic, AMBERLITE® weakly basic), cholestyramine resin, ProPac® resins (e.g., PROP AC® SAX-10, PROPAC® WAX-10, PROPAC® WCX-10); TSK-GEL® resins (e.g, TSKgel DEAE-NPR; TSKgel DEAE-5PW); and ACCLAIM® resins.

[171] In embodiments, the anion exchange chromatography is performed using FRACTOGEL® TMAE (trimethylaminoethyl, a quarternary ammonia resin). [172] In some embodiments, subjecting the sample of arylsulfatase A to the anion exchange chromatography is performed at a temperature about 23°C or less, about 18°C or less, or about 16°C or less, e.g., about 23°C, about 20°C, about 18°C, or about 16°C.

[173] Typical mobile phases for anionic 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% polar solution. In certain embodiments, the mobile phase comprises between about 1% to about 100%, about 5% to about 95%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% polar solution at any given time during the course of the separation.

[174] In certain embodiments, rASA is loaded at a binding capacity 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., between about 12 AU/L resin and about 15 AU/L resin, or between about 15 AU/L resin and about 19 AU/L resin. In some embodiments, the sample of arylsulfatase A is loaded onto the anion exchange chromatography column at a binding capacity 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 the anion exchange chromatography column at a binding capacity ranging between about 4.5-20 g/L resin (e.g., ranging between 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).

[175] The aqueous solution comprising the ASA and contaminant(s) can be loaded onto the anionic resin as a mobile phase using a loading buffer that has a salt concentration and/or a pH such that the polypeptide and the contaminant 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 wherein the salt concentration is increased. Finally, the ASA can be eluted by an elution buffer of increasing salt concentration.

Optionally, elution of the enzyme may also be mediated by gradually or stepwise decreasing the pH. The fractions containing ASA activity can be collected and combined for further purification.

[176] In some embodiments, loading the sample of arylsulfatase A onto the anion exchange chromatography column is performed with 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 from about 1 mM to about 25 mM, e.g., from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, or from about 5 mM to about 10 mM. In some embodiments, salt concentration in the mobile phase is a gradient (e.g., linear or non-linear gradient). In some embodiments, salt concentration in the mobile phase is constant. In some embodiments, salt concentration in the mobile phase may increase or decrease stepwise. In some embodiments, loading the sample of arylsulfatase A onto the anion exchange chromatography column is performed at a pH from about 5 to about 9, e.g., from about 6 to about 8, e.g., about 7.

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

[178] In one embodiment, the elution buffer contains sodium phosphate. For example, the sodium phosphate concentration of the elution buffer is from about 20 mM to about 50 mM, e.g., from about 25 mM to about 45 mM, e.g., 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 from about 200 mM to about 300 mM, e.g., from about 240 mM to about 280 mM. In some embodiments, eluting the arylsulfatase A from the anion exchange chromatography column is performed at a pH from about 5 to about 9, e.g., from about 6 to about 8, e.g., about 7.

[179] In some embodiments, eluting the arylsulfatase A from the anion exchange chromatography column includes one or more steps of elution peak collection. For example, the elution peak collection starts from about 50 mAU at the ascending side to about 50 mAU at the descending side, e.g., from about 100 mAU at the ascending side to about 50 mAU at the descending side, from about 200 mAU at the ascending side to about 50 mAU at the descending side, from about 50 mAU at the ascending side to about 100 mAU at the descending side, from about 50 mAU at the ascending side to about 200 mAU at the descending side, or from about 100 mAU at the ascending side to about 100 mAU at the descending side, e.g., as determined by spectrophotometry, e.g., at 280 nM.

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

[181] The loading buffer, washing buffer, and elution buffer described herein can include one or more buffering agents. For example, the buffering agent can be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is between about 1 mM and about 500 mM, e.g., between about 10 mM and about 250 mM, between about 20 mM and about 100 mM, between about 1 mM and 5 mM, between about 5 mM and 10 mM, between about 10 mM and 50 mM, or between about 50 mM and about 100 mM, e.g., about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM. [182] Yield, activity and purity following anion exchange chromatography may vary. In some embodiments, the arylsulfatase A activity yield is at least about 75%, e.g., at least about 85%, e.g., between about 85% and about 99%, or between about 90% and about 99%. In some embodiments, the protein yield (AU or Absorbance Units) is from about 10% to 50%, e.g., from about 20% to about 35%, or from about 25% to about 30%, e.g., as determined by spectrophotometry, e.g., at 280 nm. In some embodiments (e.g., those using a TMAE column as described below), the elution pool protein activity yield (AU or Absorbance Units) is from about 70% to 400%, e.g., from about 80% to about 390%, or from about 90% to about 350%, or from about 100% to 150%, greater than at least 95%, e.g., as determined by spectrophotometry, e.g., at 280 nm. In some embodiments (e.g., those using a TMAE column as described below), the host cell protein (HCP) log reduction value (LRV) is between about 0.5 and about 1.1, e.g., between about 0.6 and 0.9, or between about 0.7 and 0.8. In some embodiments (e.g., those 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 higher, as determined by, for example, capillary electrophoresis-SDS PAGE. In preferred embodiments, the activity yield, HCP LRV and purity (as determined by capillary electrophoresis-SDS PAGE) following anion exchange chromatography are at least about 90%, at least about 0.6 and at least about 80%, respectively.

[183] In preferred embodiments of the invention, an anionic exchange column with a high loading capacity is used. In certain embodiments of the invention, the column is characterized by a loading range between 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 greater than 4.3 g/L (e.g., is or greater than about 10 g/L, 12.5 g/L, 15 g/L, 17.5 g/L, or 20 g/L). In certain embodiments, the binding capacity of the resin is between 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 certain embodiments, the loading capacity is greater than about 80 AU/L. In some embodiments, the high load capacity column is a TMAE column. In particular 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, a Eshmuno Q column, a UNOsphere Q column, or a GigaCap Q column.

[184] In particular embodiments of the invention, a TMAE column is preequilibrated with a buffer comprising about 20 mM MES-Tris and 1000 mM NaCl at a pH of 7.0. In certain embodiments, the column is equilibrated with a buffer comprising 50 mM MES-Tris at a pH of 7.0. In some embodiments, the load 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 may be optimized and assessed by A280 absorbance as described herein.

[185] In particular embodiments utilizing a TMAE column (e.g., a Fractogel TMAE column), very little product is lost in the flow through during loading, even at loading capacities greater than 15 g/L. The capability of increasing loading capacity while minimizing flow-through loss is a significant improvement in purification methodology. In particular 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%).

[186] After loading, in some embodiments, a TMAE column is washed at least once. In particular embodiments, the column is washed twice. A first or second wash buffer may comprise an optimized level of sodium chloride. In some embodiments, the amount of sodium chloride is a first or second wash buffer is between 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, a first wash buffer comprises 50 mM MES-Tris at pH 7.0. In some embodiments, a second wash buffer comprises, 20 mM MES-Tris, 100 mM NaCl at pH 7.0. Further optimization of wash conditions, particularly second wash conditions, is encompassed within embodiments of the present invention. For example, increasing the salt concentration of a second wash may improve host cell protein (HCP) log reduction values (LRV) and overall purity, but decrease both activity and A280 yield. As described herein, particular washing conditions must be balanced with the elution conditions described below in order to provide the optimal combination of purity, activity and yield.

[187] In embodiments of the invention, recombinant ASA bound to a TMAE column is eluted with an elution buffer. In some embodiment, the amount of sodium chloride in the elution buffer is optimized. In particular embodiments, the amount of sodium chloride in the elution buffer is between about 150-300 mM (e.g. about 150-290 mM, about 150-280 mM, about 150-270 mM, about 150-260 mM, about 150-250 mM, about 150-240 mM, about 150-230 mM, about 150-220 mM, about 150-210 mM, about 170-290 mM, about 170-280 mM, about 170-270 mM, about 170-260 mM, about 170-250 mM, about 170-240 mM, about 170-230 mM, about 170-220 mM, about 170-210 mM 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 a particular example, the elution buffer comprises 50 mM MES-Tris and IM NaCl at a pH of 7.0. In some embodiments, the A280 yield following elution is greater than 60% of the load (e.g., about 60%, about 70%, about 80% or higher). Further optimization of elution conditions is encompassed within embodiments of the present invention. For example, increase elution salt concentration (i.e., conductivity) provides better yield but results in poorer purity and HCP removal. And as noted above, particular washing conditions must be balanced with the elution conditions in order to provide the optimal combination of purity, activity and yield.

Cation Exchange Chromatography

[188] In some embodiments, the method further includes 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 a typical embodiment, the cation exchange chromatography comprises sulfopropyl (SP) cation exchange chromatography, but other cation chromatography membranes or resins can be used, for example, a MUSTANG™ S membrane, an S- SEPHAROSE™ resin, or a 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 optimized temperature, e.g., as described herein, to enhance target binding and/or decrease impurity binding. For example, the cation exchange chromatography can be performed at a temperature of about 23°C, 18°C, 16°C, or less.

[189] In one embodiment, the cation exchange chromatography includes 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, e.g., 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.

[190] The aqueous solution comprising the arylsulfatase A and contaminant(s) can be loaded onto the cationic resin using a loading buffer that has a salt concentration and/or a pH such that the polypeptide and the contaminant bind to the cation exchange resin. The resin can then be washed with one or more column volumes of equilibration butter or loading buffer, and optionally followed by one or more column volumes of wash buffer wherein the salt concentration is increased. Finally, the arylsulfatase A can be eluted in an elution buffer. The fractions containing arylsulfatase A activity can be collected and combined for further purification.

[191] In a typical embodiment, the NaCl concentration and/or pH of the loading buffer, washing buffer, and/or elution buffer, can be optimized, e.g., as described herein, to enhance target binding and/or decrease 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.

[192] In some embodiments, subjecting the sample of arylsulfatase A to a 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 columns can be equilibrated with more than 3, e.g., 5 to 10 column volumes of 0.01 M NaAc, 0.01 M NaCl, 0.03 M acetic acid, pH 4.2.

