Purified arylsulfatase A and compositions thereof

Abstract

The present invention provides, inter alia, methods of treating metachromatic white matter dystrophy disease (MLD) and compositions comprising a recombinant arylsulfatase A (ASA) protein using enzyme replacement therapy.

Description

[0001] Cross - reference to related applications

[0002] This application is a divisional application of a Chinese patent application with application number 201880088888.5, filing date December 19, 2018, and invention title "Purified Arylsulfatase A and Compositions Thereof". The original application is the Chinese national phase application of a PCT patent application with international application number PCT / US2018 / 066583. This PCT patent application claims priority to U.S. Provisional Patent Application Serial No. 62 / 607,831 filed on December 19, 2017, U.S. Provisional Patent Application Serial No. 62 / 608,569 filed on December 20, 2017, U.S. Provisional Patent Application Serial No. 62 / 625,814 filed on February 2, 2018, and U.S. Provisional Patent Application Serial No. 62 / 760,597 filed on November 13, 2018. The entire content of each of these patent applications is hereby incorporated by reference. Background of the Invention

[0003] Metachromatic leukodystrophy disease (MLD) is an autosomal recessive disorder caused by a deficiency of arylsulfatase A (ASA). ASA, encoded by the ARSA gene in humans, is an enzyme that breaks down cerebroside 3 - sulfate or sulfogalactosylceramide (sulfatide) into cerebroside and sulfate. In the absence of the enzyme, sulfatides accumulate in the nervous system (e.g., myelin, neurons, and glial cells) and to a lesser extent in visceral organs. The consequences of these molecular and cellular events are progressive demyelination and axonal loss within the CNS and PNS, which are clinically accompanied by severe motor and cognitive dysfunction.

[0004] The defining clinical feature of the disorder is central nervous system (CNS) degeneration, which leads to cognitive impairment (e.g., especially mental retardation, neuropathy, and blindness).

[0005] MLD itself can manifest in young children (late infantile form), where affected children typically start showing symptoms after the first year of life (e.g., at about 15 - 24 months) and generally do not survive beyond 5 years of age. MLD itself can manifest in children (juvenile form), where affected children typically show cognitive impairment at about 3 - 10 years of age and lifespan can vary (e.g., within the range of 10 - 15 years after symptom onset). MLD itself can manifest in adults (adult - onset form) and can occur in individuals of any age (e.g., typically 16 years and older), and the progression of the disease can vary widely.

[0006] Enzyme replacement therapy (ERT) is an approved therapy for treating MLD that involves administering exogenous replacement ASA enzyme, particularly recombinant arylsulfatase A (rhASA) (e.g., recombinant human arylsulfatase A (rhASA)), to MLD patients. Summary of the Invention

[0007] The present invention particularly provides a composition comprising a purified recombinant ASA protein for enzyme replacement therapy and a method for preparing the same.

[0008] As described in the Examples section, exemplary recombinant ASA proteins purified using certain methods described herein meet the purity requirements for sale in the United States and many other countries.

[0009] In addition, the recombinant ASA proteins purified according to the present invention have different characteristics, such as a glycan profile (e.g., a threshold amount of mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein)), which can promote bioavailability, enhance targeting, and / or increase the efficacy of the recombinant ASA protein.

[0010] In one aspect, the present invention provides a method for treating metachromatic leukodystrophy (MLD), the method comprising intrathecal administration to a subject in need thereof of a therapeutically effective dose of a preparation comprising a purified recombinant human arylsulfatase A (rhASA) protein, wherein the purified rhASA protein is characterized by a proteoglycan profile comprising one or more peaks (i.e., species) corresponding to a neutral recombinant ASA protein (neutral ASA protein), a sialylated recombinant ASA protein (sialic acid ASA protein), a mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), an N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and a mixed recombinant ASA protein (mixed ASA protein), and wherein administration of the preparation results in stabilization or reduction of the progression of at least one symptom of MLD.

[0011] In one embodiment, the therapeutically effective dose of purified rhASA is 100 mg.

[0012] In one embodiment, the therapeutically effective dose of purified rhASA is 150 mg.

[0013] In one embodiment, the preparation is administered once a week.

[0014] In one embodiment, the subject has late infantile MLD.

[0015] In one embodiment, stabilization or reduction of the progression of MLD is measured using changes in the Gross Motor Function Classification (GMFC-MLD).

[0016] In one embodiment, the formulation is administered such that the change in GMFC-MLD level after 2 years of treatment in a subject with late infantile MLD compared to baseline is ≤4, ≤3, or ≤2 levels.

[0017] In one embodiment, the formulation is administered such that the GMFC-MLD score is maintained after two years of treatment.

[0018] In one embodiment, maintaining the GMFC-MLD score means that the change in GMFC-MLD after two years of treatment compared to baseline is no greater than 2 levels.

[0019] In one embodiment, the baseline is the evaluated score of GMFC-MLD before the first administration of the formulation.

[0020] In one embodiment, stabilization or reduction of the progression of MLD is measured using the change in the Gross Motor Function Measure - MLD (GMFM-MLD).

[0021] In one embodiment, the formulation is administered such that the GMFM-MLD score is maintained after two years of treatment.

[0022] In one embodiment, the GMFM-MLD score is maintained at >40.

[0023] In one embodiment, stabilization or reduction of the progression of MLD is measured by the level of sulfatide in cerebrospinal fluid.

[0024] In one embodiment, stabilization or reduction of the progression of MLD is measured by the brain N-acetylaspartate / creatine ratio (NAA / cr).

[0025] In one embodiment, stabilization or reduction of the progression of MLD is measured using the Expressive Language Function Classification - MLD level (ELFC-MLD).

[0026] In one embodiment, the baseline GMFC-MLD level of the subject is 1 - 2.

[0027] In one embodiment, the baseline GMFC-MLD level of the subject is 3.

[0028] In one embodiment, the baseline GMFC-MLD level of the subject is 4.

[0029] In one embodiment, the subject is pre-symptomatic. In one embodiment, the pre-symptomatic subject is less than 18 months of age. In one embodiment, the pre-symptomatic subject is evaluated using the Alberta Infant Motor Scale.

[0030] In some embodiments, the rhASA protein has an amino acid sequence that is at least 70% identical to SEQ ID NO:1.

[0031] In one aspect, the present invention provides a method for treating late infantile MLD, the method comprising: administering to a subject a preparation comprising a purified recombinant arylsulfatase A (rhASA) protein having an amino acid sequence that is at least 70% identical to SEQ ID NO:1, wherein the purified rhASA protein is characterized by a proteoglycan profile that comprises one or more peaks (i.e., species) corresponding to neutral recombinant ASA protein (neutral ASA protein), sialylated recombinant ASA protein (sialic acid ASA protein), mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and mixed recombinant ASA protein (mixed ASA protein), and the preparation is administered intrathecally at a dose of 150 mg at intervals of once a week.

[0032] In one embodiment, the preparation contains less than about 150 ng / mg of host cell protein (HCP).

[0033] In some embodiments, at least about 23% of the purified recombinant ASA protein, which accounts for the total purified recombinant ASA protein, corresponds to mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein).

[0034] In one embodiment, the proteoglycan profile of the purified recombinant ASA protein comprises: about 15% to about 25% neutral recombinant ASA protein (neutral ASA protein), about 35% to about 45% sialylated recombinant ASA protein (sialic acid ASA protein), about 23% to about 33% mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), about 1% to about 10% N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and about 5% to about 15% mixed recombinant ASA protein (mixed ASA protein).

[0035] In one embodiment, the proteoglycan profile of the purified recombinant ASA protein comprises: about 18% to about 22% neutral ASA protein, about 37% to about 41% sialic acid ASA protein, about 26% to about 29% M6P ASA protein, about 4% to about 6% capped M6P ASA protein, and about 7% to about 9% mixed ASA protein.

[0036] In one embodiment, the administration of the preparation produces minimal side effects (AE).

[0037] In one aspect, the present invention provides a novel process for purifying recombinant ASA protein, which can offer advantages such as cost and time reduction by improving process efficiency (e.g., reducing the total number of steps for preparing drug substance (DS) or drug product (DP)) or by allowing the preparation of compositions with beneficial characteristics (e.g., a composition of purified recombinant ASA protein containing a threshold population of mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), which may have improved bioavailability, targeting, or efficacy) or a DS composition containing a surfactant (e.g., polysorbate 20 (P20)), which may have improved storage (e.g., refrigerated) stability or result in improved process efficiency for preparing DS or DP).

[0038] In one aspect, the present invention provides a composition comprising a purified recombinant arylsulfatase A (rhASA) protein having an amino acid sequence that is at least 70% identical to SEQ ID NO:1, wherein the purified rhASA protein is characterized by one or more proteoglycan species: neutral recombinant ASA protein (neutral ASA protein), sialylated recombinant ASA protein (sialic acid ASA protein), mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), or mixed recombinant ASA protein (mixed ASA protein). In some embodiments, the mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein) is present in an amount of at least about 23% of the total purified rhASA protein content.

[0039] In one embodiment, the present invention provides a composition comprising a purified recombinant arylsulfatase A (rhASA) protein having an amino acid sequence that is at least 70% identical to SEQ ID NO:1, wherein the purified rhASA protein is present as a mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein) in an amount of at least about 23% of the total purified rhASA protein content; and the purified recombinant ASA protein contains less than about 150 ng / mg of host cell protein (HCP).

[0040] In one aspect, the present invention provides a composition comprising a purified recombinant human arylsulfatase A (rhASA) protein having an amino acid sequence that is at least 70% identical to SEQ ID NO:1, wherein at least about 23% of the total purified recombinant ASA protein corresponds to a mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein) characterized by a proteoglycan profile; and the composition contains less than about 150 ng / mg of host cell protein (HCP).

[0041] In one embodiment, the M6P ASA protein is present in the composition in an amount of at least about 26% of the total purified rhASA protein content. In one embodiment, the M6P ASA protein is present in an amount of at least about 28% of the total purified rhASA protein content. In one embodiment, the M6P ASA protein is present in the composition in an amount of about 20% to about 33% of the total purified rhASA protein content.

[0042] In one embodiment, the total purified rhASA protein comprises a neutral recombinant ASA protein (neutral ASA protein), a sialylated recombinant ASA protein (sialic acid ASA protein), an N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), or a mixed recombinant ASA protein (mixed ASA protein), or any combination thereof.

[0043] In one embodiment, the total purified rhASA protein comprises: about 23% to about 33% of mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), about 15% to about 25% of neutral recombinant ASA protein (neutral ASA protein), about 35% to about 45% of sialylated recombinant ASA protein (sialic acid ASA protein), about 1% to about 10% of N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and about 5% to about 15% of mixed recombinant ASA protein (mixed ASA protein).

[0044] In one embodiment, the neutral ASA protein is present in an amount of about 16% to about 22% of the total purified rhASA protein.

[0045] In one embodiment, the neutral ASA protein is present in an amount of about 20% to about 25% of the total purified rhASA protein.

[0046] In one embodiment, the sialic acid ASA protein is present in an amount of about 35% to about 40% of the total purified rhASA protein.

[0047] In one embodiment, the sialic acid ASA protein is present in an amount of about 37% to about 42% of the total purified rhASA protein.

[0048] In one embodiment, the capped M6P ASA protein is present in an amount of about 3% to about 5% of the total purified rhASA protein.

[0049] In one embodiment, the capped M6P ASA protein is present in an amount of about 4% to about 6% of the total purified rhASA protein.

[0050] In one embodiment, the capped M6P ASA protein is present in an amount of about 4.5% to about 5.5% of the total purified rhASA protein.

[0051] In one embodiment, the mixed ASA protein is present in an amount of about 7% to about 10% of the total purified rhASA protein.

[0052] In one embodiment, the mixed ASA protein is present in an amount of about 7.5% to about 8.5% of the total purified rhASA protein.

[0053] In one embodiment, the total purified rhASA protein comprises: about 16% to about 23% neutral ASA protein, about 37% to about 42% sialic acid ASA protein, about 23% to about 27% M6P ASA protein, about 4% to about 8% capped M6P ASA protein, and about 7% to about 10% mixed ASA protein. In one embodiment, the total purified rhASA protein comprises: about 20% to about 23% neutral ASA protein, about 35% to about 39% sialic acid ASA protein, about 26% to about 32% M6P ASA protein, about 3% to about 5% capped M6P ASA protein, and about 7% to about 9% mixed ASA protein.

[0054] In one embodiment, the total purified rhASA protein comprises: about 18% to about 22% neutral ASA protein, about 37% to about 41% sialic acid ASA protein, about 26% to about 29% M6P ASA protein, about 4% to about 6% capped M6P ASA protein, and about 7% to about 9% mixed ASA protein.

[0055] In one embodiment, the total purified rhASA protein is present at a concentration of about 20 mg / mL to about 45 mg / mL. In one embodiment, the total purified rhASA protein is present at a concentration of about 25 mg / mL to about 34 mg / mL or about 28 mg / mL to about 32 mg / mL. In one embodiment, the total purified rhASA protein is present at a concentration of about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, or about 34 mg / mL.

[0056] In one embodiment, the purified rhASA protein has a specific activity of about 50 to about 130 U / mL. In one embodiment, the purified rhASA protein has a specific activity of about 70 to about 100 U / mg. In one embodiment, the purified rhASA protein has a specific activity of about 80 to about 90 U / mg. In one embodiment, the purified rhASA protein has a specific activity of about 75 to about 95 U / mg.

[0057] In one embodiment, the purified rhASA protein contains less than about 140 ng / mg of host cell protein (HCP). In one embodiment, the purified rhASA protein contains less than about 100 ng / mg of HCP. In one embodiment, the purified rhASA protein contains less than about 80 ng / mg of HCP. In one embodiment, the purified rhASA protein contains less than about 60 ng / mg of HCP. In one embodiment, the purified rhASA protein contains less than about 100 pg / mg of host cell DNA (HCD). In one embodiment, the purified rhASA protein contains less than about 50 pg / mg of HCD. In one embodiment, the purified rhASA protein contains less than about 10 pg / mg of HCD. In one embodiment, the purified rhASA protein contains less than about 5 pg / mg of HCD, less than about 4 pg / mg of HCD, less than about 3 pg / mg of HCD, less than about 2 pg / mg of HCD or less than about 1 pg / mg of HCD.

[0058] In one embodiment, the purified rhASA protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:1. In one embodiment, the purified rhASA protein has an amino acid sequence that is at least 90% identical to SEQ ID NO:1. In one embodiment, the purified rhASA protein has an amino acid sequence that is at least 95% identical to SEQ ID NO:1.

[0059] In one embodiment, the purified rhASA protein has the same amino acid sequence as SEQ ID NO:1.

[0060] In one embodiment, the composition comprises about 0.001% to about 0.01% of polysorbate-20 (P20). In one aspect, the present invention provides a formulation that comprises the composition as detailed above and a physiologically acceptable carrier. In one embodiment, the formulation is suitable for intravenous administration.

[0061] In one embodiment, the formulation is suitable for intrathecal administration. In one embodiment, the formulation is suitable for subcutaneous administration.

[0062] In one aspect, the present invention provides a method for purifying recombinant arylsulfatase A (ASA) protein, the method comprising: purifying recombinant arylsulfatase A (ASA) protein from an impure preparation by performing one or more chromatographic steps; combining the eluates from the one or more chromatographic steps; optionally adjusting the pH of the combined eluate to a pH of about 6.0 to about 8.0; optionally subjecting the combined eluate or the pH-adjusted eluate to ultrafiltration and / or diafiltration; obtaining an eluate containing the purified recombinant arylsulfatase A (ASA) protein; and adding a surfactant to the eluate containing the purified recombinant arylsulfatase A (ASA) protein.

[0063] In one embodiment, the method further comprises the step of adjusting the pH of the combined eluate to a pH of about 6.0 to about 8.0. In one embodiment, the step of subjecting the pH-adjusted eluate to ultrafiltration and / or diafiltration.

[0064] In one embodiment, the addition of the surfactant is carried out before refrigerating the eluate containing the purified recombinant ASA protein.

[0065] In one embodiment, the surfactant is present at a concentration of about 0.0001% (v / v) to about 0.01% (v / v). In one embodiment, the surfactant is present at a concentration of about 0.001% (v / v) to about 0.01% (v / v). In one embodiment, the surfactant is polysorbate-20 (P20).

[0066] In one embodiment, the step of performing one or more chromatographic steps comprises sequentially performing anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography i.e., phenyl chromatography, and cation exchange chromatography. In some embodiments, the anion exchange chromatography uses a column with a TMAE resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The figures described below, which together form the drawings, are for illustrative purposes only and not for limitation.