[193] In some embodiments, the sample can be loaded in the buffer from the previous step of the purification process, or the sample can 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 from about 1 mM to about 25 mM, e.g., from about 5 mM to about 20 mM, e.g., 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 from about 10 mM to about 100 mM, e.g., about 20 mM, about 40 mM, or about 60 mM. In some embodiments, loading the sample of arylsulfatase A onto the cation exchange chromatography column is performed at a pH from about 3.0 and about 6.0, e.g., from about 4.0 and about 5.0, e.g., about 4.0, about 4.3, or about 4.5. In some embodiments, the sample of arylsulfatase A is loaded onto the cation exchange chromatography column at a binding capacity 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., between about 10 AU/L resin and about 14 AU/L resin, or between about 10 AU/L resin and about 12 AU/L resin.

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

[195] In some embodiments, eluting the 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 from about 25 mM to about 75 mM, e.g., from about 45 mM to about 60 mM, e.g., about 45 mM, about 50 mM, about 55 mM, or about 55 mM. In some embodiments, eluting the arylsulfatase A from the cation exchange chromatography column is performed at a pH from about 3.0 and about 6.0, e.g., from about 4.0 and about 5.0, e.g., about 4.0, about 4.3, or about 4.5. Thus, as one particular example, the sample can be eluted in a buffer comprising 0.02 M NaAc, 0.05 M NaCl, pH 4.5. Alternatively, the sample can be eluted in any other elution buffer described herein for cation exchange chromatography. [196] In some embodiments, eluting the arylsulfatase A from the cation exchange chromatography column includes one or more steps of elution peak collection. For example, the elution peak collection starts from about 50 mAU at the ascending side to about 50 mAU at the descending side, e.g., from about 100 mAU at the ascending side to about 50 mAU at the descending side, from about 200 mAU at the ascending side to about 50 mAU at the descending side, from about 50 mAU at the ascending side to about 100 mAU at the descending side, from about 50 mAU at the ascending side to about 200 mAU at the descending side, or from about 100 mAU at the ascending side to about 100 mAU at the descending side, e.g., as determined by spectrophotometry, e.g., at 280 nM. Collected eluate peaks may be pooled.

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

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

[199] The yield following cation exchange chromatography may vary. In some embodiments, the arylsulfatase A activity yield is at least about 75%, e.g., at least about 80%, e.g., between about 80% and about 105%. In some embodiments, the protein yield (AU or Absorbance Units) is from about 65% to 100%, e.g., from about 70% to about 95%, e.g., as determined by spectrophotometry, e.g., at 280 nm.

[200] The purity and activity following cation exchange chromatography is greatly improved. In some embodiments, the host cell protein (HCP) log reduction value (LRV) is between about 1.0 and about 2.5, e.g., between about 1.5 and about 2.0 or between about 1.7 and about 1.9. The specific activity of the purified arylsulfatase A can be at least from 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 determined by a method 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 by, e.g., 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 certain embodiments, decreasing the salt concentration of the loading buffer and lowering its pH enhances binding ASA to the cation exchange column but does not impact impurity binding. In other words, an optimal balance of salt concentration and pH, as set forth above, can increase yield after cation exchange chromatography without adversely affecting purity.

[201] In some embodiments, the pH of a cation exchange eluate pool may be adjusted. In certain embodiments, the pH is adjusted immediately prior to viral filtration. Cation exchange eluate (e.g., SP eluate) may be pH adjusted to about 5.5, about 6.0 about 6.5 or about 7.0 using a pH adjustment buffer comprising 0.25M sodium phosphate, 1.33M sodium chloride, 0.34M sodium citrate, pH 7.0. In certain embodiments, the pH-adjusted SP eluate pool is viral filtered on a Planova 20N filter. In some embodiments, the yield relative to input following viral filtration of pH-adjusted cation exchange eluate is between 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 viral filtration is significant as it verifies that pH adjustment to about 6.0 allows octamers of ASA (which are about 20 nm in diameter) to dissociate into dimeric form. Thus, the pore size of a viral filter may be selected to ensure that only the dimeric form is filtered (i.e., that the octameric form may be retained by the filter, or cause viral filter plugging). For examples, a viral filter with a pore size of 20 nm will retain the octameric form of ASA, but not the dimeric form.

[202] In some embodiments, ion exchange chromatography is followed by mixed mode chromatography.

Mixed-Mode Chromatography

[203] The purification methods described herein can include one or more steps of mixed-mode chromatography. Mixed-mode chromatography is a type of chromatography in which several modes of separation are applied to resolve a mixture of different molecules, typically in liquid chromatography. For example, a mixed-mode separation can include combinational phases with ion-exchange and reversed phase characteristics at the same time. These stationary phases with more than one interaction type are available from several column manufacturers. In some embodiments, Capto MMC Impres column is used for mixed mode chromatography.

[204] In some embodiments, the present invention features a method of purifying arylsulfatase A from a sample, where the method includes, 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., mixed mode chromatography described herein, such as a method including ceramic hydroxyapatite (HA) chromatography, e.g., 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 Media, CHT™ Ceramic Hydroxyapatite Type II Media, BIO-GEL® HT Hydroxyapatite, and BIO-GEL® HTP Hydroxyapatite.

[205] 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 the mixed mode exchange chromatography is performed at a temperature about 23°C or less, about 18°C or less, or about 16°C or less, e.g., about 23°C, about 20°C, about 18°C, or about 16°C.

[206] In some embodiments, loading the sample of arylsulfatase A onto the mixed mode chromatography column is performed with a loading buffer. In one embodiment, the loading buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is from about 1 mM to about 10 mM, e.g., from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, e.g., 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 from about 100 mM to about 400 mM, e.g., from about 200 to about 300 mM, e.g., about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[207] In some embodiments, loading the sample of arylsulfatase A onto the mixed mode chromatography column is performed at a pH from about 5 to about 9, e.g., from about 6 to about 8, e.g., about 7.

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

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

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

[211] In some embodiments, washing the mixed mode chromatography column is performed at a pH from about 5 to about 9, e.g., from about 6 to about 8, e.g., about 7.

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

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

[214] In some embodiments, the purification of ASA by mixed mode chromatography succeeds the purification by ion-exchange chromatography e.g., anion exchange chromatography). In some embodiments, it is contemplated, however, that these steps could be performed in the reverse order.

[215] Yield following mixed mode chromatography may vary. In some embodiments, the arylsulfatase A activity yield is at least about 80%, e.g., at least about 90%, e.g., between about 80% and about 115%. In some embodiments, the protein yield (AU or Absorbance Units) is from about 30% to 80%, e.g., from about 35% to about 75%, or from about 50% to about 70%, e.g., as determined by spectrophotometry, e.g., at 280 nm.

[216] Purity following mixed mode chromatography is greatly improved. In some embodiments, the specific activity of the purified arylsulfatase A is at least from 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 determined by a method described herein. In some embodiment, 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 by, e.g., 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 host cell protein (HCP) log reduction value (LRV) is between about 0.3 and about 0.6, e.g., between about 0.4 and 0. 5.

[217] In some embodiments, mixed mode chromatography is followed by hydrophobic interaction chromatography.

Hydrophobic Interaction Chromatography (HIC)

[218] The purification methods described herein can include subjecting the sample of arylsulfatase A to hydrophobic interaction chromatography (HIC). In one embodiment, the hydrophobic interaction chromatography includes phenyl chromatography. In some embodiments, HIC is carried out using a Capto Phenyl Impres™ column. [219] In other embodiments, the hydrophobic interaction chromatography includes butyl chromatography or octyl chromatography. In some embodiments, subjecting the sample of arylsulfatase A to HIC is performed at a temperature about 23°C or less, about 18°C or less, or about 16°C or less, e.g., 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.

[220] Hydrophobic interaction chromatography utilizes the attraction of a given molecule for a polar or non-polar environment, and in terms of protein, this propensity is governed by the hydrophobicity or hydrophilicity of residues on the exposed, outer surface of a protein. Thus, proteins are fractionated based upon their varying degrees of attraction to a hydrophobic matrix, typically an inert support with alkyl linker arms of 2-18 carbons in chain length. 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). Thus, the HIC column is typically a butyl SEPHAROSE™ column or a phenyl SEPHAROSE™ column, most typically a phenyl SEPHAROSE™ column.

[221] In some embodiments, the hydrophobic interaction chromatography includes 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).

[222] In some embodiments, subjecting the 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 the arylsulfatase A from the column. Loading, washing and elution in HIC basically follow the same principle as described above for the ion-exchange chromatography, but often nearly opposite conditions to those used in ion exchange chromatography are applied. Thus, the HIC process involves the use of a high salt loading buffer, which unravels the protein to expose hydrophobic sites. The protein is retained by the hydrophobic ligands on the column, and is exposed to a gradient of buffers containing decreasing salt concentrations. As the salt concentration decreases, the protein returns to its native conformation and eventually elutes from the column. Alternatively proteins may be eluted with PEG.

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

[224] The use of phenyl SEPHAROSE™ as solid phase in the HIC is typical in the present disclosure. Again, it is readily apparent that, when it comes to the exact conditions as well as the buffers and combinations of buffers used for the loading, washing and elution processes, a large number of different possibilities exist. In a typical embodiment, the column can be equilibrated in a buffer which contains 0.05 M NaPO4, 1 M NaCl, pH 5.5. As of convenience the sample can be loaded in the buffer from the previous step of the purification process, or the sample can be loaded using a loading buffer.

[225] In some embodiments, washing the HIC column is performed with one or more washing buffers. For example, washing the HIC column can include two or more (e.g., a first and a second) washing steps, each using a different washing buffer. In some embodiments, the washing buffer contains sodium chloride. For example, the sodium chloride concentration of the washing buffer is from about 100 mM to about 1.5 M, e.g., from about 250 mM to about 1 M, e.g., about 250 mM, about 500 mM, about 750 mM, or about 1 M. In another embodiment, the washing buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is from about 10 mM to about 100 mM, e.g., about 25 mM, about 50 mM, or about 75 mM. In some embodiments, washing the HIC column is performed at a pH from about 5 to about 7, e.g., from about 5.5 to about 6.5, e.g., about 5.5, about 6.0, or about 6.5. For example, washing can be performed using 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 can be equilibrated, loaded, and washed with any other equilibration, loading, and washing buffers described herein for HIC. [226] In some embodiments, eluting the aryl sulfatase A from the HIC column is performed with an elution buffer. In some embodiments, the elution buffer contains sodium chloride. For example, the sodium chloride concentration of the elution buffer is from about 30 mM to about 100 mM, e.g., from about 45 mM to about 85 mM, e.g., about 50 mM, about 60 mM, about 70 mM, or about 80 mM. In some embodiments, eluting the arylsulfatase A from the HIC column is performed at a pH from about 5 to about 9, e.g., from about 6 to about 8, e.g., 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.

[227] In some embodiments, eluting the arylsulfatase A from the HIC column includes one or more steps of elution peak collection. For example, the elution peak collection starts from about 50 mAU at the ascending side to about 50 mAU at the descending side, e.g., from about 100 mAU at the ascending side to about 50 mAU at the descending side, from about 200 mAU at the ascending side to about 50 mAU at the descending side, from about 50 mAU at the ascending side to about 100 mAU at the descending side, from about 50 mAU at the ascending side to about 200 mAU at the descending side, or from about 100 mAU at the ascending side to about 100 mAU at the descending side, e.g., as determined by spectrophotometry, e.g., at 280 nM.

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

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

[230] Yield following HIC may vary. In some embodiments, the arylsulfatase A activity yield is at least about 60%, e.g., at least about 70%, e.g., between about 70% and about 100%. In some embodiments, the protein yield (AU or Absorbance Units) is from about 45% to 100%, e.g., from about 50% to about 95%, or from about 55% to about 90%, e.g., as determined by spectrophotometry, e.g., at 280 nm.

[231] Purity following HIC is greatly improved. In some embodiments, the specific activity of the purified arylsulfatase A is at least from 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 determined by a method described herein.

[232] 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 by, e.g., 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 host cell protein (HCP) log reduction value (LRV) is between about 0.6 and about 1.2, e.g., between about 0.7 and 0.95.

Ultrafiltration/Diafiltration

[233] In some embodiments, the purification methods described herein can include one or more steps of downstream ultrafiltration and/or diafiltration. 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. In some embodiments, a 10 kDa Hydrosart™ membrane is used.

[234] Ultrafiltration refers to a membrane separation process, driven by a pressure gradient, in which the membrane fractionates 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 permeate components and increase further the concentration of retained components. Ultrafiltration is often combined with diafiltration into ultrafiltration/diafiltration (UFDF) purification steps.

[235] Embodiments of the invention utilize at least one, at least two, at least three or more downstream UFDF purification steps. One or more diafiltrations may occur within UFDF step (e.g., UFDFDF). In some embodiments, the protein yield (AU or Absorbance Units) following downstream UFDF, relative the amount from the preceding purification step, is from about 90% to 105%, e.g., from about 95% to about 100%, e.g., from about 97% to about 99%, as determined by spectrophotometry, e.g., at 280 nm. In some embodiments, essentially no protein is lost during UFDF.

[236] In some embodiments of the 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 determined by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and/or reverse phase-high performance liquid chromatography (RP-HPLC). In some embodiment, 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 by, e.g., 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. The specific activity of the rASA is at least 100 U/mg, e.g., 100 U/mg to 200 U/mg, e.g., as determined by a sulfatase release assay, as described below.

[237] In some embodiments, arylsulfatase A is purified by separation from contaminants according to their size in an acidic environment by tangential flow filtration. Arylsulfatase A forms an octamer at low pH with a theoretical molecular weight of 480 kDa and will therefore be retained by a relatively open membrane while most of the contaminants will pass this 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).

[238] In a typical embodiment, the diafiltration buffer comprises 0.01 M sodium phosphate-citrate, 0.137 M NaCl, pH 6.0.

[239] In some embodiments, as the starting material for this process is a suspension of arylsulfatase A as eluted from the chromatography column in the previous step of the process, the pH in this suspension is adjusted to 4-5 by addition of 0.2-1 M Na-acetate pH 4.5. Diafiltration is then performed against 1-10 buffer volumes of Na-acetate pH 4.0-5.5 in a manner well known to somebody skilled in the art. The filtration can be performed with the application of several different filter types with nominal weight cut-off values ranging from 20-450 kDa, however it is typical to use a filter with a cut-off value ranging from 100 - 300 kDa. For further processing of the arylsulfatase A containing solution the pH is adjusted to a value within the range between 7 and 8 by addition of Tris-base to a final concentration of approximately 20-50 mM. [240] As an alternative to the acidic tangential flow filtration as described above, separation of ASA from the contaminants can be obtained with acidic gel filtration using essentially the same conditions and compositions of buffers. The filtration is performed at low pH through a gel filtration column, which has been equilibrated with a solution at low pH, for example, a 0.2-0.9 M solution of Na-acetate at pH 4-5. As an option, the solution of arylsulfatase A can be concentrated by tangential flow filtration through a 20-50 kDa filter prior to the gel filtration. The extent of concentration may vary considerably so that arylsulfatase A may be concentrated from about 0.1 mg/ml to about 50 mg/ml, preferably to about 5 mg/ml.

[241] 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 Na-acetate, pH 5.4-5.7.

[242] In embodiments, a surfactant such as polysorbate-20 (P20) is added to the compositions comprising purified ASA protein prior to cold storage. In embodiments, the composition comprises a surfactant such as P20 in a concentration of about 0.0001 %(v/v) to about 0.01 %(v/v), about 0.001 %(v/v) to about 0.01 %(v/v), about 0.001%(v/v), about 0.002 %(v/v), about 0.003 %(v/v), about 0.004 %(v/v), about 0.005 %(v/v), about 0.006 %(v/v), about 0.007 %(v/v), about 0.008 %(v/v), about 0.009 %(v/v), or about 0.01 %(v/v).

Characterization of Purified ASA Proteins

[243] Purified recombinant ASA protein is characterized using various methods, including, but not limited to parameters described below.

Specific Activity

[244] Purified recombinant ASA protein may also be characterized by evaluating functional and/or biological activity. The enzyme activity of a recombinant ASA composition may be determined using methods known in the art. Typically the methods involve detecting the removal of sulfate from a synthetic substrate, which is known as sulphate release assay. One example of an enzyme activity assay involves the use of ion chromatography. This method quantifies the amount of sulfate ions that are enzymatically released by recombinant ASA from a substrate. The substrate may be a natural substrate or a synthetic substrate. In some cases, the substrate is heparin sulfate, dermatan sulfate, or a functional equivalent thereof. Typically, the released sulfate ion is analyzed by ion chromatography with a conductivity detector. In this example, the results may be expressed as U/mg of protein where 1 Unit is defined as the quantity of enzyme required to release 1 pmole sulfate ion per hour from the substrate. In some embodiments, the purified recombinant ASA has a specific activity of at least about 100 U/mg, 110 U/mg, 120 U/mg, 130 U/mg, 140 U/mg, 150 U/mg, 160 U/mg, 170 U/mg, 180 U/mg, 190 U/mg, 200 U/mg. In some embodiments, the purified recombinant ASA has a specific activity ranging from about 100-200 U/mg (e.g., about 100-190 U/mg, 100-180 U/mg, 100-170 U/mg, 100-160 U/mg, 100-150 U/mg, 100-140 U/mg, 100-130 U/mg, 100-120 U/mg, 100-110 U/mg). In some embodiments, the recombinant ASA protein has a specific activity of between about 101 U/mg to about 134 U/mg as determined by an in vitro assay.

[245] In another example, enzyme activity of a recombinant ASA composition may be determined by measuring the removal of sulfate from a 4-methylumbelliferyl-sulfate (4- MUF-sulfate) substrate to form the fluorescent methylumbelliferone. In this example, the fluorescence signal generated by a test sample can be used to calculate enzyme activity (in mU/mL) using a standard of 4-MUF. One milliunit of activity is defined as the quantity of enzyme required to convert 1 nanomole of 4-MUF-sulfate to 4-MUF in 1 minute at 37 °C. Specific activity may then calculated by dividing the enzyme activity by the protein concentration.

[246] In some embodiments, activity is determined by hydrolysis of the synthetic, chromogenic substrate, para-Nitrocatechol sulphate (pNCS) which has an end product, paraNitrocatechol (pNC) that absorbs light at 515 nm. The following equation may be used to calculate the enzyme activity in pmol pNCS hydrolyzed / min x ml (=Units/ml):

Vtot (ml) X pA = Units/ml (1) pM /1000 x Vsample (ml) x Incubation time (min) where: pA = absorbance of sample - absorbance of blank

Vtot (ml) = total reaction volume in ml (in this case 0.15 ml) Vsample (ml) = added sample volume in ml (in this case 0.05 ml) pM = the molar extinction coefficient for the product pNC, which in this case is 12 400 M-l cm-1.

Equation 1 can be simplified as: pA x (0.15 / (12 400/1000 x 0.05 x 30)) = X pmol / (minute x ml) (=Units/ml) (1)

To calculate the specific activity in pmol pNC consumed/(minute x mg) (= Units/mg), equation 1 is divided by the protein concentration of the sample:

Eq. 1 / Protein cone, (mg/ml) = Y pmol / (minute x mg) = Units/mg (2)

[247] In any example, the protein concentration of a recombinant ASA composition may be determined by any suitable method known in the art for determining protein concentrations. In some cases, the protein concentration is determined by an ultraviolet light absorbance assay. Such absorbance assays are typically conducted at about a 280 nm wavelength (A280).