[0068] Figures 1A - 1FRepresentative images of immunohistochemical staining of LAMP-1 in MLD mice treated with rhASA from process A or process B are shown, as well as corresponding morphometric analyses of the white matter of the spinal cord (A), cerebellum (B), fimbria of the hippocampus (C), cerebral peduncle (D), cerebral cortex (E), and striatum (F) of immunotolerant MLD mice treated with 0.04 mg or 0.21 mg of rhASA from process A (iii, v) or process B (iv, vi) or control (ii). Untreated C57 / B16 mice were used as WT controls (i). LAMP-1, lysosome-associated membrane protein 1; MLD, metachromatic leukodystrophy; NS, not significant; rhASA, recombinant human arylsulfatase A; WT, wild type.

[0069] Figure 2A and Figure 2B show the concentration-time curves of process A and process B rhASA in ( Figure 2A ) serum and ( Figure 2B ) CSF in juvenile cynomolgus monkeys after a single intrathecal dose of 6.0 mg.

[0070] Figure 3A and Figure 3B show the biodistribution of process A and process B rhASA in rats. Representative whole-body autoradioluminograms show the radioactive tissue distribution 4 hours after a single intrathecal dose of Figure 3A ) process A and ( Figure 3B ) process B 125 I]-rhASA 0.62 mg in male Sprague Dawley rats.

[0071] Figure 4 show the urinary excretion, fecal excretion, and total excretion curves of process A and process B 125 I]-rhASA 0.62 mg in male Sprague Dawley rats after a single intrathecal dose (n = 2 and n = 3, respectively).

[0072] Figure 5 show the dose escalation and extension study design of an open-label, dose-escalating safety assessment of rhASA. CSF, cerebrospinal fluid; GMFM-88, Gross Motor Function Measure-88; IDDD, intrathecal drug delivery device; IT, intrathecal; MLD, metachromatic leukodystrophy; rhASA, recombinant human arylsulfatase A.

[0073] Figure 6The clinical PK / PD model of rhASA concentrations in CSF, CNS, and serum and the effect on CSF sulfatide concentrations are shown. CL, drug clearance; CMT, compartment; CNS, central nervous system; CSF, cerebrospinal fluid; k in , the formation rate of sulfatide; k out , the consumption rate constant of sulfatide; K trans , transport rate constant; PK / PD, pharmacokinetics / pharmacodynamics; Q CSF , intercompartmental clearance; rhASA, recombinant human arylsulfatase A.

[0074] Figure 7A , Figure 7B and Figure 7C Goodness-of-fit plots for the population-predicted and individual-predicted rhASA concentration in CSF, sulfatide concentration in CSF, and GMFM-88 total score, respectively, are shown.

[0075] Figure 8 Representative simulated rhASA trough concentrations in cerebrospinal fluid (CSF) (left panel) and central nervous system (CNS) (right panel) are shown by dosing regimen and baseline age. Regimen 1: 100 mg every other week; Regimen 2: 100 mg weekly for 12 weeks (initially weekly dosing), then 100 mg every other week; Regimen 3: 150 mg weekly for 12 weeks (initially weekly dosing), then 150 mg every other week; Regimen 4: age-adjusted weekly dosing for 12 weeks (initially weekly dosing), then every other week (Regimen 4: 80 mg, <8 months; 120 mg, 8–<30 months; 150 mg, ≥30 months).

[0076] Figure 9 Individual-level predicted GMFM-88 total scores are shown for the four dosing schedules: Schedule 1, 100 mg every other week (EOW); Schedule 2, 100 mg weekly (EW) for 12 weeks, followed by 100 mg EOW; Schedule 3, 150 mg weekly for 12 weeks, followed by 150 mg EOW; Schedule 4, age-adjusted weekly dosing for 12 weeks, followed by EOW (80 mg, <8 months; 120 mg, 8–<30 months; 150 mg, ≥30 months).

[0077] Figure 10Shows the dose escalation and extension study design of the open-label, dose-escalation safety assessment of rhASA. All patients in the cohort continued to escalate to higher doses based on DSMB review only after they had received at least two previous doses. DSMB, Data Safety Monitoring Board; EOS, end of study; EOW, every other week; GMFM-88, Gross Motor Function Measure 88; IDDD, intrathecal drug delivery device; MLD, metachromatic leukodystrophy; MRI, magnetic resonance imaging; rhASA, recombinant human arylsulfatase A.

[0078] Figure 11 Shows the individual GMFM-88 total scores by patient age during the study for patients determined to be responders and non-responders to treatment.

[0079] Figure 12 Shows the individual MLD severity scores at baseline and week 40 for patients who responded to treatment and had available data

[0080] Figure 13 Shows the individual N-acetylaspartate (NAA) / creatine ratios in the fronto-parietal white matter at baseline and week 40 for patients who responded to treatment and had available data.

[0081] Figure 14 Shows the mean (±SD) concentration of rhASA in cerebrospinal fluid over time.

[0082] Figure 15 Shows the mean GMFM-88 total scores over time for Group 1 (Procedure A: 10 mg), Group 2 (Procedure A: 30 mg), Group 3 (Procedure A: 100 mg), Group 4 (Procedure B: 100 mg). Error bars represent the standard error of the mean.

[0083] Figure 16A and Figure 16B Shows the mean CSF sulfatide and CSF desulfatide levels for Group 1 (Procedure A: 10 mg), Group 2 (Procedure A: 30 mg), Group 3 (Procedure A: 100 mg), Group 4 (Procedure B: 100 mg), respectively. Error bars represent the standard deviation of the mean. The upper limits of normal for CSF sulfatide (0.113 μg / mL) and CSF desulfatide (0.0277 ng / mL) were the highest concentrations observed in CSF samples from 60 children without leukodystrophy who had undergone lumbar puncture (and thus may have had other psychiatric conditions). CSF, cerebrospinal fluid; LLOQ / 2, lower limit of quantification.

[0084] Figure 17A and Figure 17B Shows in the right frontal white matter ( Figure 17A)NAA / creatine ratio and ( Figure 17B ) Average change over time in the choline / creatine ratio compared to baseline. Baseline data were available for 16 patients and end-of-study data were available for 12 patients. Error bars represent standard deviation from the mean.

[0085] Figure 18A and Figure 18B show the ( Figure 18A ) NAA / creatine ratio and ( Figure 18B ) Average change over time in the choline / creatine ratio compared to baseline in the right fronto-parietal white matter. Error bars represent standard deviation from the mean.

[0086] Figure 19A and Figure 19B show the ( Figure 19A ) NAA / creatine ratio and ( Figure 19B ) Average change over time in the choline / creatine ratio compared to baseline in the right parieto-occipital white matter. Error bars represent standard deviation from the mean.

[0087] Figure 20A and Figure 20B show the ( Figure 20A ) NAA / creatine ratio and ( Figure 20B ) Average change over time in the choline / creatine ratio compared to baseline in the occipital midline white matter. Error bars represent standard deviation from the mean.

[0088] Figure 21 show the total average MLD severity score over time. Error bars represent standard error of the mean.

[0089] Figure 22 show the individual GMFM-88 total scores by month of age in sibling patients from the rhASA clinical study.

[0090] Definition

[0091] To make the present invention easier to understand, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0092] About or approximately: As used herein, when applied to one or more target values, the terms "about" or "approximately" refer to a value similar to the reference value. In certain embodiments, the terms "about" or "approximately" refer to a range of values falling 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 value, unless otherwise stated or otherwise apparent from the context (unless the number exceeds 100% of the possible value).

[0093] Bioactive: As used herein, the phrase "bioactive" refers to the characteristic of any agent that is active in a biological system and, in particular, in an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered bioactive. In certain embodiments, when a protein or polypeptide is bioactive, a portion of the protein or polypeptide that shares at least one biological activity of the protein or polypeptide is commonly referred to as a "bioactive" portion.

[0094] 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 to the mannose-6-phosphate (M6P) tag on the precursors of acid hydrolases in the Golgi apparatus, which are destined for transport to lysosomes. In addition to mannose-6-phosphate, CI-MPR also binds to other proteins, including IGF-II. CI-MPR is also referred to as the "M6P / IGF-II receptor", "CI-MPR / IGF-II receptor", "IGF-II receptor", or "IGF2 receptor". These terms and their abbreviations are used interchangeably herein.

[0095] Chromatography: As used herein, the term "chromatography" refers to a technique for separating mixtures. Generally, a mixture is dissolved in a fluid called the "mobile phase", which will carry the mixture through a structure containing another material called the "stationary phase". Column chromatography is a separation technique in which the stationary bed is inside a tube, i.e., a column.

[0096] Diluent: As used herein, the term "diluent" refers to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) diluting substance that can be used to prepare a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solutions.

[0097] Elution: As used herein, the term "elution" refers to the process of extracting one substance from another by washing with a solvent. For example, in ion exchange chromatography, elution is the process of washing the loaded resin to remove the captured ions.

[0098] Eluate: As used herein, the term "eluate" refers to the combination of the mobile phase "carrier" and the analyte material that typically emerges from chromatography as a result of elution.

[0099] Enzyme Replacement Therapy (ERT): As used herein, the term "Enzyme Replacement Therapy (ERT)" refers to any therapeutic strategy for correcting an enzyme deficiency by providing the missing enzyme. Once administered, the enzyme is taken up by cells and transported to lysosomes, where the enzyme acts to eliminate the materials that have accumulated in the lysosomes due to the enzyme deficiency. Generally, for lysosomal enzyme replacement therapy to be effective, the therapeutic enzyme is delivered to the lysosomes in the appropriate cells in the target tissue, where the storage defect is evident. The purification methods described herein can be used to purify and formulate recombinant arylsulfatase A as a drug substance for ERT in MLD.

[0100] 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., a buffer) into equilibrium with another liquid, typically to achieve a stable and uniform distribution of the components of the liquid (e.g., the buffer). For example, in some embodiments, a chromatography column can be equilibrated by passing one or more column volumes of the desired liquid (e.g., a buffer) through the column.

[0101] Improve, increase, or decrease: As used herein, the terms "improve", "increase", or "decrease" or grammatical equivalents indicate a value relative to a baseline measurement, such as a measurement in the same individual before the start of the treatment described herein, or a measurement in a control individual (or control individuals) in the absence of the treatment described herein. A "control individual" is an individual who has the same form of lysosomal storage disease as the individual to be treated and is approximately the same age as the individual to be treated (to ensure that the disease stage in the treated individual and the control individual is comparable).

[0102] Impurity: As used herein, the term "impurity" refers to a substance that has a chemical composition different from that of the target material or compound in a limited quantity of liquid, gas, or solid. Impurities are also referred to as contaminants.

[0103] Loading: As used herein, the term "loading" refers to, in chromatography, adding a liquid or solid containing a sample to a column. In some embodiments, specific components of the sample loaded onto the column are then captured as the loaded sample passes through the column. In some embodiments, specific components of the sample loaded onto the column are not captured by the column as the loaded sample passes through the column, or "flow through" the column.

[0104] Polypeptide: As used herein, "polypeptide", generally speaking, is a string of at least two amino acids attached to each other by peptide bonds. In some embodiments, the polypeptide may include at least 3 - 5 amino acids, each of which is attached to other amino acids by at least one peptide bond. Those of ordinary skill in the art will understand that polypeptides sometimes include "non-natural" amino acids or optional other entities that can still be incorporated into the polypeptide chain.

[0105] Library: As used herein, the term "library" with respect to chromatography refers to the combination of one or more fractions of the fluid that has passed through the column. For example, in some embodiments, one or more fractions (e.g., "peak fractions") containing the desired components of a sample that has been separated by chromatography can be "pooled" together to produce a single "pooled" fraction.

[0106] Alternative enzyme: As used herein, the term "alternative enzyme" refers to any enzyme that can act to at least partially replace an enzyme that is lacking or absent in the disease to be treated. In some embodiments, the term "alternative enzyme" refers to any enzyme that can act to at least partially replace a defective or absent lysosomal enzyme in a lysosomal storage disease to be treated. In some embodiments, the alternative enzyme (e.g., rhASA) is capable of reducing the substances accumulated in mammalian lysosomes, or can rescue or improve the symptoms of one or more lysosomal storage diseases (e.g., MLD). Alternative enzymes suitable for the present invention include both wild-type or modified lysosomal enzymes, and can be produced using recombinant methods and synthetic methods or purified from natural sources. The alternative enzyme can be a recombinant, synthetic, gene-activated or natural enzyme.

[0107] Soluble: As used herein, the term "soluble" refers to the ability of a therapeutic agent to form a homogeneous solution. In some embodiments, the solubility of the therapeutic agent in the solution in which it is administered and through 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 target site of action. Several factors can affect the solubility of a therapeutic agent. For example, relevant factors that can affect protein solubility include ionic strength, amino acid sequence, and the presence of other co-solvents or salts (e.g., calcium salts). In some embodiments, the therapeutic agents according to the present invention are soluble in their respective pharmaceutical compositions.

[0108] Stability: As used herein, the term "stable" refers to the ability of a therapeutic agent (e.g., a recombinase) to maintain its therapeutic efficacy (e.g., all or most of its intended biological activity and / or physicochemical integrity) over an extended period of time. The stability of a therapeutic agent and the ability of a pharmaceutical composition to maintain the stability of such a therapeutic agent can be evaluated over an extended period of time (e.g., at least 1, 3, 6, 12, 18, 24, 30, 36 months or longer). In the case of a formulation, a stable formulation is one in which the therapeutic agent substantially maintains its physical and / or chemical integrity and biological activity upon storage and during processing (e.g., freeze / thaw, mechanical mixing, and lyophilization). For protein stability, it can be measured by the formation of high molecular weight (HMW) aggregates, loss of enzyme activity, generation of peptide fragments, and shifts in charge distribution.

[0109] Virus treatment: As used herein, the term "virus treatment" refers to "virus removal", where only the virus is removed from a sample (e.g., virus filtration), or "virus inactivation", where the virus remains in the sample but is in a non-infectious form. In some embodiments, virus removal can be effected using, for example, nanofiltration and / or chromatography techniques. In some embodiments, virus inactivation can be effected using, for example, solvent inactivation, detergent inactivation, pasteurization, acidic pH inactivation, and / or ultraviolet inactivation. Detailed Description

[0110] The present invention particularly provides methods for enzyme replacement therapy and compositions comprising purified recombinant ASA (rhASA) protein. The present invention is at least in part based on the discovery that rhASA is safe and well tolerated in infants and children; thus supporting dose escalation to achieve higher efficacy. The present invention is in part directed to improved recombinant rhASA treatment doses and dosing intervals for the treatment of MLD to improve motor function, or at least reduce or arrest the progressive decline in motor function in subjects with ASA deficiency.

[0111] In one aspect, the present invention relates to a method for treating MLD, the method comprising the step of administering a therapeutically effective dose of rhASA at a suitable dosing interval to reduce or arrest the progression of MLD.

[0112] In some embodiments, the therapeutically effective dose is 100 mg or higher. In some embodiments, a suitable dosing interval is once every other week. In some embodiments, a suitable dosing interval is once a week. In some embodiments, the method comprises administering a formulation comprising rhASA at a dose of 100 mg or higher once every other week. In some embodiments, the formulation comprising rhASA is administered to a subject at a dose of 100 mg or higher once a week (EW). In some embodiments, the formulation comprising rhASA is administered to a subject at a dose of 150 mg once every other week (EOW). In some embodiments, the formulation comprising rhASA is administered to a subject at a dose of 150 mg once a week (EW).

[0113] In some embodiments, the subject has received at least 10 mg of the dose for a period of time prior to receiving a 100 mg dose. In some embodiments, the subject has received at least 30 mg of the dose for a period of time prior to receiving a 100 mg dose. In some embodiments, the subject has received a 100 mg dose for a period of time and then continues to receive 100 mg of rhASA. In some embodiments, the duration is 20 weeks, 30 weeks, 35 weeks or 40 weeks. In some embodiments, the duration is 40 weeks.

[0114] In some embodiments, the subject receiving at least 100 mg of the dose is a responder previously treated with 10 mg. In some embodiments, the subject receiving at least 100 mg of the dose is a responder previously treated with 30 mg. In some embodiments, the subject receiving at least 100 mg of the dose is a non-responder previously treated with 10 mg. In some embodiments, the subject receiving at least 100 mg of the dose is a non-responder previously treated with 30 mg. In some embodiments, the subject receiving at least 100 mg of the dose did not exhibit any adverse effects (AE) to the previous treatment with 10 mg. In some embodiments, the subject receiving at least 100 mg of the dose did not exhibit any AE to the previous treatment with 30 mg. In some embodiments, the subject receiving at least 100 mg of the dose did not exhibit any AE to the previous treatment with 100 mg.