[248] In some embodiments, purified recombinant ASA has a specific activity on a 4-methylumbelliferone substrate in a range of about 1.0 x 10³ mU/mg to 100.0 x 10³ mU/mg. In some embodiments, purified recombinant ASA has a specific activity on a 4- methylumbelliferone substrate of about 1.0 x 10³ mU/mg, about 2.0 x 10³ mU/mg, about 3.0 x 10³ mU/mg, about 4.0 x 10³ mU/mg, about 5.0 x 10³ mU/mg, about 10.0 x 10³ mU/mg, about 15.0 x 10³ mU/mg, about 20.0 x 10³ mU/mg, about 25.0 x 10³ mU/mg, about 30.0 x 10³ mU/mg, about 35.0 x 10³ mU/mg, about 40.0 x 10³ mU/mg, about 45.0 x 10³ mU/mg, about 50.0 x 10³ mU/mg, or more.

Formylglycine content and Bioactivity

[249] In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein having the amino acid sequence of SEQ ID NO: 1, wherein at least 70% of the recombinant ASA protein in the composition comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). Increased formylglycine improves bioactivity of ASA enzyme. Formylgly cine-generating enzyme (FGE) is responsible for catalyzing the conversion of specific cysteine residues on the ASA protein to formylglycine, which is a post-translational modification that is essential for catalytic activity. In some embodiments, the process of the present invention purifies ASA from CHO cells with the addition of copper to the medium. Addition of copper has beneficial effects on specific activity and % formylglycine conversion. In the absence of copper, the specific activity and % FG conversion were less than observed in ASA purified from human cells. However, when copper cofactor was included in the medium, enzyme activity increased. The increased bioactivity of purified ASA is beneficial for improving efficacy, dosing and costs in enzyme replacement therapy, as fewer doses or less enzyme is needed for therapy due to increased bioactivity and efficacy.

[250] In some embodiments, the recombinant ASA protein comprises between about 75% to greater than 95% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises at least 95%, at least 96%, at least 97%, at least 98%, at least 99% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises 100% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises between about 92% to about 98% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

[251] In some embodiments, the recombinant ASA protein comprises between about 78% to about 86% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly). In some embodiments, the recombinant ASA protein comprises at least 78%, at least 80%, at least 82%, at least 84% or at least 86% conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

Glycan Mapping

[252] In some embodiments, a purified recombinant ASA protein may be characterized by its proteoglycan composition, typically referred to as glycan mapping. Without wishing to be bound by any theory, it is thought that glycan linkage along with the shape and complexity of the branch structure may impact in vivo clearance, lysosomal targeting, bioavailability, and/or efficacy.

[253] Typically, a glycan map may be determined by enzymatic digestion and subsequent chromatographic analysis. Various enzymes may 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) may be detected by chromatographic analysis. For example, phosphoglycans may be detected by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) or size exclusion High Performance Liquid Chromatography (HPLC). In some embodiments, the proteoglycan map is determined using liquid chromatography with UV and mass spectrometry detection (LC-UV-MS). The quantity of glycan (e.g., phosphoglycan) represented by each peak on a glycan map may be calculated using a standard curve of glycan (e.g., phosphoglycan), according to methods known in the art and disclosed herein.

[254] In embodiments, the purified recombinant ASA protein is present as species comprising: neutral recombinant ASA protein, sialylated recombinant ASA protein, mannose-6-phosphated recombinant ASA protein, N-acetyl-glucosamine mannose-6- phosphated recombinant ASA protein, or hybrid recombinant ASA protein, or any combination thereof.

[255] In some embodiments, a purified recombinant ASA protein according to the present invention is characterized with a glycan map comprising at least nine peak groups indicative of neutral (peak group 1 and 2), sialylated (peak group 3, 4 and 6), capped mannose-6-phosphated (peak group 5 and 8), mono-mannose-6-phosphated (peak group 7 and 9) ASA protein, respectively.

[256] The relative amount of glycan corresponding to a group may be determined based on the peak group area relative to the corresponding peak group area in a predetermined reference standard.

[257] In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 1% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or greater of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P).

[258] In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein between about 6.9-8.9% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 6.9%, at least 7.9% or at least 8.9% of total glycans in N-linked glycosylation sites is di-mannose-6- phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein between about 7-21% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P). In some aspects, provided herein is a composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20% or at least 21% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di- M6P).

[259] In some embodiments, the N-linked glycosylation sites comprise one or more ofN140, N166, and/or N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise N140 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise N166 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise one or more of N140, N166 or N332 of SEQ ID NO: 1. In some embodiments, the N-linked glycosylation sites comprise one or more of N140, N166 and N332 of SEQ ID NO: 1.

[260] In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of: about 1% to about 12% capped M6P glycan, about 20% to about 40% total M6P glycan, about 5% to about 21% of di-M6P glycan, about 3% to about 18% hybrid glycan, about 3% to about 26% neutral glycan, and about 28% to about 43% sialic acid moieties per molecule of ASA protein. In some embodiments, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of about 7% to about 11% capped M6P glycan, about 21% to about 40% total M6P protein, about 7% to about 21% of di-M6P ASA protein, about 10% to about 18% hybrid glycan, about 3% to about 21% neutral glycan, and about 28% to about 42% sialic acid moieties per molecule of ASA protein. In some embodiments, about 1% to about 10% capped M6P glycan, provided herein is a composition, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of about 24% to about 32% total M6P glycan, about 3% to about 11% hybrid glycan, about 16% to about 26% neutral glycan, and about 34% to about 43% sialic acid moieties per molecule of ASA protein.

[261] In some embodiments, the composition comprises at least about 5% di-M6P glycan. In some embodiments, the composition comprises at least about 10% di-M6P glycan. In some embodiments, the composition comprises at least about 15% di-M6P glycan.

[262] In some embodiments, the composition comprises a ratio of mono-M6P to di- M6P of between about 2: 1 to 1 : 1.

[263] In embodiments, a peak group area of neutral recombinant ASA protein is the range characteristic of any of the exemplary formulations described herein, with a variation of about 1% to about 10% in either direction for each endpoint of the exemplified range (e.g., a variation of about 1% to about 5% for each endpoint of the exemplified range).

[264] Various reference standards for glycan mapping are known in the art and can be used to practice the present invention.

[265] It is contemplated that the glycosylation pattern of a purified recombinant ASA protein e.g., a composition comprising purified recombinant ASA protein having a ratio of mono-M6P to di-M6P of between about 2: 1 to 1 : 1) may impact the bioavailability, targeting, or efficacy of the protein. It is contemplated that the increased di-M6P leads to better cellular uptake and lysosomal targeting increasing efficacy of enzyme replacement therapy.

Purity

[266] The purity of purified recombinant ASA protein is typically measured by the level of various impurities (e.g., host cell protein or host cell DNA) present in the final product. For example, the level of host cell protein (HCP) may be measured by ELISA or SDS-PAGE. In some embodiments, the purified recombinant ASA protein contains less than 70 ng HCP/mg ASA protein (e.g., less than 70, 60, 50, 40, 30, 20, 10, 5 ng HCP/mg ASA protein). In embodiments, purified recombinant ASA protein contains less than about 70 ng HCP/mg ASA. In embodiments, purified recombinant ASA protein contains less than about 60 ng HCP/mg ASA. In embodiments, purified recombinant ASA protein contains less than about 50 ng HCP/mg ASA. In embodiments, purified recombinant ASA protein contains less than about 40 ng HCP/mg ASA. In embodiments, purified recombinant ASA protein contains less than about 30 ng HCP/mg ASA. In embodiments, purified recombinant ASA protein contains less than about 20 ng HCP/mg ASA.

[267] In some embodiments, the purified recombinant ASA protein contains less than about 40 pg/mg, 35 pg/mg, 30 pg/mg, 25 pg/mg, 20 pg/mg, 15 pg/mg, or 10 pg/mg Host Cell DNA (HCD). In embodiments, purified recombinant ASA protein contains less than about 10 pg/mg, 5 pg/mg, 1 pg/mg HCD per ASA protein. In embodiments, purified recombinant ASA protein contains less than about 1 pg/mg HCD per ASA protein.

[268] In some embodiments, the purified recombinant ASA protein, when subject to SDS-PAGE with Coomassie Brilliant Blue staining, has no new bands with intensity greater than the 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% assay control.

[269] In some embodiments, the purified recombinant ASA protein, when subject to SDS-PAGE with Western blotting against HCP, has no bands with intensity greater than the 15 kDa HCP band assay control, and no new bands with intensity greater than the 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or 1.0% assay control. In embodiments, no more than three HCP bands are detected.

[270] In some embodiments, the purified recombinant ASA protein, when subject to SDS-PAGE with silver staining, has no new bands with intensity greater than the 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% assay control.

[271] In some embodiments, the host cell protein (HCP) log reduction value (LRV) is between about 0.3 and about 0.6, e.g., between about 0.4 and 0.5. Various assay controls may be used, in particular, those acceptable to regulatory agencies such as FDA.

[272] The purity of purified recombinant ASA protein may also be determined by one or more of size exclusion chromatography -high performance liquid chromatography (SEC-HPLC), capillary electrophoresis-SDS PAGE (CE-SDS PAGE), and/or reverse phase- high performance liquid chromatography (RP-HPLC) (e.g., using columns of octadecyl (C18)-bonded silica, and carried out at an acidic pH with TFA as a counter-ion). In some embodiments of the invention, the major peak in the chromatogram is ASA. Parameters that may be altered or optimized to increase resolution include gradient conditions, organic modifier, counter ion, temperature, column pore size and particle size, solvent composition and flow rate. Purity levels may be discerned by main peak percentage, as known to those of skill in the art. For example, purity may be determined by integrating the main and side peaks observed and calculating the main peak’s percentage of the total area.