[0115] In some embodiments, the formulation comprising rhASA is administered at a dose of 100 mg or higher, once a week (EW) for 12 weeks, and then administered once every other week (EOW). In some embodiments, the formulation comprising rhASA is administered at a dose of 150 mg, once a week (EW) for 12 weeks, and then administered once every other week (EOW). In some embodiments, the subject did not exhibit adverse effects to the administration.

[0116] The present invention is based in part on the discovery that the therapeutic application of rhASA is effective in treating a broader range of MLD, particularly late infantile MLD. In one aspect, the present invention provides a method of treating late infantile MLD. In some embodiments, the method comprises administering to a subject having late infantile MLD a preparation comprising rhASA. In some embodiments, the method comprises administering to a subject having late infantile MLD an effective therapeutic dose of a preparation comprising rhASA at a suitable dosing interval to reduce or arrest the progression of late infantile MLD. In some embodiments, the method comprises intrathecal administration. In some embodiments, an effective therapeutic dose for treating late infantile MLD is 150 mg. In some embodiments, a suitable dosing interval for treating late infantile MLD is once a week.

[0117] In some embodiments, the subject having late infantile MLD is 72 months of age or less. In some embodiments, the subject having late infantile MLD is 48 months of age or less. In some embodiments, the subject having late infantile MLD is 24 months of age or less. In some embodiments, the subject having late infantile MLD is 18 months of age or less. The subject is selected and classified based on the motor function determined by the Gross Motor Function Classification (GMFC).

[0118] In some embodiments, the method comprises intrathecally administering to a subject having late infantile MLD a preparation comprising rhASA at a dose of 150 mg once EW. In some embodiments, the administration continues for at least two years.

[0119] The selection of a subject having late infantile MLD for treatment with a preparation comprising rhASA involves several factors based on age, disease state, and motor function characterization. In some embodiments, treatment is administered to early symptomatic subjects. In some embodiments, the subject having early symptomatic late infantile MLD is 18 - 48 months of age. In some embodiments, the subject having early symptomatic late infantile MLD has a gross motor function classification of MLD[GMFC - MLD] levels 1 - 2.

[0120] In some embodiments, treatment for late infantile MLD is administered to mid - symptomatic subjects. In some embodiments, the late symptomatic subject is 18 - 72 months of age. In some embodiments, the late symptomatic subject has a GMFC MLD level 4.

[0121] In some embodiments, a treatment for late infantile MLD is administered to pre-symptomatic or minimally symptomatic subjects. In some embodiments, the pre-symptomatic or minimally symptomatic subject is 18 months of age or younger, or is a pre-symptomatic sibling of a participant with the same ASA allele constitution. In one embodiment, pre-symptomatic subjects are evaluated using the Alberta Infant Motor Scale (Piper M and Darrah J, “Motor Assessment of a Developing Infant,” published February 9, 1994, page 222; ISBN: 9780721643076, Saunders).

[0122] In some embodiments, the invention provides a purified rhASA composition for administration to a subject having MLD.

[0123] Treatment of metachromatic leukodystrophy disease (MLD)

[0124] The compositions and methods of the invention can be used to effectively treat individuals having or predisposed to MLD by delivery to the CNS. Certain methods of the invention for treating MLD generally comprise the steps of intrathecally administering recombinant arylsulfatase A (ASA) to a subject in need of treatment at a therapeutically effective dose and at a dosing interval for a treatment period sufficient to stabilize or slow the progression of a motor dysfunction as measured by the GMFC-MLD level (indicating a decline in motor function). In many methods provided according to the present disclosure, no serious side effects associated with the administration of recombinant arylsulfatase A were observed in the subjects.

[0125] Motor function

[0126] In some embodiments, the neurological impairment in MLD patients is characterized by a decline in motor function such as gross motor function. In some embodiments, treatment efficacy includes a measurement involving one or more motor functions.

[0127] In some embodiments, methods for treating metachromatic leukodystrophy (MLD) syndrome are provided, the method comprising the steps of intrathecally administering recombinant arylsulfatase A (ASA) to a subject in need of treatment at a therapeutically effective dose and at a dosing interval for a treatment period sufficient to improve, stabilize or reduce the decline of one or more motor functions relative to baseline. In some embodiments, the administration of the recombinant ASA enzyme also results in an improvement, stabilization or reduction of the decline of one or more cognitive functions, adaptive functions and / or executive functions.

[0128] One or more motor functions may include, for example, gross motor function. It should be understood that gross motor function can be evaluated by any suitable method. For example, in some embodiments, the change in gross motor function relative to a baseline of motor function is measured using the total raw score or percentage of a gross motor function test scale such as the Gross Motor Function Measure 88 (GMFM-88) or the Gross Motor Function Classification (GMFC).

[0129] Gross Motor Function Measure 88 (GMFM-88)

[0130] In some embodiments, the neurological impairment in MLD patients is characterized by a decline in gross motor function. It should be understood that gross motor function can be evaluated by any suitable method. For example, in some embodiments, the change in gross motor function relative to a baseline of motor function is measured using the total raw score of the Gross Motor Function Measure 88 (GMFM-88). In some embodiments, the treated subject has a baseline GMFM-88 score greater than 40%. In some embodiments, the treated subject has a baseline GMFM-88 score less than 40%.

[0131] In some embodiments, administration of the recombinant ASA enzyme according to the methods disclosed herein results in less decline in motor function than is typically observed without administration. For example, in some embodiments, administration of the recombinant ASA enzyme according to the methods of the invention results in a decline in the GMFM-88 score of less than 10%, 20%, 30%, 40%, or 50%.

[0132] In some embodiments, the baseline GMFM-88 score of the treated subject is 35. In some embodiments, the baseline GMFM-88 score of the treated subject is higher than 35. In some embodiments, the baseline GMFM-88 score of the treated subject is 40. In some embodiments, the baseline GMFM-88 score of the treated subject is higher than 40. In some embodiments, administration of the recombinant ASA enzyme according to the methods disclosed herein maintains motor function relative to the baseline. In some embodiments, administration of the recombinant ASA enzyme according to the methods disclosed herein maintains the motor function measured by the GMFM-88 score.

[0133] In some embodiments, administration of the recombinant ASA enzyme according to the methods disclosed herein substantially stabilizes motor function, such as a score such as the GMFM-88 score. "Substantially stabilizes" means that there is no decline or deterioration over a period of time, such as over a treatment period and / or over a period during which decline or deterioration would typically be expected in the absence of treatment.

[0134] In some embodiments, administration of a recombinant ASA enzyme according to the methods disclosed herein results in an improvement in motor function, such as an improvement in scores such as the GMFM-88 score.

[0135] It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the enzyme replacement therapy or overseeing the administration of the enzyme replacement therapy, and the dosage ranges described herein are only exemplary and are not intended to limit the scope or practice of the invention.

[0136] Gross Motor Function Classification (GMFC-MLD)

[0137] The Gross Motor Function Classification (GMFC-MLD) can be used as a tool for the standardized assessment of gross motor function in MLD. The GMFC-MLD consists of seven levels, representing all clinically relevant stages from normal (level 0) to complete loss of gross motor function (level 6). In some embodiments, the GMFC-MLD is used to measure gross motor function.

[0138] In some embodiments, the baseline GMFC level of the treated subject is less than or equal to 2 (able to assist with standing and walking). In some embodiments, administration of a recombinant ASA enzyme according to the methods disclosed herein results in the maintenance of motor function relative to baseline as evaluated by the GMFC level. In some embodiments, administration of a recombinant ASA enzyme according to the methods disclosed herein results in the maintenance of a GMFC level equal to or less than 2.

[0139] In some embodiments, administration of a recombinant ASA enzyme according to the methods of the invention results in an increase (indicating a decline in motor function) in its GMFC-MLD level compared to baseline of no more than 2 at 2 years. In some embodiments, administration of a recombinant ASA enzyme according to the methods of the invention results in an increase (indicating a decline in motor function) in its GMFC-MLD level compared to baseline of no more than 3 at 2 years. In some embodiments, as evaluated by a change in GMFC-MLD compared to baseline of no greater than 2 levels, administration of a recombinant ASA enzyme according to the methods of the invention results in the maintenance of gross motor function.

[0140] Therapeutic administration

[0141] The purified recombinant ASA protein can be administered to MLD patients according to known methods. For example, the purified recombinant ASA protein can be delivered intravenously, subcutaneously, intramuscularly, parenterally, transdermally, or transmucosally (e.g., orally or nasally).

[0142] In some embodiments, recombinant ASA or a pharmaceutical composition containing recombinant ASA is administered to a subject by intravenous administration.

[0143] In some embodiments, recombinant ASA or a pharmaceutical composition containing recombinant ASA is administered to a subject by intrathecal administration. As used herein, the term "intrathecal administration" or "intrathecal injection" refers to injection into the spinal canal (the intrathecal space surrounding the spinal cord). A variety of techniques can be used, including but not limited to injection into the lateral ventricle by drilling or cisternal or lumbar puncture, etc. In some embodiments, "intrathecal administration" or "intrathecal delivery" according to the present invention refers to IT administration or delivery via the lumbar region or lumbar area, i.e., lumbar IT administration or delivery. As used herein, the term "lumbar area" or "lumbar region" refers to the area between the third and fourth lumbar (lumbar dorsal) vertebrae, and more inclusively refers to the L2-S1 region of the spinal column. In some embodiments, recombinant ASA or a pharmaceutical composition containing recombinant ASA is administered to a subject by intrathecal administration, as described in PCT International Publications WO2011 / 163648 and WO2011 / 163650, which are incorporated herein by reference in their entirety.

[0144] In some embodiments, recombinant ASA or a pharmaceutical composition containing recombinant ASA is administered to a subject by subcutaneous (i.e., under the skin) administration. For such purposes, syringe injection formulations can be used. However, other devices for administering formulations are also available, such as injection devices (e.g., Inject-Ease and Genject devices); syringe pens (such as GenPen); needleless devices (e.g., MediJector and BioJector); and subcutaneous patch delivery systems.

[0145] In some embodiments, intrathecal administration can be used in combination with other routes of administration (e.g., intravenous, subcutaneous, intramuscular, parenteral, transdermal, or transmucosal (e.g., oral or nasal)).

[0146] The present invention contemplates single or multiple administrations of a therapeutically effective amount of recombinant ASA or a pharmaceutical composition containing ASA as described herein. Recombinant ASA or a pharmaceutical composition containing recombinant ASA can be administered at regular intervals, depending on the nature, severity, and extent of the subject's medical condition (e.g., lysosomal storage disease). In some embodiments, a therapeutically effective amount of recombinant ASA or a pharmaceutical composition containing recombinant ASA can be administered periodically at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, once every two months, once a month, once every two weeks, once a week, once a day, or continuously).

[0147] CNS Delivery

[0148] The various stable formulations described herein are generally suitable for CNS delivery of therapeutic agents. The stable formulations according to the invention can be used for CNS delivery via various techniques and routes, including but not limited to intrastromal, intracerebral, intracerebroventricular (ICV), intrathecal (e.g., IT-lumbar, IT-cisterna magna) administration, as well as any other techniques and routes for direct or indirect injection into the CNS and / or CSF. The term "cisterna magna" refers to the space around and below the cerebellum via an opening between the skull and the top of the spine. Generally, injection via the cisterna magna is also referred to as "cisterna magna delivery".

[0149] Intrathecal delivery

[0150] In some embodiments, the replacement enzyme is delivered to the CNS in the formulations described herein. In some embodiments, the replacement enzyme is delivered to the CNS by administration into the cerebrospinal fluid (CSF) of a subject in need of treatment. In some embodiments, intrathecal administration is used to deliver the desired replacement enzyme (e.g., ASA protein) into the CSF. As used herein, intrathecal administration (also referred to as intrathecal injection) refers to injection into the spinal canal (the intrathecal space around the spinal cord). Various techniques can be used, including but not limited to lumbar puncture. Exemplary methods are described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143 - 192, the contents of which are incorporated herein by reference.

[0151] According to the invention, the enzyme can be injected at any region around the spinal canal. In some embodiments, the enzyme is injected into the lumbar region. As used herein, the term "lumbar region" or "lumbar area" refers to the region between the third and fourth lumbar (lumbar dorsal) vertebrae, and more inclusively refers to the L2 - S1 region of the spinal column. Generally, intrathecal injection via the lumbar region or lumbar area is also referred to as "lumbar intrathecal delivery" or "lumbar intrathecal administration".

[0152] In some embodiments, a therapeutic protein, such as recombinant arylsulfatase A, is delivered by lumbar intrathecal administration, e.g., between the third and fourth lumbar (lower back) vertebrae, and more inclusively within the L2 - S1 region of the spinal column. It is contemplated that lumbar intrathecal administration or delivery is distinguished from cisterna magna delivery in that lumbar intrathecal administration or delivery according to the invention provides better and more effective delivery to the distal spinal canal, whereas, in particular, cisterna magna delivery generally does not deliver well to the distal spinal canal.

[0153] Devices for intrathecal delivery

[0154] According to the present invention, various devices can be used for intrathecal delivery. In some embodiments, a device for intrathecal administration comprises a fluid inlet port (e.g., an injectable port); a hollow body (e.g., a catheter) having a first flow aperture in fluid communication with the fluid inlet port and a second flow aperture configured for insertion into the spinal cord; and a fixation mechanism for fixing the insertion of the hollow body in the spinal cord. As a non-limiting example, suitable fixation mechanisms include one or more small blocks mounted on the surface of the hollow body and adjustable suture loops on the one or more small blocks to prevent the hollow body (e.g., the catheter) from slipping out of the spinal cord. In various embodiments, the fluid inlet port includes a reservoir. In some embodiments, the fluid inlet port includes a mechanical pump (e.g., an infusion pump). In some embodiments, the implanted catheter is connected to a reservoir (e.g., for bolus delivery) or an infusion pump. The fluid inlet port can be implanted or external.

[0155] In some embodiments, intrathecal administration can be performed by lumbar puncture (i.e., slow bolus) or via a port-catheter delivery system (i.e., infusion or bolus). In some embodiments, the catheter is inserted between the laminae of the lumbar vertebrae and the tip is spiraled upward through the thecal space to the desired level (generally L3-L4).

[0156] In some embodiments, intrathecal administration is by intermittent or continuous access to an implanted intrathecal drug delivery device (IDDD).

[0157] Relative to intravenous administration, the single dose volume suitable for intrathecal administration is generally very small. Generally, intrathecal delivery according to the present invention maintains the composition of the CSF and the balance of the intracranial pressure of the subject. In some embodiments, intrathecal delivery is performed in the absence of corresponding removal of CSF from the subject. In some embodiments, a suitable single dose volume can be, for example, less than about 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1.5 ml, 1 ml or 0.5 ml. In some embodiments, a suitable single dose volume can be about 0.5 - 5 ml, 0.5 - 4 ml, 0.5 - 3 ml, 0.5 - 2 ml, 0.5 - 1 ml, 1 - 3 ml, 1 - 5 ml, 1.5 - 3 ml, 1 - 4 ml or 0.5 - 1.5 ml. In some embodiments, intrathecal delivery according to the present invention involves the step of first removing a desired amount of CSF. In some embodiments, less than about 10 ml (e.g., less than about 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml) of CSF is first removed before intrathecal administration. In those cases, a suitable single dose volume can be, for example, more than about 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml or 20 ml.

[0158] Other devices for intrathecal administration of therapeutic compositions or formulations are described in U.S. Patent No. 6,217,552, which is incorporated herein by reference. Alternatively, the drug can be administered intrathecally, for example, by single injection or continuous infusion. It should be understood that the dosage treatment can be in the form of a single-dose administration or multiple doses.

[0159] For injection, the formulations of the present invention can be formulated in a liquid solution. Additionally, the enzyme can be formulated in a solid form and redissolved or resuspended immediately before use. Freeze-dried forms are also included. The injection can be, for example, in the form of a bolus injection of the enzyme or continuous infusion (e.g., using an infusion pump).

[0160] Non-intrathecal delivery methods

[0161] Therapeutic enzymes, such as recombinant arylsulfatase A, can be administered by non-intrathecal means, for example, by injecting into the brain of a subject through the lateral ventricle. For example, the injection can be performed through a burr hole made in the subject's skull. In another embodiment, the enzyme and / or other pharmaceutical formulations are administered into the ventricles of the subject through a surgically inserted shunt. For example, the injection can be performed in the larger lateral ventricles. In some embodiments, the injection can also be performed in the third and fourth smaller ventricles.

[0162] Alternatively, the pharmaceutical composition can be administered by injecting into the cisterna magna or lumbar region of the subject.

[0163] Certain devices can be used to effect the administration of the therapeutic composition. For example, a formulation containing the desired enzyme can be administered using an Ommaya reservoir, which is commonly used for intrathecal administration of drugs for meningeal carcinomatosis (Lancet 2:983-84, 1963). More specifically, in this method, a ventricular catheter is inserted through a hole formed in the anterior horn and connected to an Ommaya reservoir mounted under the scalp, and the reservoir is punctured subcutaneously for intrathecal delivery of the specific enzyme to be replaced, which is injected into the reservoir.

[0164] Sustained release / continuous delivery

[0165] In another embodiment of the method of the present invention, after a pharmaceutically acceptable formulation is administered to a subject, the pharmaceutically acceptable formulation provides a continuous delivery, such as "sustained release", of the enzyme or other pharmaceutical composition used in the present invention to the subject for at least one, two, three, four weeks or longer periods.

[0166] As used herein, the term "sustained delivery" refers to the continuous delivery in vivo of a pharmaceutical formulation of the invention for a period of time after administration, preferably for at least several days, a week or several weeks. Sustained delivery of the composition can be demonstrated, for example, by the sustained efficacy of an enzyme over time (e.g., sustained delivery of an enzyme can be demonstrated by a continuously decreasing amount of storage granules in a subject). Alternatively, sustained delivery of an enzyme can be demonstrated by detecting the presence of the enzyme in vivo over time.

[0167] Delivery to target tissue

[0168] As discussed above, one of the surprising and important features of the present invention is that therapeutic agents, particularly alternative enzymes administered using the inventive methods and compositions of the present invention, are able to effectively and extensively diffuse across the brain surface and penetrate various layers or regions of the brain, including deep brain regions. Additionally, the inventive methods and compositions of the present invention effectively deliver a therapeutic agent (e.g., ASA enzyme) to various tissues, neurons or cells of the spinal cord, including the lumbar region, which is difficult to target by existing CNS delivery methods such as ICV injection. Furthermore, the inventive methods and compositions of the present invention deliver a sufficient amount of a therapeutic agent (e.g., ASA enzyme) to the bloodstream as well as various peripheral organs and tissues.

[0169] Thus, in some embodiments, a therapeutic protein (e.g., ASA enzyme) is delivered to the central nervous system of a subject. In some embodiments, a therapeutic protein (e.g., ASA enzyme) is delivered to one or more target tissues of the brain, spinal cord and / or peripheral organs. As used herein, the term "target tissue" refers to any tissue affected by a lysosomal storage disease to be treated or any tissue in which a lysosomal enzyme with normal expression is lacking. In some embodiments, target tissues include those tissues in a patient suffering from or predisposed to a lysosomal storage disease in which there is, for example, a detectable or abnormally high amount of an enzyme substrate stored in the lysosomes of the tissue cells. In some embodiments, target tissues include those tissues that exhibit disease-related pathologies, symptoms or characteristics. In some embodiments, target tissues include those tissues in which a defective lysosomal enzyme is typically expressed at elevated levels. As used herein, target tissue can be brain target tissue, spinal cord target tissue and / or peripheral target tissue. Exemplary target tissues are described in detail below.

[0170] Brain target tissue

[0171] Generally, the brain can be divided into different regions, layers and tissues. For example, the meningeal tissue is the membrane system that envelops the central nervous system including the brain. The meninges contain three layers, including the dura mater, arachnoid matter and pia mater. Generally, the main functions of the meninges and cerebrospinal fluid are to protect the central nervous system. In some embodiments, a therapeutic protein according to the present invention is delivered to one or more layers of the meninges.

[0172] The brain has three main divisions, including the cerebrum, cerebellum, and brainstem. The cerebral hemispheres lie above most of the other brain structures and are covered by a cortical layer. Below the cerebrum is the brainstem, which resembles a stalk to which the cerebrum is attached. At the back of the brain, below the cerebrum and behind the brainstem is the cerebellum.

[0173] The diencephalon lies near the midline of the brain and above the midbrain, and includes the thalamus, epithalamus, hypothalamus, epithalamus, prethalamus, and pretectum. The mesencephalon, also known as the midbrain, includes the tectum, tegmentum, ventricular septal cavity, and cerebral peduncles, red nucleus, and cranial nerve III nucleus. The midbrain is involved in vision, hearing, motor control, sleep / wake, alertness, and temperature regulation.

[0174] Tissue regions of the central nervous system, including the brain, can be characterized based on tissue depth. For example, CNS (e.g., brain) tissue can be characterized as surface or superficial tissue, mid-deep tissue, and / or deep tissue.

[0175] According to the present invention, a therapeutic protein (e.g., a replacement enzyme) can be delivered to any suitable brain target tissue associated with a particular disease to be treated in a subject. In some embodiments, a therapeutic protein (e.g., a replacement enzyme) according to the present invention is delivered to a surface or superficial brain target tissue. In some embodiments, a therapeutic protein according to the present invention is delivered to a mid-deep brain target tissue. In some embodiments, a therapeutic protein according to the present invention is delivered to a deep brain target tissue. In some embodiments, a therapeutic protein according to the present invention is delivered to a combination of surface or superficial brain target tissue, mid-deep brain target tissue, and / or deep brain target tissue. In some embodiments, a therapeutic protein according to the present invention is delivered to deep brain tissue at least 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or more below (or within) the outer surface of the brain.

[0176] In some embodiments, a therapeutic agent (e.g., an enzyme) is delivered to one or more surface or superficial tissues of the cerebrum. In some embodiments, the targeted surface or superficial tissue of the cerebrum is within 4 mm of the surface of the cerebrum. In some embodiments, the targeted surface or superficial tissue of the cerebrum is selected from pia mater tissue, cerebral cortical band tissue, hippocampus, Virchow Robin space, blood vessels within the VR space, hippocampal body, hypothalamic portion on the inner surface of the brain, optic nerve and tract, olfactory bulb and processes, and combinations thereof.

[0177] In some embodiments, a therapeutic agent (such as an enzyme) is delivered to one or more deep tissues of the brain. In some embodiments, the targeted surface or superficial tissues of the brain are located 4 mm (e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm) below (or within) the brain surface. In some embodiments, the targeted deep tissues of the brain include the cerebral cortical band. In some embodiments, the targeted deep tissues of the brain include one or more of the diencephalon (such as the hypothalamus, thalamus, anterior thalamus, subthalamus, etc.), hindbrain, lentiform nucleus, basal ganglia, caudate nucleus, putamen, amygdala, globus pallidus, and combinations thereof.

[0178] In some embodiments, a therapeutic agent (such as an enzyme) is delivered to one or more tissues of the cerebellum. In certain embodiments, the targeted one or more tissues of the cerebellum are selected from tissues of the molecular layer, tissues of the Purkinje cell layer, tissues of the granular cell layer, cerebellar peduncles, and combinations thereof. In some embodiments, a therapeutic agent (such as an enzyme) is delivered to one or more deep tissues of the cerebellum, including but not limited to tissues of the Purkinje cell layer, tissues of the granular cell layer, deep cerebellar white matter tissue (e.g., deep relative to the granular cell layer), and deep cerebellar nuclear tissue.

[0179] In some embodiments, a therapeutic agent (such as an enzyme) is delivered to one or more tissues of the brainstem. In some embodiments, the targeted one or more tissues of the brainstem include brainstem white matter tissue and / or brainstem nuclear tissue.

[0180] In some embodiments, a therapeutic agent (such as an enzyme) is delivered to various brain tissues, including but not limited to gray matter, white matter, periventricular region, leptomeninges, meninges, neocortex, cerebellum, deep tissues in the cerebral cortex, molecular layer, caudate / putamen region, midbrain, pons, or deep regions of the medulla, and combinations thereof.

[0181] In some embodiments, a therapeutic agent (such as an enzyme) is delivered to various cells in the brain, including but not limited to neurons, glial cells, perivascular cells, and / or meningeal cells. In some embodiments, a therapeutic protein is delivered to oligodendrocytes in the deep white matter.

[0182] Spinal cord

[0183] Generally, regions or tissues of the spinal cord can be characterized based on the depth of the tissue. For example, spinal cord tissue can be characterized as surface or superficial tissue, mid - deep tissue, and / or deep tissue.

[0184] In some embodiments, a therapeutic agent (such as an enzyme) is delivered to one or more surface or superficial tissues of the spinal cord. In some embodiments, the targeted surface or superficial tissues of the spinal cord are located within 4 mm of the spinal cord surface. In some embodiments, the targeted surface or superficial tissues of the spinal cord include the pia mater and / or white matter tracts.

[0185] In some embodiments, a therapeutic agent (e.g., an enzyme) is delivered to one or more deep tissues of the spinal cord. In some embodiments, the targeted deep tissues of the spinal cord are located internally at a distance of 4 mm from the surface of the spinal cord. In some embodiments, the targeted deep tissues of the spinal cord contain spinal cord gray matter and / or ependymal cells.

[0186] In some embodiments, a therapeutic agent (e.g., an enzyme) is delivered to neurons of the spinal cord.

[0187] Peripheral target tissues

[0188] As used herein, a peripheral organ or tissue refers to any organ or tissue that is not part of the central nervous system (CNS). Peripheral target tissues can include, but are not limited to, the hematopoietic system, liver, kidney, heart, endothelium, bone marrow and bone marrow-derived cells, spleen, lung, lymph nodes, bone, cartilage, ovaries, and testes. In some embodiments, a therapeutic protein (e.g., a replacement enzyme) according to the present invention is delivered to one or more of the peripheral target tissues.

[0189] Biodistribution and bioavailability

[0190] In various embodiments, once delivered to the target tissue, the therapeutic agent (e.g., ASA enzyme) localizes intracellularly. For example, the therapeutic agent (e.g., an enzyme) can localize to exons, axons, lysosomes, mitochondria, or vacuoles of the target cell (e.g., neurons, e.g., Purkinje cells). For example, in some embodiments, an intrathecally administered enzyme demonstrates translocation kinetics such that the enzyme moves within the perivascular space (e.g., by a pulsation-assisted convective mechanism). Additionally, an active axonal transport mechanism related to the binding of the administered protein or enzyme to neurofilaments can also contribute to or otherwise facilitate the distribution of the intrathecally administered protein or enzyme into deeper tissues of the central nervous system.

[0191] In some embodiments, the therapeutic agent (e.g., ASA enzyme) delivered according to the present invention can reach a therapeutic or clinically effective level or activity in the various target tissues described herein. As used herein, a therapeutic or clinically effective level or activity is a level or activity that is sufficient to confer efficacy in the target tissue. Efficacy can be objective (i.e., measurable by some test or biomarker) or subjective (i.e., an indication of an effect or a feeling of an effect given by the subject). For example, a therapeutic or clinically effective level or activity can be an enzymatic level or activity that is sufficient to improve symptoms associated with a disease in the target tissue (e.g., GAG storage).

[0192] Therapeutically effective dose and administration intervals

[0193] A therapeutically effective amount is typically administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosing regimen) can vary, for example, according to the route of administration and combination with other pharmaceutical agents. Additionally, the specific therapeutically effective amount (and / or unit dose) for any particular patient can depend on various factors, including the disorder to be 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, the route of administration and / or the rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors well known in the medical arts.

[0194] In some embodiments, the therapeutically effective dose is or greater than about 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg or about 200 mg. In certain embodiments, the therapeutically effective dose is or greater than about 150 mg. In some embodiments, the therapeutically effective dose is or less than about 500 mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg or 150 mg. In certain embodiments, the therapeutically effective dose is less than about 200 mg. In some embodiments, the therapeutically effective dose ranges between about 10 - 200 mg, about 20 - 200 mg, about 30 - 200 mg, about 40 - 200 mg, about 50 - 200 mg, about 160 - 200 mg, about 70 - 200 mg, about 80 - 200 mg, about 90 - 200 mg, about 100 - 200 mg, about 125 - 200, about 150 - 200, about 50 - 300 mg, about 75 - 300 mg, about 100 - 300 mg, about 150 - 300 or about 10 - 50 mg. In some embodiments, the therapeutically effective dose ranges between about 100 mg and about 200 mg.

[0195] In some embodiments, the therapeutically effective dose ranges from about 0.005 mg / kg brain weight to 500 mg / kg brain weight, such as from about 0.005 mg / kg brain weight to 400 mg / kg brain weight, from about 0.005 mg / kg brain weight to 300 mg / kg brain weight, from about 0.005 mg / kg brain weight to 200 mg / kg brain weight, from about 0.005 mg / kg brain weight to 100 mg / kg brain weight, from about 0.005 mg / kg brain weight to 90 mg / kg brain weight, from about 0.005 mg / kg brain weight to 80 mg / kg brain weight, from about 0.005 mg / kg brain weight to 70 mg / kg brain weight, from about 0.005 mg / kg brain weight to 60 mg / kg brain weight, from about 0.005 mg / kg brain weight to 50 mg / kg brain weight, from about 0.005 mg / kg brain weight to 40 mg / kg brain weight, from about 0.005 mg / kg brain weight to 30 mg / kg brain weight, from about 0.005 mg / kg brain weight to 25 mg / kg brain weight, from about 0.005 mg / kg brain weight to 20 mg / kg brain weight, from about 0.005 mg / kg brain weight to 15 mg / kg brain weight, from about 0.005 mg / kg brain weight to 10 mg / kg brain weight.

[0196] In some embodiments, the therapeutically effective dose is greater than about 0.1 mg / kg brain weight, greater than about 0.5 mg / kg brain weight, greater than about 1.0 mg / kg brain weight, greater than about 3 mg / kg brain weight, greater than about 5 mg / kg brain weight, greater than about 10 mg / kg brain weight, greater than about 15 mg / kg brain weight, greater than about 20 mg / kg brain weight, greater than about 30 mg / kg brain weight, greater than about 40 mg / kg brain weight, greater than about 50 mg / kg brain weight, greater than about 60 mg / kg brain weight, greater than about 70 mg / kg brain weight, greater than about 80 mg / kg brain weight, greater than about 90 mg / kg brain weight, greater than about 100 mg / kg brain weight, greater than about 150 mg / kg brain weight, greater than about 200 mg / kg brain weight, greater than about 250 mg / kg brain weight, greater than about 300 mg / kg brain weight, greater than about 350 mg / kg brain weight, greater than about 400 mg / kg brain weight, greater than about 450 mg / kg brain weight, greater than about 500 mg / kg brain weight.

[0197] In some embodiments, a therapeutically effective dose may also be defined in mg / kg body weight. As will be appreciated by those skilled in the art, brain weight and body weight can be correlated. Dekaban AS. “Changes in brain weights during the span of human life: relation of brain weights to body heights and body weights,” Ann Neurol 1978; 4:345-56. Thus, in some embodiments, the dose can be converted as shown in Table 2.

[0198] Table 2

[0199]

[0200] In some embodiments, a therapeutically effective dose may also be defined in mg / 15 cc of CSF. As will be appreciated by those skilled in the art, a therapeutically effective dose based on brain weight and body weight can be converted to mg / 15 cc of CSF. For example, the CSF volume in adults is approximately 150 mL (Johanson CE et al., “Multiplicity of cerebrospinal fluid functions: New challenges in health and disease,” Cerebrospinal Fluid Res. May 14, 2008; 5:10). Thus, a single dose injection of 0.1 mg to 50 mg of protein for an adult would be a dose of approximately 0.01 mg / 15 cc of CSF (0.1 mg) to 5.0 mg / 15 cc of CSF (50 mg) in adults.

[0201] It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of enzyme replacement therapy, and the dosage ranges described herein are only exemplary and are not intended to limit the scope or practice of the invention.

[0202] In certain embodiments, the recombinant arylsulfatase A enzyme is administered at dosing intervals, such as regular intervals. For example, the dosing interval can be once a week, once every two weeks, or once a month.

[0203] In certain embodiments, administration of the recombinant arylsulfatase A enzyme reaches a treatment period. The treatment period can be pre-determined or can be adjusted based on the patient's response to treatment, including but not limited to evidence of possible adverse symptoms and / or treatment efficacy as discussed herein. For example, the treatment period can be at least 6 months, at least 9 months, at least 12 months, at least 24 months, or even greater.

[0204] In some embodiments, it is contemplated that a subject will undergo treatment over a certain treatment period, go through a period during which they do not receive treatment or receive an alternative treatment, and then undergo another treatment period.