[273] In some embodiments of the invention, addition of DTT reduces the low molecular weight impurities. The use of CHO cell lines leads to elevated impurity levels as measured by size exclusion chromatography, especially of low molecular weight (LMW) species, which are present at levels below the limit of quantitation in ASA purified from human cell lines, but are elevated in the process of the present invention. The LMW species are typically misfolded species that are unable to form the predominant dimeric species, likely formed by oxidized cysteine residues leading to disulfide scrambling and disrupting the normal dimer formation interface. In order to minimize LMW species, in the process of the present invention, an exemplary reducing agent, for example, dithiothreitol (DTT) is added reduce disulfide bonds. Addition of DTT is carried out at different concentrations (redox equivalent of 10 mol/mol to 1000 mol/mol of DTT/rhASA). In some embodiments, 10 mol DTT/mol ASA is added leading to reduced LMW species without increasing HMW species. In some embodiments, addition of DTT into the viral inactivation pool resulted in the highest reduction in LMW species.

[274] In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and as determined by the main peak percentage of SEC-HPLC is greater than or equal to 95% (e.g., about 96%, about 97%, about 98%, about 99% or higher). In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and as determined by the main peak percentage of SEC-HPLC is greater than or equal to 97% (e.g., about 97%, about 98%, about 99%, or higher).

[275] In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and as determined by main peak percentage of RP-HPLC is greater than or equal to 97% (i.e., about 97%, about 98%, about 99% or higher). In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and as determined by main peak percentage of RP-HPLC is greater than or equal to 98% (i.e., about 98%, about 99% or higher).

Charge Profile

[276] Purified recombinant ASA may be characterized by the charge profile associated with the protein. Typically, protein charge profile reflects the pattern of residue side chain charges, typically present on the surface of the protein. Charge profile may be determined by performing an ion exchange (IEX) chromatography (e.g., HPLC) assay on the protein. In some embodiments, a “charge profile” refers to a set of values representing the amount of protein that elutes from an ion exchange column at a point in time after addition to the column of a mobile phase containing an exchange ion.

[277] Typically, a suitable ion exchange column is an anion exchange column. For example, a charge profile may be determined by strong anion exchange (SAX) chromatography using a high performance liquid chromatography (HPLC) system. In general, recombinant ASA adsorbs onto the fixed positive charge of a strong anion exchange column and a gradient of increasing ionic strength using a mobile phase at a predetermined flow rate elutes 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 acid) ASA species. The concentration of proteins in the eluate is detected by ultraviolet light absorbance (at 280 nm).

[278] In some embodiments, recombinant ASA adsorbs at about pH 8.0 in 20 mM TRIS-HC1 onto the fixed positive charge of a Mini Q PE column 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.

[279] In some embodiments, a charge profile may be depicted by a chromatogram of absorbance units versus time after elution from the HPLC column. The chromatogram may comprise a set of one or more peaks, with each peak in the set identifying a subpopulation of recombinant ASAs of the composition that have similar surface charges.

Peptide Mapping

[280] In some embodiments, peptide mapping may be used to characterize amino acid composition, post-translational modifications, and/or cellular processing; such as cleavage of a signal peptide, and/or glycosylation. Typically, a recombinant protein may be broken into discrete peptide fragments, either through controlled or random breakage, to produce a pattern or peptide map. In some cases, a purified ASA protein may be first subjected to enzymatic digest prior to analytic analysis. Digestion may be performed using a peptidase, glycoside hydrolase, phosphatase, lipase or protease and/or combinations thereof, prior to analytic analysis. The structural composition of peptides may 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.

Metals Analysis

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

[282] In some embodiments, residual phosphorous is measured and compared to a reference sample. Without wishing to be bound by any particular theory, it is hypothesized that residual phosphorus contributes to maintaining drug substance pH. In some embodiments of the invention, residual phosphorous is between about 10-50 ppm (i.e., between 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 between about 5-7 (i.e., between about 5.5-7.0, about 5.5-6.5, about 5.5-6.0, about 6.0-7.0, about 6.0-6.5, about 6.0-6.4, about 6.0-6.3, about 6.0-6.2, about 6.0-6.1, about 6.1- 6.2).

[283] In some embodiments, recombinant ASA purified according to the methods disclosed herein contains calcium. Without wishing to be bound by any particular theory, it is hypothesized that calcium ions present in the active site of ASA may be necessary for enzymatic activity. In some embodiments of the invention, calcium is present at levels between about 1-20 ppm (i.e., between 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).

Pharmaceutical Composition and Administration

[284] In one aspect, provided herein is a method of treating metachromatic leukodystrophy (MLD), the method comprising administering the composition of any one of the preceding claims to a subject in need of treatment.

[285] Purified recombinant ASA protein may be administered to a MLD patient in accordance with known methods. For example, purified recombinant ASA protein may be delivered intrathecally, intraventricularly, intravenously, subcutaneously, intramuscularly, parenterally, transdermally, or transmucosally (e.g., orally or nasally)). In some embodiments, a recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by intravenous administration.

[286] In some embodiments, a recombinant ASA or a pharmaceutical composition containing the same is administered to the cerebrospinal fluid (CSF). In some embodiments, a recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by intraventricular administration. In some embodiments, a recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by intrathecal administration. As used herein, the term “intrathecal administration” or “intrathecal injection” refers to an injection into the spinal canal (intrathecal space surrounding the spinal cord). Various techniques may be used including, without limitation, lateral cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like. In some embodiments, “intrathecal administration” or “intrathecal delivery” according to the present invention refers to IT administration or delivery via the lumbar area or region, i.e., lumbar IT administration or delivery. As used herein, the term “lumbar region” or “lumbar area” refers to the area between the third and fourth lumbar (lower back) vertebrae and, more inclusively, the L2-S1 region of the spine. In some embodiments, a recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by intrathecal administration as described in PCT international publications WO2011/163648 and WO201 1/163650, incorporated herein by reference in their entirety.

[287] In some embodiments, a recombinant ASA or a pharmaceutical composition containing the same is administered to the subject by subcutaneous (i.e., beneath the skin) administration. For such purposes, the formulation may be injected using a syringe. However, other devices for administration of the formulation are available such as injection devices (e.g., the Inject-ease and Genject devices); injector pens (such as the GenPen); needleless devices (e.g., MediJector and BioJector); and subcutaneous patch delivery systems.

[288] In some embodiments, the pharmaceutical composition is administered at a dose of at least 150 mg. In some embodiments, administration of the pharmaceutical composition results in a reduction in the amount of glucosaminoglycans within the CSF of the patient. [289] In some embodiments, intrathecal or intraventricular administration may be used in conjunction with other routes of administration (e.g., intravenous, subcutaneously, intramuscularly, parenterally, transdermally, or transmucosally (e.g., orally or nasally)).

[290] The present invention contemplates single as well as multiple administrations of a therapeutically effective amount of a recombinant ASA or a pharmaceutical composition containing the same described herein. A recombinant ASA or a pharmaceutical composition containing the same can be administered at regular intervals, depending on the nature, severity and extent of the subject’s condition (e.g., a lysosomal storage disease). In some embodiments, a therapeutically effective amount of a recombinant ASA or a pharmaceutical composition containing the same may be administered periodically at regular intervals (e.g., once every year, once every six months, once every five months, once every three months, bimonthly (once every two months), monthly (once every month), biweekly (once every two weeks), weekly, daily or continuously). In some embodiments, the recombinant ASA or a pharmaceutical composition containing the same is administered at least once weekly.

[291] A recombinant ASA or a pharmaceutical composition containing the same can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and therapeutic agent can be sterile. The formulation should suit the mode of administration.

[292] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interference with their activity. In some embodiments, a water-soluble carrier suitable for intravenous administration is used.

[293] The composition or medicament, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.

[294] The composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. 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 ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.

Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[295] 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. In embodiments, the osmolality of a formulation is about 250 to about 350 mOsmol/kg (e.g., about 255 to about 320 mOsmol/kg, about 260 to about 310 mOsmol/kg, or about 280 to about 300 mOsmol/kg).

[296] In another embodiment, the ASA is formulated in a physiological buffer, such as: 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) formulation buffer II containing (in mM): Tris-HCl (10), Glycine (25 - 30), Mannitol (230 - 270), and water for injection.

[297] Arylsulfatase A purified by a method herein can be used as a medicament for reducing the sphingolipid 3-O-sulfogalactosylceramide (galactosyl sulphatide) levels within cells in the peripheral nervous system and/or within the central nervous system in a subject suffering from and/or being diagnosed with Metachromatic Leukodystrophy. The administration of ASA will lead to decreased impairment of motor-learning skills and or to increased nerve motor conduction velocity and/or nerve conduction amplitude. As used herein, the term “therapeutically effective amount” is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and/or ameliorating the 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 to thereby treat such lysosomal storage disease or the symptoms thereof (e.g., a reduction in or elimination of the presence or incidence of “zebra bodies” or cellular vacuolization following the administration of the compositions of the present invention to a subject). Generally, the amount of a therapeutic agent (e.g., a recombinant lysosomal enzyme) administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art will be readily able to determine appropriate dosages depending on these and other related factors. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.

[298] A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[299] It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the 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 claimed invention.

EXAMPLES

Example 1. Exemplary High-yielding ASA Purification Process

[300] This example illustrates an improved process for purifying rhASA at high yield, while maintaining purity of ASA. In some embodiments, the rhASA is derived for a large-scale manufacturing process.

[301] The high yield process of the present invention was produces large quantities of highly pure ASA for therapeutic use, thereby meeting large dosing requirements for treating metachromatic leukodystrophy. This Example and the Examples below describe the improved ASA purification process. In some aspects, as illustrated in this example and the examples below, the recombinant purified protein is rhASA. In some aspects the drug product is a sterile solution of recombinant human arylsulfatase A, 30 mg/mL, in 154 mM sodium chloride with 0.005% (vol/vol) polysorbate 20 (P-20) at pH 6.0.