[0205] In some embodiments, the enzymatic levels or activities achievable with the therapeutic agent (e.g., replacement enzyme) delivered according to the invention are at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the normal levels or activities of the corresponding lysosomal enzyme in the target tissue. In some embodiments, compared to a control or baseline (e.g., the endogenous level or activity without treatment), the therapeutic agent (e.g., replacement enzyme) delivered according to the invention can achieve an increase in enzyme level or activity of at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. In some embodiments, the therapeutic agent (e.g., replacement enzyme) delivered according to the invention can achieve an increase in enzyme level or activity of at least about 10 nmol / hr / mg, 20 nmol / hr / mg, 40 nmol / hr / mg, 50 nmol / hr / mg, 60 nmol / hr / mg, 70 nmol / hr / mg, 80 nmol / hr / mg, 90 nmol / hr / mg, 100 nmol / hr / mg, 150 nmol / hr / mg, 200 nmol / hr / mg, 250 nmol / hr / mg, 300 nmol / hr / mg, 350 nmol / hr / mg, 400 nmol / hr / mg, 450 nmol / hr / mg, 500 nmol / hr / mg, 550 nmol / hr / mg, or 600 nmol / hr / mg in the target tissue.

[0206] In some embodiments, the inventive method according to the present invention can be particularly used to target the lumbar region. In some embodiments, the therapeutic agent (e.g., replacement enzyme) delivered according to the present invention can achieve an increase in enzyme level or activity of at least about 500 nmol / hr / mg, 600 nmol / hr / mg, 700 nmol / hr / mg, 800 nmol / hr / mg, 900 nmol / hr / mg, 1000 nmol / hr / mg, 1500 nmol / hr / mg, 2000 nmol / hr / mg, 3000 nmol / hr / mg, 4000 nmol / hr / mg, 5000 nmol / hr / mg, 6000 nmol / hr / mg, 7000 nmol / hr / mg, 8000 nmol / hr / mg, 9000 nmol / hr / mg or 10,000 nmol / hr / mg in the lumbar region.

[0207] Generally, the therapeutic agent (e.g., replacement enzyme) delivered according to the present invention has a long enough half-life in the CSF as well as in the target tissues of the brain, spinal cord and peripheral organs. In some embodiments, the therapeutic agent (e.g., replacement enzyme) delivered according to the present invention can have a half-life of at least about 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 18 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, up to 3 days, up to 7 days, up to 14 days, up to 21 days or up to one month. In some embodiments, in some embodiments, the therapeutic agent (e.g., replacement enzyme) delivered according to the present invention can maintain a detectable level or activity in the CSF or bloodstream 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, 102 hours or one week after administration. Various methods known in the art can be used to determine the detectable level or activity.

[0208] In certain embodiments, a therapeutic agent (e.g., a replacement enzyme) delivered according to the present invention reaches a concentration of at least 30 μg / ml in the CNS tissues and cells of a subject after administration (e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes or less after intrathecal administration of the pharmaceutical composition to the subject). In certain embodiments, a therapeutic agent (e.g., a replacement enzyme) delivered according to the present invention reaches a concentration of at least 20 μg / ml, at least 15 μg / ml, at least 10 μg / ml, at least 7.5 μg / ml, at least 5 μg / ml, at least 2.5 μg / ml, at least 1.0 μg / ml or at least 0.5 μg / ml in the target tissues or cells (e.g., brain tissue or neurons) of a subject after administration to such subject (e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes or less after intrathecal administration of such pharmaceutical composition to the subject).

[0209] Therapeutic preparation

[0210] Recombinant ASA or a pharmaceutical composition containing recombinant ASA can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and the therapeutic agent can be sterile. The preparation should be suitable for the mode of administration.

[0211] 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 alcohol, polyethylene glycol, gelatin, carbohydrates (such as lactose, amylose or starch), sugars (such as mannitol, sucrose, etc.), dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, aromatic oils, fatty acid esters, hydroxyethylcellulose, polyvinylpyrrolidone, etc. and combinations thereof. When needed, the pharmaceutical preparation can be mixed with adjuvants (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts affecting osmotic pressure, buffers, coloring agents, flavoring agents and / or aromatic substances, etc.), which do not react detrimentally with the active compound or interfere with its activity. In some embodiments, a water-soluble carrier suitable for intravenous administration is used.

[0212] If desired, the composition or drug can also contain small amounts of wetting agents or emulsifying agents or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release preparation or powder. The composition can also be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral preparations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, saccharin sodium, cellulose, magnesium carbonate, etc.

[0213] The composition or drug can be formulated into a pharmaceutical composition suitable for administration to humans according to conventional procedures. For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile, isotonic, aqueous buffer. If necessary, the composition may also include solubilizing agents and local anesthetics to relieve pain at the injection site. Generally, the components are supplied separately or mixed together in unit dosage forms, such as dry lyophilized powders or anhydrous concentrates in airtight containers, such as ampoules or vials, indicating the quantity of the active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water, saline, or dextrose / water. When the composition is administered by injection, ampoules of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0214] 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 an embodiment, the osmolality of the 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).

[0215] In another embodiment, ASA is formulated in a physiological buffer, such as:

[0216] a) Formulation buffer I containing the following substances (in mM): Na2HPO4 (3.50 - 3.90), NaH2PO4 (0 - 0.5), glycine (25 - 30), mannitol (230 - 270), and water for injection; or

[0217] b) Formulation buffer II containing the following substances (in mM): Tris-HCl (10), glycine (25 - 30), mannitol (230 - 270), and water for injection.

[0218] The arylsulfatase A purified by the method of the present invention can be used as a drug for reducing the level of intracellular sulfatide 3-O-sulfo-galactosylceramide (galactosylsulfatide) in the peripheral nervous system and / or within the central nervous system in subjects suffering from and / or diagnosed with metachromatic leukodystrophy. Administration of ASA will result in a reduced impairment of motor learning skills and / or an increase in nerve motor conduction velocity and / or nerve conduction amplitude. As used herein, the term "therapeutically effective amount" is determined to a large extent based on the total amount of the therapeutic agent contained in the pharmaceutical composition of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., prevent, treat, modulate, cure, prevent and / or improve the underlying disease or condition). For example, a therapeutically effective amount can be an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, such as an amount sufficient to modulate the lysosomal enzyme receptor or its activity to treat such lysosomal storage diseases or their symptoms (e.g., a reduction or elimination of the presence or incidence of "zebra bodies" or cellular vacuolization after administration of the composition of the present invention to a subject). Generally, the amount of the therapeutic agent (e.g., recombinant lysosomal enzyme) administered to a subject in need thereof will depend on the characteristics of the subject. These characteristics include the subject's condition, disease severity, general health, age, gender, and weight. One of ordinary skill in the art will be able to readily determine the appropriate dosage based on these and other relevant factors. Additionally, objective and subjective assays can optionally be employed to determine the optimal dosage range.

[0219] In embodiments, the characteristics of the purified recombinant ASA protein (e.g., characteristic glycan profiles, such as a purified recombinant ASA protein having a threshold amount of mannose-6-phosphorylated recombinant ASA protein) and other properties, such as the characteristic maximum level of host cell protein (HCP), the characteristic maximum amount of host cell DNA (HCD), and / or specific specific activity) can contribute to the desired properties of the composition containing the purified recombinant ASA protein (e.g., improved storage stability, improved therapeutic properties, etc.).

[0220] In embodiments, the present invention provides a method for purifying recombinant ASA protein from an impure preparation (e.g., unprocessed biological material, such as a cell culture medium containing ASA) using a process involving only a single step of ultrafiltration / diafiltration after chromatography. In embodiments, this single-step UF / DF process is achieved by combining the eluate from the chromatography step and adjusting the pH of the combined eluate to 6.0 or higher than about 6.0. In some embodiments, this simplified process is combined with a high-loading chromatography step to facilitate large-scale production of recombinant ASA protein.

[0221] Aspects of the invention are described in further detail in the following subsections. The use of subsections is not intended to limit the invention. Each subsection may be applicable to any aspect of the invention. In this application, unless otherwise specified, the use of "or" means "and / or".

[0222] Arylsulfatase A

[0223] 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 typically metabolized to galactocerebroside by 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, which hydrolyzes the 3-O-sulfate bond. Deficiency of arylsulfatase A occurs in all tissues from patients with late infantile, juvenile, and adult forms of metachromatic leukodystrophy (MLD). As used herein, the arylsulfatase A protein will be referred to as "ASA" or "ARSA", and saposin B will be referred to as "Sap-B".

[0224] 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 an octamer of 480 kDa under acidic conditions (pH ≤ approximately 5.0), but dissociates into dimers at neutral pH levels. In human urine, the enzyme consists of two different subunits of 63 kDa and 54 kDa (Laidler PM et al. Biochim Biophys Acta. 1985, 827, 73 - 83). Arylsulfatase A purified from human liver, placenta, and fibroblasts also consists of two subunits that vary slightly in size and range from 55 kDa to 64 kDa (Draper RK et al. Arch Biochemica Biophys. 1976, 177, 525 - 538, Waheed A et al. HoppeSeylers Z Physiol Chem. 1982, 363, 425 - 430, Fujii T et al. Biochim Biophys Acta. 1992, 15 1122, 93 - 98). As is the case with other lysosomal enzymes, arylsulfatase A is synthesized as a glycosylated precursor on membrane - bound ribosomes. It then traverses the endoplasmic reticulum and the Golgi apparatus, where its N - linked oligosaccharides are processed to form complex - type phosphorylated and sulfated oligosaccharides (Waheed A et al. Biochim Biophys Acta. 1985, 847, 53 - 61, Braulke T et al. Biochem Biophys Res Commun. 1987, 143, 178 - 185). In normally cultured fibroblasts, a 62 - kDa precursor polypeptide is produced, which is transferred to acidic pre - lysosomal endosomes via mannose - 6 - phosphate receptor binding (Braulke T et al. J Biol Chem. 1990, 265, 6650 - 6655, Kelly BM et al. Eur J Cell Biol. 1989, 48, 71 - 78).

[0225] The methods described herein can be used to purify arylsulfatase A from any source, such as tissue or cultured cells (e.g., human cells (e.g., fibroblasts)) that recombinantly produce arylsulfatase A. Arylsulfatase A of any origin (including but not limited to humans and other animals, such as mammals) can be produced by the methods described herein.

[0226] The length of the human arylsulfatase A signal peptide (18 amino acids) is based on the consensus sequence and the specific processing site of the signal sequence. Thus, cleavage of the signal peptide from the deduced human ASA cDNA (EMBL GenBank accession numbers J04593 and X521151) occurs after residue 18 (Ala) in all cells, resulting in the mature form of human arylsulfatase A. As used herein, recombinant arylsulfatase A will be abbreviated as "rASA". The mature form of arylsulfatase A, including the mature form of human arylsulfatase A, will be referred to as "mASA", while the mature recombinant human ASA is referred to as "mrhASA".

[0227] Multiple forms of arylsulfatase A have been demonstrated by 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 MetabBiol. 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, indicating that much of the charge heterogeneity of arylsulfatase A is due to variations in the carbohydrate content of the enzyme.

[0228] 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 enzyme inhibitors at concentrations in the millimolar range or lower.

[0229] The gene structure of human arylsulfatase A has been described. As used herein, this gene will be referred to as "ARSA". However, in some cases, "ARSA" can also refer to the arylsulfatase A protein. 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 a 507 amino acid enzyme unit (Stein et al. J Biol Chem. 1989, 264, 1252-1259). Messenger RNAs of 2.1 kb, 3.7 kb and 4.8 kb have been detected in fibroblasts, and the 2.1 kb messenger RNA is apparently responsible for most of the active arylsulfatase A produced by the cells (Kreysing et al. Eur J Biochem. 1990, 191, 627-631). The ARSA sequence has been deposited in EMBL GenBank under accession number X521150. Kreysing et al. described the differences between the published cDNA and the coding portion of ARSA (Eur J Biochem. 1990, 191, 627-631). The cDNA sequences initially described by Stein et al. (J Biol Chem. 1989, 264, 1252-1259) and Kreysing et al. (Eur J Biochem. 1990, 191, 627-631) have been deposited in EMBL GenBank under the following accession numbers J04593 and X521151, respectively.

[0230] Several polymorphisms and over 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). Disease - related mutations in the ARSA gene can be divided into two major categories, which have a good correlation with the clinical phenotypes of MLD. When introduced into cultured animal cell lines, one category (I) does not produce active enzyme, does not produce immunoreactive protein, and does not express ASA activity. The other category (A) produces a small amount of cross - reactive material and low levels of functional enzyme in cultured cells. Homozygous individuals with category (I) mutations, or individuals with two different mutations from this category, exhibit late - infantile MLD. Most individuals with one category (I) mutation and one category (A) mutation develop into the juvenile - onset form, while individuals with two category (A) mutations usually exhibit adult MLD. Some mutations have been found relatively frequently, while other mutations have only been detected in single families. Specific mutations can be traced through members of many families, but routine carrier screening is not yet feasible.

[0231] In addition to the above - mentioned disease - related mutations, several polymorphisms have been found in the ARSA gene. Very low ASA activity has been found in clinically normal parents of some MLD patients and also in the general population. This so - called pseudo - deficiency of ASA has been associated with common polymorphisms in the ARSA gene (Gieselmann et al., Dev Neurosci. 1991, 13, 222 - 227).

[0232] Recombinant ASA protein

[0233] As used herein, the term "recombinant ASA protein" refers to any molecule or part of a molecule that can substitute for at least part of the activity of the 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 directed to purifying a recombinant ASA protein that is a polypeptide having an amino acid sequence that is substantially similar or identical to the mature human ASA protein.

[0234] Typically, human ASA is produced as a precursor molecule that is processed into a mature form. This process generally occurs by removal of an 18 - amino acid signal peptide. Typically, the precursor form is also referred to as the full - length precursor or full - length ASA protein and contains 507 amino acids. The 18 N - terminal amino acids are cleaved, generating a mature form that is 489 amino acids in length. Thus, ASA protein activity is generally not expected to require the 18 N - terminal amino acids. The amino acid sequences of the mature form (SEQ ID NO:1) and the full - length precursor (SEQ ID NO:2) of a typical wild - type or naturally occurring human ASA protein are shown in Table 1.

[0235] Table 1. Human Arylsulfatase A

[0236]

[0237]

[0238] Thus, in some embodiments, the recombinant ASA protein purified by the embodiments of the present invention is the mature human ASA protein (SEQ ID NO:1). In some embodiments, the recombinant ASA protein purified by the embodiments of the present invention can be a homolog or analog of the mature human ASA protein. For example, a homolog or analog of the mature human ASA protein can be a modified mature human ASA protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to the wild - type or naturally occurring ASA protein (e.g., SEQ ID NO:1), while retaining substantially the ASA protein activity. Thus, in some embodiments, the recombinant ASA protein purified by the embodiments of the present invention is substantially homologous to the mature human ASA protein (SEQ ID NO:1). In some embodiments, the recombinant ASA protein purified by the embodiments of the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO:1. In some embodiments, the recombinant ASA protein purified by the embodiments of the present invention is substantially identical to the mature human ASA protein (SEQ ID NO:1). In some embodiments, the recombinant ASA protein purified by the embodiments of the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:1. In some embodiments, the recombinant ASA protein purified by the embodiments of the present invention contains a fragment or a portion of the mature human ASA protein.

[0239] Alternatively, the recombinant ASA protein purified by an embodiment of the present invention is a full-length ASA protein. In some embodiments, the recombinant ASA protein can be a homolog or analog of the full-length human ASA protein. For example, a homolog or analog of the full-length human ASA protein can be a modified full-length human ASA protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to the wild-type or naturally occurring full-length ASA protein (e.g., SEQ ID NO:2), while retaining the basic ASA protein activity. Thus, in some embodiments, the recombinant ASA protein purified by an embodiment of the present invention is substantially homologous to the full-length human ASA protein (SEQ ID NO:2). In some embodiments, the recombinant ASA protein purified by an embodiment of the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO:2. In some embodiments, the recombinant ASA protein purified by an embodiment of the present invention is substantially identical to SEQ ID NO:2. In some embodiments, the recombinant ASA protein purified by an embodiment of the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2. In some embodiments, the recombinant ASA protein purified by an embodiment of the present invention contains a fragment or a portion of the full-length human ASA protein. As used herein, the full-length ASA protein generally contains a signal peptide sequence.

[0240] Homologs or analogs of the human ASA protein can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such methods being found, for example, in references that compile such methods. In some embodiments, conservative amino acid substitutions include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In some embodiments, a "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.