[302] Briefly, CHO cell-lines are used to achieve high titer (e.g., about to 0.2 g/L) thereby increasing Target Viable Cell Density (VCD) and improving ASA yield in the process of the present invention. In some embodiments, CHO cell lines lack glutamine synthetase and cells are grown in the presence of exogenous L-glutamine in the medium. In some embodiments, copper is added to the medium.

[303] An expression vector comprising rhASA (GenBank Identifier: 7262293) and formylglycine generating enzyme (FGE) (GenBank identifier: 30840148) expression cassettes are used to increase formylglycine at position 51 of rhASA by FGE. Formylglycine at position 51 of rhASAis a post-translational modification required for ASA enzymatic activity.

[304] The steps of the process of the present invention are shown in FIG. 2A and FIG. 2B. As shown in FIG. 2A, the process involves purification from CHO cells (such as CHOZN® GS-/- ) at a high cell density, and includes steps for clarification, and purification and polishing processes that use different resins to limit CHO host cell protein and impurity species. Recombinant ASA is produced in a large scale from CHO cells starting from thawing a vial of CHO cells and expanding cells in a shake flask, followed by wave bag, then in a 500 liter seed reactor to a 1500 liter production reactor, where media exchange is carried out by perfusion for 25 days at 1.0 VVD (volume of media per bioreactor volume per day) to maintain optimal viable cell density, followed by clarification and a single column affinity chromatography step daily. Following this, Sartopore filtration is carried out, followed by optional storage of intermediate purified product. Specifically, as shown in FIG. 2A, the upstream process includes an affinity chromatography capture step, which improves process yield. As shown in FIG. 2B, the process further includes a DTT reduction step after viral inactivation, for purification of dimeric rhASA. Further, changes in downstream chromatography steps ensure that impurities are minimized to below acceptable levels. For example, in some embodiments, a ultra-high binding capacity anion exchange resin with wide pH and flow rate working range is used (e.g. Nuvia Q), followed by a weak cation exchange or mixed mode resin (e.g. Capto MMC ImpRes) and subsequently a hydrophobic interaction chromatography resin for high-resolution intermediate and polishing steps (e.g. Capto Phenyl ImpRes). Exemplary characteristics of the ASA protein purified by the present invention are as shown in Table 2A and Table 2B below.

[305] Table 2A. Exemplary characteristics of purified ASA protein

Table 2B. Exemplary characteristics of purified ASA protein at large-scale

[306] Overall, the ASA purification process of the present invention using a CHO cell line and affinity purification results in high yield of 5200 grams of recombinant ASA per 2000L of cell culture (more than 20 times the amount of recombinant ASA produced compared to other processes), high % FG leading to high biological activity and potency, and product impurity below acceptable limits, thereby lowering costs and increasing efficiency of a manufacturing process yielding a highly pure, safe and high quality ASA product for therapeutic use. Example 2. Effect of adding a reducing agent, dithiothreitol (DTT) in the purification process.

[307] This example illustrates the effects of adding a reducing agent, for example, dithiothreitol (DTT) to the ASA purification process.

[308] The use of CHO cell lines leads to elevated levels of impurity from size exclusion chromatography, especially of low molecular weight (LMW) species present at levels below the limit of quantitation in ASA purified from human cell lines. The LMW species are typically misfolded species that are unable to form the predominant dimeric species. Without wishing to be bound by any particular theory, it is contemplated that they are likely formed by oxidized cysteine residues leading to disulfide scrambling and disrupting the normal dimer formation interface.

[309] In order to minimize LMW species in the CHO cell-based process, an exemplary reducing agent, for example, dithiothreitol (DTT) was added to reduce disulfide bonds. The effect of addition of DTT was tested at different steps of the purification process (unclarified, clarified, viral inactivation pool, and Nuvia Q eluate) as well as at different concentrations (redox equivalent of 10 mol/mol to 1000 mol/mol of DTT/rhASA).

[310] As shown in FIG. 6A-FIG. 6C, the addition of DTT into the viral inactivation pool resulted in the highest reduction in LMW species. Without wishing to be bound by any particular theory, it is contemplated that the benefit of DTT addition at this step is likely due to the higher level of control in DTT addition and the increased purity of the rhASA drug substance.

[311] Further, the results showed that DTT addition at 10 mol/mol DTT/rhASA redox units has benefits in decreasing LMW without the undesirable side-effect of concomitantly increasing the HMW species.

[312] Higher levels of DTT lead to increased HMW species. The levels of HMW are somewhat mitigated by additional downstream purification via mixed mode chromatography (MMC).

[313] Overall, the addition of DTT after the viral inactivation step resulted in reduced LMW species in the CHO cell-based process. A DTT level of 10 mol/mol DTT/rhASA redox units was found to limit LMW without increasing HMW species.

Example 3. Effect of adding Copper to the medium for growing CHO cells

[314] This example illustrates the effects of addition of copper to the medium growing CHO cells on product quality. Formylgly cine-generating enzyme (FGE) is responsible for catalyzing the conversion of specific cysteine residues on the ASA protein to formylglycine, which is a post-translational modification that is essential for catalytic activity.

[315] Briefly, a study was conducted in small-scale bioreactors to evaluate the impact of addition of copper as a cofactor on ASA enzyme activity in the CHO cell-based process.

[316] As shown in Table 3, the results showed that addition of copper had beneficial effects on specific activity and % formylglycine conversion. In the absence of copper, the specific activity and % FG conversion were less in ASA purified from CHO cells than observed in ASA purified from human cells. However, when copper was included in the medium, enzyme activity increased. Without wishing to be bound by any particular theory, it is contemplated that copper acts as a cofactor for the proper function of formylglycine- generating enzyme (FGE).

Table 3. Effects of addition of copper on specific activity and % FG of ASA

[317] These results demonstrate that ASA produced by the CHO cell-based process had increased specific activity and increased FG% with the addition of copper to the medium.

Example 4. Stability of purified rhASA

[318] This example illustrates the stability of purified rhASA produced by the method of the present invention at long-term (<65°C), accelerated (5±3°C) and stress storage conditions (25 ±2°C).

[319] Briefly, drug substance from a pilot run 200 liter bioreactor of the CHO cellbased process was tested for stability and compared with ASA produced from human cells. Stability was compared over three months at long-term (<65°C), accelerated (5±3°C) and stress storage conditions (25 ±2°C). [320] Protein concentration, pH, main peak and high molecular weight peak (BMW) by size exclusion chromatography, and specific activity was measured to evaluate stability at long-term conditions (<-65°C), accelerated conditions (5±3°C) and stress conditions (25±2°C).

[321] FIG. 3 A is a graph of stability of protein concentration following long-term storage (<-65°C). FIG. 3B is a graph of stability of pH following long-term storage (<-65°C). FIG. 3C is a graph of size exclusion chromatography (main peak) following long-term storage. FIG. 3D is a graph of size exclusion chromatography (high molecular weight peak) following long-term storage. (<-65°C). FIG. 3E is a graph showing specific activity of rhASA following long-term storage (<-65°C).

[322] FIG. 4A is a graph of stability of protein concentration following storage under accelerated conditions (5±3°C). FIG. 4B is a graph of stability of pH following storage under accelerated conditions (5±3°C). FIG. 4C is a graph of size exclusion chromatography (main peak) under accelerated conditions (5±3°C). FIG. 4D is a graph of size exclusion chromatography (high molecular weight peak) under accelerated conditions (5±3°C). FIG. 4E is a graph showing specific activity of rhASA under accelerated conditions (5±3°C).

[323] FIG.5 A is a graph of stability of protein concentration following storage under stress conditions (25±2°C). FIG. 5B is a graph of stability of pH following storage under stress conditions (25±2°C). FIG. 5C is a graph of size exclusion chromatography (main peak) under stress conditions (25±2°C). FIG. 5D is a graph of size exclusion chromatography (high molecular weight peak) under stress conditions (25±2°C). FIG. 5E is a graph showing specific activity of rhASA under accelerated conditions (25±2°C).

[324] The results showed rhASA produced from CHO cells by the methods described herein were stable across multiple conditions.

Example 5. In vivo pharmacodynamic studies of rhASA in an MLD mouse model

[325] In this example, ASA produced by the process of the present invention is evaluated for pharmacodynamics in an immunotolerant metachromatic leukodystrophy (MLD) mouse model.

[326] An ASA knockout (ASA-/-) mouse model for MLD is used which shows similar storage patterns of cerebroside-3 -sulfate in various neuronal and non-neuronal tissues. Histopathology of brain tissue demonstrates a reduction of axonal cross-sectional area and astrogliosis in mice that are one year of age. In humans, sulfatide accumulation leads to widespread demyelination in peripheral and central nervous systems (PNS and CNS), and shows severe white matter damage.

[327] Briefly, ASA knockout mice are administered rhASA purified by a CHO cellbased process or rhASA purified from human cells at 4-month and 6 month-old following once weekly dosing for one month.

[328] As described in Table 4, MLD mice are treated with a vehicle control (Group A), rhASA purified by the CHO cell-based process (Groups B and C) or rhASA purified by the human cell-based process (Groups D and E). Untreated control MLD mice (Group F) and WT mice (Group G) serve as age-matched controls. Mice receiving rhASA treatment are treated weekly with either 0.04 (Groups B and D) or 0.21 mg (Groups C and E) of rhASA weekly in a dose volume of 10 pl of test substance and 1 pl of dye.

[329] Intrathecal lumbar (IT-L) injections are carried out using 32-gauge needle attached to a gas tight 10 ul Hamilton syringe in a slow bolus of 5-10 seconds per injection. Imaging is carried out to determine if the infrared dye is distributed throughout the central nervous system.

[330] Clinical signs are recorded daily and body weights assessed weekly. Blood and CSF will be collected. Blood (serum) will be collected at 2, 4, 8, 12 and 24 hours postadministration. CSF is collected pre-dose and at necropsy (about 24 hours after last dose).

[331] Tissue distribution of rhASA is assessed by immunohistochemistry (IHC) in brain, spinal cord, liver and kidney, followed by morphometry to quantify ASA levels in tissue.

[332] LAMP-1 tissue distribution is assessed by imaging, followed by morphometry to determine ASA efficacy.

[333] Table 4. Pharmacodynamic study design in an immunotolerant MLD mouse model

[334] The tissue distribution of rhASA in brain, spinal cord, liver and kidney will be assessed by immunohistochemical (IHC) followed by morphometry using Aperio ImageScope software to quantify ASA tissue levels. The tissue efficacy of rhASA will be assessed by IHC followed by morphometry using Aperio ImageScope software to quantify LAMP-1 tissue levels.

Example 6. In vivo pharmacodynamic studies of rhASA in a non-human primate

[335] In this example, ASA produced by the process of the present invention is evaluated for pharmacodynamics in a non-human primate model.

[336] In this example, cynomolgus (cyno) monkeys are administered 6 mg of ASA purified by a CHO-cell based process or a human cell-based process at the same dosing concentration (mg/ml). Group A will be dosed with ASA purified by a human cell-based process on Day 1, and with the process of the present invention on Day 8. Group B will be dosed with ASA purified by the process of the present invention on Day 1 and with ASA purified by a human cell-based process on Day 8.

[337] Subsequently CSF will be collected pre-dose and for 6-8 timepoints up to 24 hours post-administration.

[338] Clinical signs are monitored daily and body weights measured prior to dosing. Blood and CSF are collected prior to dosing and immediately prior to sacrifice. After the last blood and CSF samples were collected, the animals are sacrificed, necropsied and selected organs harvested and saved for collection. Half of the tissue from 6-8 different brain regions, spinal cord, liver and kidney is snap frozen, and the other half of the same tissue is assessed by 10% neutral buffered formalin (NBF) for immunohistochemistry.

Table 5. Pharmacodynamic studies of rhASA in a non-human primate

Table 6. Tissue distribution post intrathecal (IT) administration

Example 7. Pharmacokinetic and biodistribution studies of rhASA in a non-human primate

[339] In this example, ASA produced by the process of the present invention was evaluated for serum and CSF pharmacokinetics and tissue distribution in non-human primates (NHPs) after single intrathecal lumbar (IT-L) injection.

[340] In this example, cynomolgus monkeys (3/group) were dosed with a single IT- L injection with vehicle control (TAK-611 Placebo) or with ASA at 9 mg/dose (Human cellbased process or CHO cell-based process) or 3 mg/dose (CHO cell-based process) at a dose volume of 1 mL. Male and female monkeys were assigned to groups of 1/sex/group (vehicle control) or 3/sex/group (Human cell-based process or CHO cell-based process) and designated for terminal necropsy 1, 6, or 24 hours after dosing.

[341] Table 7. PK and biodistribution in non-human primates

[342] Increased positive immunohistochemical staining for arylsulfatase A (ARSA) was observed in nearly all tissues evaluated with the exception of kidney, but it was most intense and extensive in the tissues of the central and peripheral nervous systems. Increased staining was generally confined to the cerebrospinal fluid (CSF) and immediately adjacent tissues, including the surrounding meningeal connective tissue, the superficial neuropil of the brain and spinal cord, the fibrotic scar surrounding the catheter site, and the nerve roots and ganglia. Although modest variability was occasionally present, the staining was generally comparable in intensity in the animals from each dose group regardless of time of necropsy (1, 6 and 24 hours post dose). No differences in staining intensity and distribution were noted between the 9 mg/dose human cell-based process and CHO cell-based process, but staining was generally reduced in the 3 mg/dose CHO cell-based process monkeys, consistent with the reduced dose of ASA administered.

[343] Overall, intrathecal lumbar bolus administration of ASA from human cellbased or CHO cell-based process resulted in an increase in immunohistochemical staining. Example 8. Comparability of ASA drug substance manufactured from human cell-based process and CHO cell-based process

[344] In this example, ASA drug substance produced by the human cell-based process was compared with ASA drug substance produced by the CHO cell-based process.

[345] Table 8. Comparison of ASA drug substance manufactured from human cellbased process and CHO cell-based process

[346] Overall, the ASA drug substance from a CHO cell-based process and a human cell-based process has comparable properties.

Example 9. Comparability of ASA drug product manufactured from human cell-based process and CHO cell-based process

[347] In this example, ASA drug product produced by the human cell-based process was compared with ASA drug product produced by the CHO cell-based process. [348] Table 9. Exemplary characteristics of purified ASA protein from human cellbased process

[349] Table 10. Exemplary characteristics of purified ASA protein from CHO cell- based process

[350] Overall, the ASA drug product from a CHO cell-based process and a human cell-based process has comparable properties, including at a large scale.

Example 10. In vivo efficacy of ASA in reducing short-chain sulfatides

[351] This example illustrates in vivo efficacy of ASA based on the reduction of short-chain sulfatides in the brain of hASAC69S/ASA-/- mice after IT-lumbar administration of human cell-based and CHO cell-based purification processes. [352] An ASA knockout (ASA-/-) mouse model for MLD was used which shows similar storage patterns of cerebroside-3 -sulfate in various neuronal and non-neuronal tissues. In humans, sulfatide accumulation leads to widespread demyelination in peripheral and central nervous systems (PNS and CNS), and shows severe white matter damage.

[353] Briefly, ASA knockout mice are administered rhASA purified by a CHO cellbased process or rhASA purified from human cells, n = 10 for ASA treated groups (5 males and 5 females); n = 6 for Vehicle and WT groups (3 males and 3 females). Intrathecal lumbar (IT-L) injections were carried out using 32-gauge needle attached to a gas tight 10 pl Hamilton syringe in a slow bolus of 5-10 seconds per injection.

[354] FIG. 7A is a graph that shows the reduction in short-chain sulfatides with 16 carbon non-hydroxylated fatty acids in ASA treated animals. FIG. 7B is a graph that shows the reduction in short-chain sulfatides with 18 carbon non-hydroxylated fatty acids in ASA treated animals. FIG. 7C is a graph that shows the reduction in total short-chain sulfatide fatty acids in ASA treated animals. FIG. 7D is a graph that shows C18:00-OH levels which represent short-chain sulfatides with 18 carbon hydroxylated fatty acids. FIG. 7E is a graph that shows C20:0 levels which represents short-chain sulfatides with 20 carbon non- hydroxylated fatty acids.

[355] Vertical lines represent the standard error of mean (SEM). One star (*) represents p-value < 0.05, 2 stars (**) represents p-value < 0.01, three stars (***), p-value <0.001, four stars (****). p-value < 0.0001. As shown in FIG. 7A-7E, C denotes recombinant human arylsulfatase A produced by human cell-based process and D denotes arylsulfatase A produced by CHO cell-based process. The numerical quantities represent millgram quantities, e.g., 0.04 mg; 0.21 mg.; WT: C57/BL6 Wild-Type; ns: not significant.

[356] Overall, the results showed that ASA administration results in reduced accumulation of fatty acids in a dose-dependent manner, with slightly greater reductions seen in CHO cell-based process.

[357] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of examples only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

Claims

CLAIMS What is claimed is:

1. A composition comprising recombinant arylsulfatase A having the amino acid sequence of SEQ ID NO: 1, wherein at least 70% of the recombinant ASA protein in the composition comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

2. The composition of claim 1, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

3. The composition of claim 2, wherein at least 97% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

4. A composition comprising recombinant arylsulfatase A (ASA) protein, wherein at least 1% of total glycans in N-linked glycosylation sites is di-mannose-6-phosphate (di-M6P).

5. The composition of any one of the preceding claims, wherein the N-linked glycosylation sites comprise one or more of N140, N166, and/or N332 of SEQ ID NO: 1.

6. The composition of any one of the preceding claims, wherein the recombinant ASA protein has a specific activity of 50 to 130 U/mg as determined by an in vitro assay.

7. The composition of any one of the preceding claims, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more peak groupsindicative of: capped M6P, total M6P, di-M6P, hybrid, neutral and sialylated glycans.

8. The composition of claim 7, wherein the proteoglycan map is determined by High Performance Anion Exchange Chromatography with Fluorescence Detection (HPAEC-FLD).

9. The composition of claim 8, wherein the HPAEC-FLD uses a 2-aminobenzamide (2 -AB) labeling method.

10. The composition of any one of the preceding claims, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising

(a) about 1% to about 12% capped M6P glycan, about 20% to about 40% total M6P glycans, about 5% to about 21% of di-M6P glycan, about 3% to about 18% hybrid glycan, about 3% to about 26% neutral glycan, and/or about 28% to about 43% sialic acid glycan moieties per molecule of ASA protein;

(b) about 7% to about 11% capped M6P glycan, about 21% to about 40% total glycan, about 7% to about 21% of di-M6P glycan, about 10% to about 18% hybrid glycan, about 3% to about 21% neutral glycan, and about 28% to about 42% sialic acid glycan moieties per molecule of ASA protein;

(c) about 1% to about 10% capped M6P glycan, about 24% to about 32% total M6P glycan, about 3% to about 11% hybrid glycan, about 16% to about 26% neutral glycan, and about 34% to about 43% sialic acid moieties per molecule of ASA protein.

11. The composition of any one of the preceding claims, wherein the amount of di-M6P glycan is at least 5%.

12. The composition of any one of the preceding claims, wherein the amount of diM6P glycan is at least 10%.

13. The composition of any one of the preceding claims, wherein the amount of di-M6P glycan is at least 15%.

14. The composition of any one of the preceding claims, wherein the ratio of a mono- M6P to di-M6P is between about 2: 1 to 1 : 1.

15. The composition of any one of the preceding claims, wherein the recombinant ASA protein contains less than 70 ng/mg Host Cell Protein (HCP).

16. The composition of any one of the preceding claims, wherein the recombinant ASA protein is at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to SEQ ID NO: 1.

17. The composition of any one of the preceding claims, wherein the recombinant ASA protein is present in the composition at a concentration of at least 30 mg/ml.