[0241] In some embodiments, the recombinant ASA protein can contain a moiety that binds to a receptor on the surface of a target cell to facilitate cellular uptake and / or lysosomal targeting. For example, such a receptor can be the cation-independent mannose-6-phosphate receptor (CI-MPR), which binds mannose-6-phosphate (M6P) residues. Additionally, CI-MPR also binds other proteins, including IGF-II. In some embodiments, the recombinant ASA protein contains M6P residues on the protein surface. Specifically, the recombinant ASA protein can contain a bisphosphorylated oligosaccharide that has a higher binding affinity for CI-MPR. In some embodiments, a suitable enzyme contains at most about on average of at least about 20% bisphosphorylated oligosaccharide / enzyme. In other embodiments, a suitable enzyme can contain about 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% bisphosphorylated oligosaccharide / enzyme.

[0242] In some embodiments, the recombinant ASA enzyme can be fused to a lysosomal targeting moiety capable of binding to a receptor on the surface of a target cell. Suitable lysosomal targeting moieties can be IGF-I, IGF-II, RAP, p97 and their variants, homologs or fragments (e.g., including those peptides having a sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to the wild-type mature human IGF-I, IGF-II, RAP, p97 peptide sequences). The lysosomal targeting moiety can be conjugated or fused to the ASA protein or enzyme at the N-terminus, C-terminus or internally.

[0243] Purification of Recombinant Arylsulfatase A

[0244] Embodiments of the present invention include a purification process for producing arylsulfatase A (“ASA”), particularly recombinant human ASA (“rhASA”) drug substance.

[0245] A variety of techniques, in whole or in part, optionally modified as described herein, can be used to produce a purified ASA drug substance.

[0246] For example, exemplary purification processes include one or more of the following steps (e.g., 1, 2, 3, 4, 5, 6 or all 7 of these steps):

[0247] ● Thaw and combine unpurified bulk rhASA (UPB).

[0248] ● Capture and filter the UPB by ultrafiltration and diafiltration (“UFDF” or “capture UFDF”).

[0249] ● After filtration, the captured material can be virus inactivated prior to chromatographic purification.

[0250] ● A method includes successively using four chromatographic columns: an anion exchange column (e.g., a Fractogel TMAE HiCap resin column), a ceramic hydroxyapatite type I (HA) column, a hydrophobic interaction column (HIC; e.g., a phenyl sepharose FF column), and a cation exchange (e.g., SP) column.

[0251] ● Combine the SP eluate and adjust the pH of the combined eluate (e.g., adjust to a pH of about 5.5 to about 6.5 (e.g., about 6.0)).

[0252] ● Then subject the resulting pH-adjusted cation exchange eluate to virus filtration, followed by a single concentration and diafiltration step to reach the final target protein concentration.

[0253] ● Add polysorbate 20 (P20) to the eluate (e.g., before frozen storage).

[0254] As used herein, a "contaminant" is a substance different from the desired polypeptide product, such as arylsulfatase A (ASA). A contaminant can be a variant of the desired polypeptide (e.g., a deamidated variant or an asparaginyl variant of the desired polypeptide) or another molecule, such as a polypeptide, a nucleic acid, and an endotoxin.

[0255] As used herein, "purifying" a polypeptide from a composition or sample comprising the polypeptide and one or more contaminants means increasing the purity of the polypeptide in the composition or sample by (fully or partially) removing at least one contaminant from the composition or sample.

[0256] A "purification step" can be part of an entire purification method, resulting in a composition that, based on the total weight of the composition, contains at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight of the target polypeptide.

[0257] The purity of arylsulfatase A can be measured by, for example, one or more of the following: host cell protein (HCP) Western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse-phase HPLC, and size exclusion HPLC.

[0258] For example, the arylsulfatase A used in the compositions and methods described herein can also be characterized by a characteristic glycan profile (e.g., any of the exemplary glycan profiles described herein).

[0259] In some embodiments, the specific activity of the purified arylsulfatase A is at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg, as determined, for example, by the methods described herein. In some embodiments, the purified recombinant ASA has a specific activity in the range of about 50 - 200 U / mg (such as about 50 - 190 U / mg, 50 - 180 U / mg, 50 - 170 U / mg, 50 - 160 U / mg, 50 - 150 U / mg, 50 - 140 U / mg, 50 - 130 U / mg, 50 - 120 U / mg, 50 - 110 U / mg, 50 - 100 U / mg, 60 - 140 U / mg, 60 - 130 U / mg, 60 - 120 U / mg, 60 - 110 U / mg, 60 - 100 U / mg, 70 - 140 U / mg, 70 - 130 U / mg, 70 - 120 U / mg, 70 - 110 U / mg, 70 - 100 U / mg, 80 - 140 U / mg, 80 - 130 U / mg, 80 - 120 U / mg, 80 - 110 U / mg, 80 - 100 U / mg, 90 - 140 U / mg, 90 - 130 U / mg, 90 - 120 U / mg, 90 - 110 U / mg, 90 - 100 U / mg, 100 - 140 U / mg, 100 - 130 U / mg, 100 - 120 U / mg, 100 - 110 U / mg, 110 - 140 U / mg, 110 - 130 U / mg, 110 - 120 U / mg, 120 - 140 U / mg, 120 - 130 U / mg or 130 - 140 U / mg), as determined, for example, by the methods described herein.

[0260] The raw material for the purification method is any impure preparation. For example, the impure preparation can be an unprocessed cell culture medium containing a recombinant ASA protein secreted from cells (e.g., mammalian cells) that produce the ASA protein or a crude cell lysate containing the ASA protein. In some embodiments, the impure preparation can be a partially processed cell culture medium or cell lysate. For example, the cell culture medium or cell lysate can be concentrated, diluted, and treated by virus inactivation, virus processing, or virus removal. In some embodiments, virus removal can be achieved using nanofiltration and / or chromatography techniques, etc. In some embodiments, virus inactivation can be achieved using solvent inactivation, detergent inactivation, pasteurization, acidic pH inactivation, and / or ultraviolet inactivation, etc. The cell culture medium or cell lysate can also be treated with protease, DNase, and / or RNase to reduce the levels of host cell proteins and / or nucleic acids (e.g., DNA or RNA). In some embodiments, the unprocessed or partially processed biological material (e.g., cell culture medium or cell lysate) can be frozen and stored at a desired temperature (e.g., 2 - 8°C, -4°C, -25°C, -75°C) for a period of time and then thawed for purification. As used herein, the impure preparation is also referred to as the raw material or the loading material.

[0261] The purification methods described herein can include, but are not limited to, one or more of the following steps: depth filtration, virus inactivation, ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography), mixed mode chromatography, hydrophobic interaction chromatography, ultrafiltration / diafiltration, and virus removal filtration. In some embodiments, the purification methods described herein further include affinity chromatography.

[0262] In the chromatography step, the appropriate volume of the resin used when packed into the chromatography column is reflected by the dimensions of the column (i.e., the diameter of the column and the height of the resin) and varies depending on, for example, the amount of protein in the applied solution and the binding capacity of the resin used. However, in order to obtain the production and purification of ASA on an industrial scale, increasing the scale of the production process as well as the purification process is also within the scope of the present disclosure. Thus, parameters such as the size, diameter, and flow rate of the column can be increased to match 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, the flow rate ranges from about 40 - 400 cm / hour (e.g., between about 100 cm / hour and 150 cm / hour) or is from about 5 - 100 ml.

[0263] Ultrafiltration - Capture

[0264] In some embodiments of the present invention, the purification methods disclosed herein include one or more steps of upstream ultrafiltration to capture ASA (e.g., human recombinant ASA) produced by a perfusion bioreactor. As used herein, ultrafiltration refers to membrane filtration, buffer exchange, and overall fractionation, with a filter pore size for membrane filtration of from 0.001 μm to 0.1 μm, which can be used for concentrating and desalting dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules). The ultrafiltration methods used in the embodiments of the present invention include tangential flow ultrafiltration or crossflow filtration.

[0265] As used herein, tangential flow filtration and ultrafiltration refer to an arrangement in which, as a portion passes through the membrane (permeate), the feed stream passes parallel to the membrane surface while the remainder (retentate) is recycled back to the feed reservoir. In some embodiments, the pore size of the tangential flow ultrafiltration filter is selected to allow recombinant ASA to permeate through the filter. In other embodiments, the pore size is selected so as to retain substantially all of the ASA in the feed passing through the filter. As described elsewhere, ASA exists as an octamer of 480 kDa at acidic conditions (pH ≤ about 5.0), but dissociates into dimers at neutral pH levels. Thus, the pH of the feed can be adjusted in combination with the selection of an appropriate pore size to retain ASA on the membrane or allow it to pass through as permeate.

[0266] A pore size with a cut-off molecular weight of at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 300 kDa, at least 400 kDa, or at least 500 kDa can be selected. For example, a filter with a pore size of at least 10 kDa will retain most proteins in the feed with a molecular weight of about 11 kDa or higher. As another example, a filter with a pore size of at least 400 kDa will retain most proteins with a molecular weight higher than 400 kDa. In some embodiments, the feed retention rate is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher. Similarly, the pore size can be selected to separate permeates of a specific size. For example, a filter with a pore size of at least 500 kDa will allow most proteins with a molecular weight less than 500 kDa to permeate through. In some embodiments, the permeation rate is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher.

[0267] The filtration area or capacity can also be optimized for the methods disclosed herein. Considerations affecting the selection of the filtration area include robustness, cost, feed flow rate (i.e., crossflow velocity), transmembrane pressure, permeate flux rate, equipment fitness, and throughput. In some embodiments, the permeate flux is 50 - 100 liters per square meter per hour2 ("LMH"). In some embodiments, the feed flow rate is about 250 - 600 LMH, including the end values. In some embodiments, the feed flow rate is about 250 - 350 LMH, including the end values. In some embodiments, the feed flow rate is about 175 - 245 LMH, including the end values. In some embodiments, the feed flow rate is about 170 - 230 LMH, including the end values. In some embodiments, the feed flow rate is about 120 - 160 LMH, including the end values. In some embodiments, the feed flow rate is about 15 - 30 LMH, including the end values. In some embodiments, the feed flow rate is about 11 - 21 LMH, including the end values. In some embodiments, the filtration area is about 0.02 m 2 , about 0.14 m 2 , about 0.7 or about 3.5 m 2 . In certain embodiments, the transmembrane pressure is about 55 - 60 psi, including the end values. In some embodiments, the transmembrane pressure is about 15 - 25 psi, including the end values. In some embodiments, the transmembrane pressure is about 10 - 20 psi, including the end values. In some embodiments, the transmembrane pressure is about 5 - 15 psi.

[0268] The ultrafiltration filters used in the embodiments of the present invention may include membrane materials known to those skilled in the art, including but not limited to polyethersulfone and stabilized cellulose.

[0269] An exemplary filter cartridge for use in the embodiments of the present invention is Sartorius Another exemplary filter cartridge for use in the embodiments of the present invention is Sartorius 30kD standard membrane. Yet another exemplary filter cartridge for use in the embodiments of the present invention is the Cuno depth filter ZB. In some embodiments of the present invention, the Cuno Zeta Plus depth filter is used.

[0270] Depth filtration

[0271] The purification methods described herein may include one or more steps of depth filtration. Depth filters are various filters that use a porous filtration medium to retain particles throughout the medium rather than only on the surface of the medium. They contain a filtration medium with a graded density that allows larger particles to be intercepted near the surface of the filter, while smaller particles penetrate the larger opening areas of the filter surface and are only intercepted in the smaller openings closer to the center of the filter. Although certain embodiments employ depth filtration steps only during the upstream recovery stage (i.e., before subsequent chromatographic purification steps), other embodiments employ depth filters during one or more additional purification stages. In some embodiments of the present invention, the Cuno Zeta Plus depth filter is used.

[0272] After depth filtration, filtration at 0.45 microns (±2 μm) can be optionally carried out to remove particulates and reduce the bioburden in the preparation for downstream processing.

[0273] Virus inactivation

[0274] The purification methods described herein may include one or more steps of virus inactivation. In some embodiments, virus inactivation includes solvents and / or detergents. The solvent or detergent may include, for example, polysorbate 20, polysorbate 80, tributyl phosphate (TnBP), or combinations thereof. Virus inactivation may involve incubation in the solvent or detergent for 3 - 24 hours. In another embodiment, virus inactivation includes virus filtration, such as by filtration using a Planova TM filter.

[0275] It should be understood that these methods are intended to produce a preparation of an enzyme that is substantially free of infectious virus and may be referred to as a "virus-safe product". Additionally, it is contemplated that the various methods may be used independently or in combination.

[0276] Virus inactivation can be achieved by adding one or more "virus inactivators" to the solution containing the enzyme. In some embodiments, the virus inactivation step is carried out prior to the chromatographic purification step (i.e., before loading the impure preparation onto the first chromatographic column) in order to ensure that no amount or concentration of the reagent is present in the final product that would compromise the safety of the product when used as a drug or when the product is used in the preparation of a drug; other embodiments employ depth filters during one or more additional purification stages. For example, in some embodiments, the inventive method according to the present invention further includes a step of virus removal after the last chromatographic column.

[0277] The term "virus inactivator" is intended to denote a reagent (e.g., a detergent) or method that can be used to inactivate both lipid-enveloped viruses and non-lipid-enveloped viruses. The term "virus inactivator" should be understood to cover both combinations of such reagents and / or methods (where appropriate) and also only one type of such reagent or method.

[0278] Typical virus inactivators are detergents and / or solvents, most typically a detergent-solvent mixture. It should be understood that the virus inactivator is optionally a mixture of one or more detergents and one or more solvents. A variety of detergents and solvents can be used for virus inactivation. The detergents can be selected from nonionic detergents and ionic detergents and are chosen to be substantially non-denaturing. Generally, nonionic detergents are used because they facilitate the removal of the detergent from the rASA preparation in subsequent purification steps. Suitable detergents are described, for example, by Shanbrom et al. in U.S. Patent Nos. 4,314,997 and 4,315,919. Typical detergents are those sold under the trademarks Triton X-100 and Tween 20 or Tween 80. Preferred solvents for the virus inactivator are dialkyl phosphates or trialkyl phosphates, as described by Neurath and Horowitz in U.S. Patent No. 4,764,369. A typical solvent is tributyl phosphate (TnBP). A particularly preferred virus inactivator for the practice of the present invention is Tween 80, but alternatively, other reagents or combinations of reagents can be used. The typical reagent is added in such a volume that the concentration of Tween-80 in the ASA-containing solution is in the range of about 0.5 wt% - 4.0 wt%, preferably a concentration of about 1 wt%. Then TnBP can be added to a final concentration of 0.3% based on the new volume of the ASA-containing sample.

[0279] The virus inactivation step is carried out under conditions that inactivate enveloped viruses, thereby producing a substantially virus-safe rhASA-containing solution. Generally, such conditions include a temperature of 4 - 37°C, such as 19°C - 28°C, 23°C - 27°C, typically about 25°C, and an incubation time found to be effective through validation studies. Generally, an incubation time of 1 - 24 hours is sufficient, preferably 10 - 18 hours, such as about 14 hours, to ensure adequate virus inactivation. However, the appropriate conditions (temperature and incubation time) depend on the virus inactivator employed, the pH, and the protein concentration and lipid content of the solution.

[0280] It is expected that other methods of virus removal or inactivation can also be employed to produce virus-safe products, such as the addition of methylene blue followed by inactivation by ultraviolet radiation.

[0281] The purification methods described herein may include one or more steps of virus removal filtration. Generally, virus filtration is performed after enzyme purification by one or more chromatographic steps. In some embodiments, the virus filtration step is performed by passing the ASA-containing solution resulting from the purification step through a sterile filter and then passing the sterile-filtered solution through a nanofilter. A "sterile filter" means a filter that will substantially remove all microorganisms capable of reproducing and / or causing infection. Although typically the pore size of the filter is about 0.1 micron, the pore size range can also be between about 0.05 and 0.3 microns. It is feasible to replace the virus filtration of the sample during the purification process with contacting or combining the sample with a detergent.

[0282] Some embodiments of the invention include at least two steps of virus inactivation and / or filtration. For example, virus inactivation prior to column chromatography can be combined with virus removal after all chromatographic steps are completed. Virus removal after chromatography can be performed before or after one or more steps of ultrafiltration / diafiltration (UFDF) (e.g., tangential flow ultrafiltration). In one specific example, an eluate is obtained from the last step of chromatographic purification (e.g., cation exchange (SP) chromatography), and the pH of the eluate pool is adjusted to about 5.5, about 6.0, about 6.5, or about 7.0, and then virus filtration is performed. Thus, in certain embodiments, virus filtration is performed before a single step of UFDF (e.g., using a Planova TM filter). In another example, an eluate is obtained from the last step of chromatographic purification (e.g., cation exchange (SP) chromatography), subjected to the first step of UFDF without pH adjustment, and then virus filtration and the second step of UFDF are performed.

[0283] In certain embodiments, the pH-adjusted cation exchange eluate pool is subjected to virus filtration on a Planova 20N filter. In certain embodiments, after virus filtration of the pH-adjusted cation exchange eluate, the yield relative to the input is about 90%-100%; that is, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher as evaluated by A280 absorbance. Thus, in some embodiments, there is substantially no loss of recombinant ASA during virus filtration. The yield of virus filtration is very important because it verifies that adjusting the pH to about 6.0 allows the dissociation of ASA octamers (with a diameter of about 20 nm) into dimer form. Thus, the pore size of the virus filter can be selected to ensure that only the dimer form is filtered (i.e., the octamer form can be retained by the filter or cause clogging of the virus filter). For example, a virus filter with a pore size of 20 nm will retain the octamer form of ASA and not the dimer form.

[0284] Affinity chromatography

[0285] The purification methods described herein may include one or more steps of affinity chromatography (e.g., immunoaffinity chromatography, immobilized metal ion affinity chromatography, and / or immobilized ligand affinity chromatography).

[0286] Briefly, affinity chromatography is a chromatographic technique that relies on highly specific interactions, such as those between a receptor and a ligand, an antigen and an antibody, or an enzyme and a substrate. As will be known to those skilled in the art, the selective molecules employed in the affinity chromatography steps of the purification methods described herein can be based on various properties of the recombinantly produced ASA that can be exploited by the selective molecules (e.g., three-dimensional structure, glycosylation, etc.). Exemplary selective molecules (or capture reagents) that can be utilized in the affinity chromatography step include Protein A, Protein G, antibodies, metal ions (e.g., nickel), specific substrates, ligands, or antigens. In some embodiments, the selective molecule suitable for the 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 are commercially available or can be obtained by immunization of non-human animals (e.g., mice, rats, rabbits, chickens, goats, sheep, horses, or other animals suitable for producing antibodies against human proteins).

[0287] Typically, the target molecule (e.g., recombinant ASA) is retained on a solid or stationary phase or medium through interaction with the selective molecule, while other unwanted molecules are not retained as they are not bound by the selective molecule. The solid medium can then be removed from the mixture, optionally washed, and the target molecule is released from the retention by elution. In some embodiments, the affinity column can be eluted by changing the ionic strength in a gradient. For example, salt concentration, pH, pI, and ionic strength can be used to separate or form a gradient for separation.

[0288] In some embodiments, the recombinant ASA protein can be produced with a tag to facilitate purification by affinity chromatography. As will be known to those skilled in the art, protein tags can include, for example, glutathione-S-transferase (GST), hexahistidine (His), maltose-binding protein (MBP), etc. In some embodiments, lectins are used in affinity chromatography to separate components in a sample. For example, certain lectins specifically bind to specific carbohydrate molecules and can be used to separate glycoproteins from non-glycosylated proteins, or to separate one glycoform from another.

[0289] Ion exchange chromatography

[0290] The purification methods described herein may include one or more steps of ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography).

[0291] As is known to those skilled in the art, ion exchangers (e.g., anion exchangers and / or cation exchangers) can be based on various materials with respect to the matrix and with respect to the attached charged groups. For example, the following matrices can be used, where the materials mentioned are more or less cross-linked: agarose-based (such as SEPHAROSE TM CL-6B, SEPHAROSE TM FastFlow and SEPHAROSE TM High Performance), cellulose-based (such as DEAE ), dextran-based (such as ), silica-based, and synthetic polymer-based.

[0292] Ion exchange resins can be prepared according to known methods. Generally, before loading a sample or composition containing a polypeptide and one or more contaminants onto the resin, an equilibration buffer that allows the resin to bind its counterion can be passed through the ion exchange resin. Conveniently, the equilibration buffer can be the same as the loading buffer, but this is not necessary.

[0293] In an optional embodiment of the present invention, after eluting the polypeptide, the ion exchange resin can be regenerated with a regeneration buffer so that the column can be reused. Generally, the salt concentration and / or pH of the regeneration buffer can be such that substantially all of the contaminants and the target polypeptide are eluted from the ion exchange resin. Generally, the regeneration buffer has a very high salt concentration in order to elute the contaminants and the polypeptide from the ion exchange resin.

[0294] Anion exchange chromatography

[0295] Embodiments of the present invention include, for example, providing a sample of arylsulfatase A (e.g., recombinant arylsulfatase A) and subjecting the sample to anion exchange chromatography, such as the anion exchange chromatography described herein. For anion exchange resins, the charged groups 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. Anion exchange chromatography can use, for example, Q SEPHAROSE TM Fast Flow, Q SEPHAROSE TM High Performance, QSEPHAROSE TM XL, CAPTO TM Q, DEAE, TOYOPEARL Q, TMAE (trimethylaminoethyl, quaternary ammonium resin), ESHMUNO TM Q, NUVIA TM Q or UNOSPHERE TM Q is used. Other anion exchangers can be used within the scope of the present invention, including but not limited to quaternary amine resins or "Q resins" (e.g., CAPTOTM-Q, QAE ); diethylaminoethane (DEAE) resins (e.g., DEAE benzoylated naphthylated DEAE, diethylaminoethyl ); resins; resins, resins (e.g., IRA–67, strongly basic, weakly basic), cholestyramine resin, resins (e.g., SAX–10, WAX–10, WCX–10), resins (e.g., TSKgel DEAE–NPR; TSKgel DEAE–5PW) and resins.

[0296] In an embodiment, anion exchange chromatography is performed using TMAE (trimethylaminoethyl, quaternary ammonium resin).

[0297] In some embodiments, subjecting the arylsulfatase A sample to anion exchange chromatography is carried out at a temperature of about 23 °C or lower, about 18 °C or lower, or about 16 °C or lower, e.g., about 23 °C, about 20 °C, about 18 °C, or about 16 °C.

[0298] Typical mobile phases for anion exchange chromatography include relatively polar solutions such as water, acetonitrile, organic alcohols (such as methanol, ethanol, and isopropanol), or solutions containing 2-(N-morpholino)-ethanesulfonic acid (MES). Thus, in certain embodiments, the mobile phase comprises 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, at any given time during the separation process, the mobile phase comprises 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.

[0299] In certain embodiments, rhASA is loaded at a binding capacity of about 23 AU / L resin or lower, such as about 19 AU / L resin or lower, about 15 AU / L resin or lower, or about 12 AU / L resin or lower, such as 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, an arylsulfatase A sample is loaded onto an anion exchange chromatography column at a binding capacity of at least about 4.5 g / L resin (e.g., at least about 5 g / L resin, 6 g / L resin, 7 g / L resin, 8 g / L resin, 9 g / L resin, 10 g / L resin, 11 g / L resin, 12 g / L resin, 13 g / L resin, 14 g / L resin, or 15 g / L resin). In some embodiments, an arylsulfatase A sample is loaded onto an anion exchange chromatography column at a binding capacity in the range of about 4.5 - 20 g / L resin (e.g., in the range of about 5 - 20 g / L resin; 5 - 19 g / L resin, 5 - 18 g / L resin, 5 - 17 g / L resin, 5 - 16 g / L resin, 5 - 15 g / L resin, 7.5 - 20 g / L resin, 7.5 - 19 g / L resin, 7.5 - 18 g / L resin, 7.5 - 17 g / L resin, 7.5 - 16 g / L resin, 7.5 - 15 g / L resin, 10 - 20 g / L resin, 10 - 19 g / L resin, 10 - 18 g / L resin, 10 - 17 g / L resin, 10 - 16 g / L resin, or 10 - 15 g / L resin).

[0300] A loading buffer with a salt concentration and / or pH that allows the polypeptide and contaminants to bind to an anion exchange resin can be used to load an aqueous solution containing ASA and contaminants as the mobile phase onto an anion resin. The resin can then be washed with one or more column volumes of the loading buffer and then with one or more column volumes of a wash buffer, where the salt concentration is increased. Finally, ASA can be eluted with an elution buffer with increasing salt concentration. Optionally, elution of the enzyme can also be mediated by gradually or stepwise decreasing the pH. Fractions with ASA activity can be collected and combined for further purification.

[0301] In some embodiments, loading an arylsulfatase A sample onto an 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, such as 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, the salt concentration in the mobile phase is a gradient (e.g., linear or non-linear gradient). In some embodiments, the salt concentration in the mobile phase is constant. In some embodiments, the salt concentration in the mobile phase can be increased or decreased stepwise. In some embodiments, loading an arylsulfatase A sample onto an anion exchange chromatography column is performed at a pH of about 5 to about 9, such as about 6 to about 8, such as about 7.

[0302] In some embodiments, washing an anion exchange chromatography column is performed with one or more wash buffers. For example, washing the anion exchange column can include two or more (e.g., first and second) washing steps, each using a different wash buffer. In one embodiment, the wash buffer does not contain sodium chloride. In another embodiment, the wash buffer contains sodium chloride. For example, the sodium chloride concentration of the wash buffer is from about 50 mM to about 200 mM, such as 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, such as about 80 mM, about 100 mM, about 120 mM, or about 140 mM. In some embodiments, washing an anion exchange chromatography column is performed at a pH of about 5 to about 9, such as about 6 to about 8, such as about 7.

[0303] In one embodiment, the elution buffer contains sodium phosphate. For example, the sodium phosphate concentration in the elution buffer is from about 20 mM to about 50 mM, such as from about 25 mM to about 45 mM, such as about 30 mM, about 35 mM or about 40 mM. In another embodiment, the elution buffer does not contain sodium chloride. In another embodiment, the elution buffer contains sodium chloride. For example, the sodium chloride concentration in the elution buffer is from about 200 mM to about 300 mM, such as from about 240 mM to about 280 mM. In some embodiments, the elution of arylsulfatase A from the anion exchange column is carried out at a pH of about 5 to about 9, such as about 6 to about 8, such as about 7.

[0304] In some embodiments, the elution of arylsulfatase A from the anion exchange column includes one or more elution peak collection steps. For example, the elution peak collection starts from about 50 mAU on the rising side to about 50 mAU on the falling side, such as starting from about 100 mAU on the rising side to about 50 mAU on the falling side, starting from about 200 mAU on the rising side to about 50 mAU on the falling side, starting from about 50 mAU on the rising side to about 100 mAU on the falling side, starting from about 50 mAU on the rising side to about 200 mAU on the falling side, or starting from about 100 mAU on the rising side to about 100 mAU on the falling side, such as determined by spectrophotometry, for example, at 280 nM.

[0305] It will be apparent to those of ordinary skill in the art that many different buffers can be used in the loading, washing, and elution steps. However, the column is typically washed 1–10 times with a buffer containing 0.05 M MES-Tris (pH 7.0) to equilibrate the column. For 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, and then washed with a washing buffer containing 0.02 MES-Tris, 0.12 M NaCl (pH 7.0). Alternatively, the column can be equilibrated, loaded, and washed with any other equilibration buffer, loading buffer, and washing buffer for anion exchange chromatography described herein. The sample can be eluted in a buffer containing 0.02 MES-Tris, 0.26 M NaCl (pH 7.0). Alternatively, the sample can be eluted in any other elution buffer for anion exchange chromatography described herein.

[0306] The loading buffer, washing buffer, and elution buffer described herein may include one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is between about 1 mM and about 500 mM, such as 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, such as about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0307] The yield, activity, and purity after anion exchange chromatography may vary. In some embodiments, the activity yield of arylsulfatase A is at least about 75%, such as at least about 85%, such as between about 85% and about 99%, or between about 90% and about 99%. In some embodiments, the protein yield (AU or absorbance units), as measured by spectrophotometry, for example at 280 nm, is about 10% to 50%, such as about 20% to about 35%, or about 25% to about 30%. In some embodiments (e.g., those using the TMAE column described below), the eluted library protein activity yield (AU or absorbance units), as measured by spectrophotometry, for example at 280 nm, is about 70% to 400%, such as about 80% to about 390%, or about 90% to about 350%, or about 100% to 150%, greater than at least 95%. In some embodiments (e.g., those using the TMAE column described below), the log reduction value (LRV) of host cell protein (HCP) is between about 0.5 and about 1.1, such as between about 0.6 and 0.9, or between about 0.7 and 0.8. In some embodiments (e.g., those using the TMAE column described below), the purity, as determined by capillary electrophoresis - SDS PAGE, is at least 75%, such as at least 80%, at least 85%, at least 90%, or higher. In a preferred embodiment, the activity yield, HCP LRV, and purity (as determined by capillary electrophoresis - SDS PAGE) after anion exchange chromatography are at least about 90%, at least about 0.6, and at least about 80%, respectively.

[0308] In a preferred embodiment of the present invention, an anion exchange column with a high loading capacity is used. In certain embodiments of the present 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., equal to 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 loading capacity column is a TMAE column. In a particular embodiment, the column is selected from the group consisting of: TMAE column, Nuvia Q column, Q Sepharose Fast Flow column, Capto Q column, Q Sepharose XL column, Eshmuno Q column, UNOsphere Q column or GigaCap Q column.

[0309] In a particular embodiment of the present invention, the TMAE column is pre - equilibrated with a buffer solution having a pH of 7.0 and containing about 20 mM MES - Tris and 1000 mM NaCl. In certain embodiments, the column is equilibrated with a buffer solution having a pH of 7.0 and containing 50 mM MES - Tris. In some embodiments, the loading flow rate of the TMAE column is about 75 - 125 cm / hour (i.e., about 75 - 115 cm / hour, about 75 - 110 cm / hour, about 75 - 105 cm / hour, about 75 - 100 cm / hour, about 85 - 115 cm / hour, about 85 - 110 cm / hour, about 85 - 105 cm / hour, about 85 - 100 cm / hour, about 95 - 115 cm / hour, about 95 - 110 cm / hour, about 95 - 105 cm / hour, about 95 - 100 cm / hour, about 100 - 120 cm / hour, about 100 - 115 cm / hour, about 100 - 110 cm / hour, about 100 cm / hour). As described herein, the loading conditions can be optimized and evaluated by A280 absorbance.

[0310] In certain embodiments utilizing a TMAE column (e.g., a Fractogel TMAE column), even at a loading capacity greater than 15 g / L, little product is lost in the flow-through during the loading process. The ability to increase the loading capacity while minimizing flow-through losses is a significant improvement in the purification method. In certain embodiments of the present invention, the amount of product lost in the flow-through is less than 30% of the loaded amount (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%).

[0311] After loading, in some embodiments, the TMAE column is washed at least once. In certain embodiments, the column is washed twice. The first wash buffer or the second wash buffer may contain an optimized level of sodium chloride. In some embodiments, the amount of sodium chloride in the first wash buffer or the 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, the first wash buffer contains 50 mM MES-Tris, pH 7.0. In some embodiments, the second wash buffer contains 20 mM MES-Tris, 100 mM NaCl, pH 7.0. Further optimization of the wash conditions, particularly the second wash conditions, is encompassed in the embodiments of the present invention. For example, increasing the salt concentration of the second wash can improve the log reduction value (LRV) and overall purity of host cell proteins (HCPs), but will simultaneously reduce activity and A280 yield. As described herein, specific wash conditions must be balanced with the elution conditions described below in order to provide an optimal combination of purity, activity, and yield.

[0312] In embodiments of the present invention, the recombinant ASA bound to the TMAE column is eluted with an elution buffer. In some embodiments, the amount of sodium chloride in the elution buffer is optimized. In certain embodiments, the amount of sodium chloride in the elution buffer is between about 150 - 300 mM (such as 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 mM, about 220 mM, or about 260 mM). In a specific example, the elution buffer contains 50 mM MES-Tris and 1 M NaCl, with a pH of 7.0. In some embodiments, the A280 yield after elution is greater than 60% of the loaded amount (e.g., about 60%, about 70%, about 80% or higher). Further optimization of the elution conditions is encompassed in embodiments of the present invention. For example, increasing the elution salt concentration (i.e., conductivity) can provide a better yield but results in poorer purity and HCP removal. And as described above, specific washing conditions must be coordinated with the elution conditions in order to provide an optimal combination of purity, activity, and yield.