18. The composition of claim 17, wherein the recombinant ASA protein is present in the composition at a concentration of at least 35 mg/ml.

19. The composition of claim 17, wherein the recombinant ASA protein is present in the composition at a concentration of 40 mg/ml.

20. The composition of any one of the preceding claims, wherein the recombinant ASA protein is purified from CHO cells.

21. The composition of any one of the preceding claims, wherein the recombinant ASA is purified from human cells.

22. A method of treating metachromatic leukodystrophy (MLD), comprising administering the composition of any one of the preceding claims to a subject in need of treatment.

23. The method of claim 22, wherein the composition is administered at a dose of at least 150 mg.

24. The method of claim 22 or 23, wherein the pharmaceutical composition is administered to the CSF.

25. The method of any one of claims 22-24, wherein the pharmaceutical composition is administered by intrathecal or intraventricular injection.

26. The method of any one of claims 22-25, wherein the pharmaceutical composition is administered at least once weekly.

27. The method of any one of claims 22-26, wherein administration of the pharmaceutical composition results in a reduction in the amount of glucosaminoglycans within the CSF of the patient.

28. A method of purifying recombinant arylsulfatase A (ASA) protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 70 ng/mg HCP.

29. The method of claim 28, wherein the method comprises 5 chromatography steps or less.

30. The method of claim 28 or 29, wherein the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography, and wherein the purified recombinant ASA protein contains less than 70 ng/mg HCP.

31. The method of any one of claims 28-30, wherein the affinity chromatography is carried out using a single column.

32. The method of any one of claims 28-31, wherein the affinity chromatography column is cleaned using a chaotropic agent.

33. The method of claim 32, wherein the affinity chromatography column is cleaned using guanidium or urea.

34. The method of claim 33, wherein the affinity chromatography column is cleaned every two, three or more cycles.

35. The method of claim 34, wherein the affinity chromatography column is cleaned every three cycles.

36. The method of any one of claims 28-35, wherein the method comprises affinity chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography in sequential order.

37. The method of any one of claims 28-36, wherein the method further comprises a step of viral inactivation.

38. The method of claim 37, wherein the viral inactivation step comprises addition of a reducing agent.

39. The method of claim 38, wherein the reducing agent is dithiothreitol (DTT).

40. The method of claim 39, wherein the 10-1000 mol of DTT is added per mol of recombinant ASA protein.

41. The method of claim 40, wherein 10 mol of DTT is added per mol of recombinant ASA protein.

42. The method of any one of claims 28-41, wherein the method comprises purifying ASA from CHO cells.

43. The method of claim 42, wherein the CHO cells lack glutamine synthetase.

44. The method of claim 42, wherein the CHO cells are grown in a medium comprising L-glutamine and copper.

45. The method of any one of claims 28-41, wherein the recombinant ASA protein contains less than 70 ng/mg HCP.

46. The method of any one of claims 28-45, wherein at least 70% of the recombinant ASA protein in the composition comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

47. The method of claim 46, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

48. The method of claim 47, wherein at least 97% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

49. The method of any one of claims 28-48, wherein at least 1% of total glycans in N- linked glycosylation sites of the recombinant ASA protein is di-mannose-6-phosphate (di- M6P).

50. The method of any one of claims 49, wherein the N-linked glycosylation sites comprise one or more of N140, N166, and/or N332 of SEQ ID NO: 1.

51. The method of any one of claims 28-50, wherein the recombinant ASA protein has a specific activity of at least 100 U/mg as determined by an in vitro assay.

52. The method of any one of claims 28-51, wherein the recombinant ASA protein is characterized by a proteoglycan map comprising one or more of:

(a) about 1% to about 12% capped M6P glycan, about 20% to about 40% total M6P glycan, about 5% to about 21% of di-M6P glycan, about 3% to about 18% hybrid glycan, about 3% to about 26% neutral glycan, and about 28% to about 43% sialic acid moieties per molecule of ASA protein;

(b) about 7% to about 11% capped M6P glycan, about 21% to about 40% total M6P glycan, about 7% to about 21% of di-M6P glycan, about 10% to about 18% hybrid glycan, about 3% to about 21% neutral glycan, or about 28% to about 42% sialic acid moieties per molecule of ASA protein;

(c) about 1% to about 10% capped M6P glycan, about 24% to about 32% total M6P glycan, about 3% to about 11% hybrid glycan, about 16% to about 26% neutral glycan, or about 34% to about 43% sialic acid moieties per molecule of ASA protein.

53. The method of any one of claims 28-52, wherein the amount of di-M6P glycan is at least 5%.

54. The method of any one of claims 28-52, wherein the amount of di-M6P glycan is at least 10%.

55. The method of any one of claims 28-52, wherein the amount of di-M6P glycan is at least 15%.

56. The method of any one of claims 28-53, wherein the ratio of a mono-M6P to di-M6P is between about 2: 1 to 1 : 1.

57. The method of any one of claims 28-56, wherein the recombinant ASA protein contains less than 70 ng/mg HCP.

58. The method of any one of claims 28-57, wherein the recombinant ASA protein has about 7% to about 11% capped M6P glycan.

59. The method of any one of claims 28-58, wherein the recombinant ASA protein has about 21% to about 40% total M6P glycan.

60. The method of any one of claims 28-59, wherein the recombinant ASA protein has about 7% to about 21% di-M6P glycan.

61. The method of any one of claims 28-60, wherein the recombinant ASA protein has about 7% to about 21% mono-M6P glycan.

62. The method of any one of claims 28-61, wherein the recombinant ASA protein has about 10% to about 18% hybrid glycan.

63. The method of any one of claims 28-62, wherein the recombinant ASA protein has about 3% to about 21% neutral glycan.

64. The method of any one of claims 28-63, wherein the recombinant ASA protein has about 28% to about 42% sialic acid moieties per molecule.

65. The method of any one of claims 52-64, wherein the proteoglycan map is determined by High Performance Anion Exchange Chromatography with Fluorescence Detection (HPAEC-FLD) uses a 2-aminobenzamide (2-AB) labeling method.

66. The method of any one of claims 28-65, wherein the recombinant ASA protein has an amino acid sequence of SEQ ID NO: 1.

67. The method of any one of claims 28-66, wherein the recombinant ASA is produced from CHO cells comprising e one or more exogenous nucleic acids encoding recombinant ASA protein and/or formylgly cine-generating enzyme (FGE).

68. The method of claim 67, wherein the one or more exogenous nucleic acids are integrated in the genome of the cells.

69. The method of claim 67, wherein the one or more exogenous nucleic acids are present on one or more extra-chromosomal constructs.

70. The method of claim 69, wherein the one or more exogenous nucleic acids are present on a single extra-chromosomal construct.

71. The method of any one of claims 28-70, wherein the cells overexpress the recombinant ASA protein.

72. The method of any one of claims 28-71, wherein the cells overexpress FGE.

73. A method for large-scale production of recombinant arylsulfatase (ASA) protein in CHO cells, comprising culturing CHO cells co-expressing a recombinant ASA protein and a formylglycine generating enzyme (FGE) in suspension in a large-scale culture vessel in medium containing copper.

74. The method of claim 73, wherein the method of purifying recombinant arylsulfatase A protein from an impure preparation comprises affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and/or hydrophobic interaction chromatography.

75. The method of claims 73 or 74, wherein the method comprises purifying recombinant arylsulfatase A protein from an impure preparation comprising affinity chromatography and one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography in sequential order.

76. The method of claim 75, wherein elution from affinity chromatography column(s) is carried out using an elution buffer comprising 50mM glycine-HCl and 50 mM NaCl at pH 3.1.

77. The method of any one of claims 73-76, wherein the method comprises addition of a reducing agent during a viral inactivation step.

78. The method of claim 77, wherein the reducing agent is dithiothreitol (DTT).

79. The method of claim 78, wherein the 10-1000 mol of DTT is added per mol of recombinant ASA protein.

80. The method of claim 79, wherein 10 mol of DTT is added per mol of recombinant ASA protein.

81. The method of any one of claims 73-80, wherein at least 70% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

82. The method of any one of claims 73-81, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

83. The method of claim 82, wherein at least 97% of the recombinant ASA protein comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly).

84. The method of any one of claims 73-83, wherein the scale is between about 200 liters to about 2000 liters.

85. The method of claim 84, wherein the scale is about 200 liters.

86. The method of claim 84, wherein the scale is about 2000 liters.

87. The method of any one of claims 73-86, wherein the method produces at least about 1000g, at least about 2000g, at least about 3000g, at least about 4000g, or at least about 5000g of recombinant ASA.

88. The method of any one of claims 73-87, wherein the method yields at least about 2g of recombinant ASA per liter of cell culture, at least about 2.1g of recombinant ASA per liter of cell culture, at least about 2.2g of recombinant ASA per liter of cell culture, at least about 2.3g of recombinant ASA per liter of cell culture, at least about 2.4g of recombinant ASA per liter of cell culture, at least about 2.5g of recombinant ASA per liter of cell culture or at least about 2.6g of recombinant ASA per liter of cell culture.

89. The method of any one of claims 73-88, wherein the method results in a process yield of at least 60%, 70%, 80% of starting recombinant ASA material.

90. A composition comprising recombinant arylsulfatase A (ASA) protein having the amino acid sequence of SEQ ID NO: 1, wherein at least 85% of the recombinant ASA protein in the composition comprises conversion of the cysteine residue corresponding to Cys51 of SEQ ID NO: 1 to formylglycine (FGly), wherein the glycan profile of the recombinant ASA protein comprises 21.6-39.4% total M6P (mono-M6P + di-M6P) glycan and 7.8-20.9% di-M6P glycan per molecule of the recombinant ASA protein, wherein said glycan profile is characterized by High Performance Anion Exchange Chromatography with Fluorescence Detection (HPAEC-FLD) using 2- aminobenzamide (2-AB) labeling method, wherein the recombinant ASA protein has a specific activity of 80-150 U/mg as determined by an in vitro assay.