[0313] Cation exchange chromatography

[0314] In some embodiments, the method further comprises subjecting the arylsulfatase A sample to cation exchange chromatography, e.g., sulfopropyl (SP) cation exchange chromatography as described herein. In some embodiments, the arylsulfatase A sample is subjected to anion exchange chromatography prior to cation exchange chromatography. In typical embodiments, cation exchange chromatography comprises sulfopropyl (SP) cation exchange chromatography, but other cation chromatography membranes or resins can be used, such as MUSTANG TM S membrane, S-SEPHAROSE TM resin or Blue SEPHAROSE TMResin. In some embodiments, the method further includes concentrating and / or filtering the arylsulfatase A sample, for example, by ultrafiltration and / or diafiltration, such as tangential flow ultrafiltration. Cation exchange chromatography can be performed at an optimal temperature, for example, as described herein, to enhance target binding and / or reduce impurity binding. For example, cation exchange chromatography can be performed at a temperature of about 23 °C, 18 °C, 16 °C or lower.

[0315] In one embodiment, the cation exchange chromatography includes sulfopropyl (SP) cation exchange chromatography. In another embodiment, the cation exchange chromatography is a polishing step. Cation exchange chromatography (e.g., sulfopropyl (SP) cation exchange chromatography) can be performed using one or more of, for example, the following: SP-650, SP-550, SP-3PW, SP-5PW, SP SEPHAROSE TM FastFlow, SP SEPHAROSE TM High Performance, SP SEPHAROSE TM XL, S membrane, HS50, UNOSPHERE TM S and MACROCAP TM S.

[0316] An aqueous solution containing arylsulfatase A and contaminants can be loaded onto a cation resin using a loading buffer having a salt concentration and / or pH such that the polypeptide and contaminants bind to the cation exchange resin. The resin can then be washed with one or more column volumes of equilibration buffer or loading buffer, and then optionally washed with one or more column volumes of wash buffer, wherein the salt concentration is increased. Finally, arylsulfatase A can be eluted in an elution buffer. Fractions having arylsulfatase A activity can be collected and combined for further purification.

[0317] In typical embodiments, for example, as described herein, the NaCl concentration and / or pH of the loading buffer, wash buffer, and / or elution buffer can be optimized to enhance target binding and / or reduce impurity binding. In some embodiments, the NaCl concentration in the loading buffer is about 20 mM, 15 mM, 10 mM or lower. In some embodiments, the pH of the loading buffer is about 4.5, 4.3, 4.0 or lower. In some embodiments, the NaCl concentration in the wash buffer is about 20 mM, 15 mM, 10 mM or lower. In some embodiments, the NaCl concentration in the elution buffer is about 55 mM, 50 mM, 45 mM, 40 mM or lower.

[0318] In some embodiments, subjecting an arylsulfatase A sample to cation exchange chromatography comprises: loading the arylsulfatase A sample onto a cation chromatography column (e.g., a sulfopropyl (SP) cation exchange column), washing the cation exchange chromatography column, and eluting the arylsulfatase A from the column. In some embodiments, the column can be equilibrated with more than 3 column volumes, e.g., 5 to 10 column volumes, of 0.01 M NaAc, 0.01 M NaCl, 0.03 M acetic acid (pH 4.2).

[0319] 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 about 1 mM to about 25 mM, e.g., 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 about 10 mM to about 100 mM, e.g., about 20 mM, about 40 mM or about 60 mM. In some embodiments, loading the arylsulfatase A sample onto the cation exchange chromatography column is carried out at a pH of about 3.0 to about 6.0, e.g., about 4.0 to about 5.0, e.g., about 4.0, about 4.3 or about 4.5. In some embodiments, the arylsulfatase A sample is loaded onto the cation exchange chromatography column with a binding capacity of about 15 AU / L resin or lower, e.g., about 14 AU / L resin or lower, or about 12 AU / L resin or lower, 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.

[0320] In some embodiments, washing the cation exchange column is performed with one or more wash buffers. For example, washing the cation exchange column can include two or more (e.g., first and second) washing steps, each using a different wash 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 buffer, loading buffer, and wash buffer for cation exchange chromatography described herein. In one embodiment, the wash buffer contains sodium chloride. For example, the sodium chloride concentration of the wash buffer is from about 1 mM to about 25 mM, such as from about 5 mM to about 20 mM, or from about 10 mM to about 15 mM, such as about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In another embodiment, the wash buffer contains sodium acetate. For example, the sodium acetate concentration of the loading buffer is from about 10 mM to about 100 mM, such as about 20 mM, about 40 mM, or about 60 mM. In some embodiments, washing the cation exchange column is performed at a pH of about 3.0 to about 6.0, such as about 4.0 to about 5.0, such as about 4.0, about 4.3, or about 4.5.

[0321] In some embodiments, eluting arylsulfatase A from the cation exchange 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, such as from about 45 mM to about 60 mM, such as about 45 mM, about 50 mM, about 55 mM, or about 55 mM. In some embodiments, eluting arylsulfatase A from the cation exchange column is performed at a pH of about 3.0 to about 6.0, such as about 4.0 to about 5.0, such as about 4.0, about 4.3, or about 4.5. Thus, as a specific example, the sample can be eluted in a buffer containing 0.02 M NaAc, 0.05 M NaCl at pH 4.5. Alternatively, the sample can be eluted in any other elution buffer for cation exchange chromatography described herein.

[0322] In some embodiments, eluting arylsulfatase A from the cation exchange column includes one or more elution peak collection steps. For example, elution peak collection starts from about 50 mAU on the rising side to about 50 mAU on the falling side, such as from about 100 mAU on the rising side to about 50 mAU on the falling side, from about 200 mAU on the rising side to about 50 mAU on the falling side, from about 50 mAU on the rising side to about 100 mAU on the falling side, from about 50 mAU on the rising side to about 200 mAU on the falling side, or from about 100 mAU on the rising side to about 100 mAU on the falling side, such as determined by spectrophotometry, e.g., at 280 nM. The collected elution peaks can be combined.

[0323] The loading buffer, washing buffer, and elution buffer described herein may include one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is between about 1 mM and about 500 mM, such as 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, such as about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0324] In some embodiments, subjecting the arylsulfatase A sample to cation exchange chromatography is carried out at a temperature of about 23 °C or lower, about 18 °C or lower, or about 16 °C or lower, such as about 23 °C, about 20 °C, about 18 °C, or about 16 °C. In some embodiments, subjecting the arylsulfatase A sample to cation exchange chromatography is carried out between about 23 °C and about 16 °C, such as at about 23 °C, about 20 °C, about 18 °C, or about 16 °C, and loading the arylsulfatase A sample onto the cation exchange chromatography column is carried out at a pH between about 4.5 and about 4.3, such as at about 4.5, about 4.4, or about 4.3. In some embodiments, subjecting the arylsulfatase A sample to cation exchange chromatography is carried out at about 23 °C, and loading the arylsulfatase A sample onto the cation exchange chromatography column is carried out at a pH of about 4.5. In some embodiments, subjecting the arylsulfatase A sample to cation exchange chromatography is carried out at about 23 °C, and loading the arylsulfatase A sample onto the cation exchange chromatography column is carried out at a pH of about 4.3. In some embodiments, subjecting the arylsulfatase A sample to cation exchange chromatography is carried out at about 18 °C, and loading the arylsulfatase A sample onto the cation exchange chromatography column is carried out at a pH of about 4.5. In some embodiments, subjecting the arylsulfatase A sample to cation exchange chromatography is carried out at about 18 °C, and loading the arylsulfatase A sample onto the cation exchange chromatography column is carried out at a pH of about 4.3.

[0325] The yield after cation exchange chromatography may vary. In some embodiments, the activity yield of arylsulfatase A is at least about 75%, such as at least about 80%, such as between about 80% and about 105%. In some embodiments, the protein yield (AU or absorbance units), for example, as determined by spectrophotometry, such as at 280 nm, is between about 65% and 100%, such as between about 70% and about 95%.

[0326] The purity and activity are significantly improved after cation exchange chromatography. In some embodiments, the log reduction value (LRV) of host cell protein (HCP) is between about 1.0 and about 2.5, such as between about 1.5 and about 2.0 or between about 1.7 and about 1.9. The specific activity of purified arylsulfatase A, as determined by the methods described herein, can be at least about 50 U / mg to about 140 U / mg, such as 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. In some embodiments, arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. The purity of arylsulfatase A can be measured by, for example, one or more of the following: host cell protein (HCP) Western blot, Coomassie staining of SDS-PAGE, silver staining of SDS-PAGE, reverse-phase HPLC, and size exclusion HPLC. In certain embodiments, reducing the salt concentration and lowering the pH of the loading buffer enhances the binding of ASA to the cation exchange column without affecting the binding of impurities. In other words, as described above, the optimal balance of salt concentration and pH can increase the yield after cation exchange chromatography without adversely affecting the purity.

[0327] In some embodiments, the pH of the cation exchange eluate pool can be adjusted. In certain embodiments, the pH is adjusted immediately before virus filtration. The pH of the cation exchange eluate (e.g., SP eluate) can be adjusted to about 5.5, about 6.0, about 6.5, or about 7.0 using a pH adjustment buffer (pH 7.0) containing 0.25 M sodium phosphate, 1.33 M sodium chloride, and 0.34 M sodium citrate. In certain embodiments, the pH-adjusted SP eluate pool is subjected to virus filtration on a Planova 20N filter. In certain embodiments, after virus filtration of the pH-adjusted cation exchange eluate, the yield relative to the input is about 90%-100%; that is, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher, as evaluated by A280 absorbance. The yield of virus filtration is very important because it verifies that adjusting the pH to about 6.0 allows the dissociation of the ASA octamer (with a diameter of about 20 nm) into the dimer form. Therefore, the pore size of the virus filter can be selected to ensure that only the dimer form is filtered (i.e., the octamer form can be retained by the filter or cause clogging of the virus filter). For example, a virus filter with a pore size of 20 nm will retain the octamer form of ASA but not the dimer form.

[0328] Mixed-mode chromatography

[0329] The purification methods described herein may include one or more steps of mixed-mode chromatography. Mixed-mode chromatography is a type of chromatography in which several separation modes are typically applied in liquid chromatography to resolve mixtures of different molecules. For example, mixed-mode separation can simultaneously include a combined phase having both ion-exchange and reverse-phase characteristics. These stationary phases having more than one type of interaction are available from several column manufacturers.

[0330] In one aspect, the present disclosure features a method for purifying arylsulfatase A from a sample, the method including, for example, providing a sample of arylsulfatase A (e.g., recombinant arylsulfatase A), and subjecting the arylsulfatase A sample to mixed-mode chromatography, such as the mixed-mode chromatography described herein, such as a method including ceramic hydroxyapatite (HA) chromatography (e.g., type I or type II hydroxyapatite chromatography). In some embodiments, the mixed-mode chromatography is performed using one or more of the following: CHT TM type I ceramic hydroxyapatite media, CHT TM type II ceramic hydroxyapatite media, HT hydroxyapatite, and HTP hydroxyapatite.

[0331] In some embodiments, subjecting the arylsulfatase A sample to mixed-mode chromatography includes: loading the arylsulfatase A sample 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 arylsulfatase A sample to mixed-mode ion-exchange chromatography is performed at a temperature of about 23 °C or lower, about 18 °C or lower, or about 16 °C or lower, such as about 23 °C, about 20 °C, about 18 °C, or about 16 °C.

[0332] In some embodiments, loading the arylsulfatase A sample onto the mixed-mode chromatography column is performed using a loading buffer. In one embodiment, the loading buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is from about 1 mM to about 10 mM, such as from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, such as 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, such as from about 200 mM to about 300 mM, such as about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0333] In some embodiments, loading the arylsulfatase A sample onto the mixed-mode chromatography column is performed at a pH of about 5 to about 9, such as about 6 to about 8, such as about 7.

[0334] In some embodiments, mixed-mode chromatography includes ceramic hydroxyapatite (HA) chromatography. Hydroxyapatite (HAP) generally 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, as well as non-specific interactions between positively charged protein amino groups and negatively charged phosphate ions on the resin. By adjusting the pH of the buffer, basic or acidic proteins can be selectively adsorbed onto the column; elution can be achieved by changing the salt concentration of the buffer. Similarly, it is apparent that many buffer compositions and combinations of buffers can be employed. However, the column is typically washed 1–10 times with a buffer (pH 7.0) containing 0.001M NaPO4, 0.02M MES-Tris, 0.26M NaCl to equilibrate the column. For convenience, the sample can be loaded in the buffer from the previous step of the purification process, or a loading buffer can be used to load the sample. The column can be washed with 1-10 column volumes of the buffer used for equilibration and then washed with a wash buffer (pH 7.0) containing 0.005M NaPO4, 0.02M MES-Tris, 0.26M NaCl. Alternatively, the column can be equilibrated, loaded, and washed with any other equilibration buffer, loading buffer, and wash buffer for mixed-mode chromatography described herein. The sample can be eluted in a buffer (pH 7.0) containing 0.04M NaPO4. Optionally, the column can be stripped by washing with 1-10 column volumes of 0.4M NaPO4 (pH 12). Alternatively, the sample can be eluted in any other elution buffer for mixed-mode chromatography described herein.

[0335] In some embodiments, washing a mixed-mode chromatography column is performed with one or more wash buffers. For example, washing a mixed-mode chromatography column can include two or more (e.g., first and second) washing steps, each using a different wash buffer.

[0336] In one embodiment, the wash buffer contains sodium phosphate. For example, the sodium phosphate concentration of the wash buffer is from about 1 mM to about 10 mM, such as from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, such as about 1 mM, about 5 mM, or about 10 mM. In another embodiment, the wash buffer contains sodium chloride. For example, the sodium chloride concentration of the wash buffer is from about 50 mM to about 600 mM, such as from about 100 mM to about 500 mM, or from about 200 mM to about 400 mM, such as about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0337] In some embodiments, washing the mixed-mode chromatography column is carried out at a pH of from about 5 to about 9, such as from about 6 to about 8, such as about 7.

[0338] In some embodiments, eluting arylsulfatase A from the mixed-mode chromatography column is carried out at a pH of from about 5 to about 9, such as from about 6 to about 8, such as about 7. In some embodiments, eluting arylsulfatase A from the mixed-mode chromatography column includes one or more elution peak collection steps. For example, the elution peak collection starts at about 50 mAU on the rising side and ends at about 50 mAU on the falling side, such as starting at about 100 mAU on the rising side and ending at about 50 mAU on the falling side, starting at about 200 mAU on the rising side and ending at about 50 mAU on the falling side, starting at about 50 mAU on the rising side and ending at about 100 mAU on the falling side, starting at about 50 mAU on the rising side and ending at about 200 mAU on the falling side, or starting at about 100 mAU on the rising side and ending at about 100 mAU on the falling side, such as determined by spectrophotometry, for example, at 280 nM.

[0339] The loading buffer, washing buffer, and elution buffer described herein may include one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is between about 1 mM and about 500 mM, such as 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, such as about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0340] In some embodiments, purifying ASA by mixed-mode chromatography is successfully purified by ion exchange chromatography (e.g., anion exchange chromatography). However, it is contemplated that these steps may be carried out in the reverse order.

[0341] The yield after mixed-mode chromatography may vary. In some embodiments, the activity yield of arylsulfatase A is at least about 80%, such as at least about 90%, such as between about 80% and about 115%. In some embodiments, the protein yield (AU or absorbance units), such as determined by spectrophotometry, for example, at 280 nm, is from about 30% to 80%, such as from about 35% to about 75%, or from about 50% to about 70%.

[0342] The purity after mixed-mode chromatography is greatly improved. In some embodiments, the specific activity of the purified arylsulfatase A, as determined by the methods described herein, is at least about 50 U / mg to about 140 U / mg, such as 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. 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, for example, one or more of the following: host cell protein (HCP) Western blot, Coomassie staining of SDS-PAGE, silver staining of SDS-PAGE, reverse-phase HPLC, and size exclusion HPLC. In some embodiments, the log reduction value (LRV) of host cell protein (HCP) is between about 0.3 and about 0.6, such as between about 0.4 and 0.5.

[0343] Hydrophobic interaction chromatography (HIC)

[0344] The purification methods described herein may include subjecting an arylsulfatase A sample to hydrophobic interaction chromatography (HIC). In one embodiment, the hydrophobic interaction chromatography includes phenyl chromatography. In other embodiments, the hydrophobic interaction chromatography includes butyl chromatography or octyl chromatography. In some embodiments, subjecting the arylsulfatase A sample to HIC is carried out at a temperature of about 23 °C or lower, about 18 °C or lower, or about 16 °C or lower, such as about 23 °C, about 20 °C, about 18 °C, or about 16 °C. In some embodiments, the arylsulfatase A sample is subjected to mixed-mode chromatography prior to HIC.

[0345] Hydrophobic interaction chromatography exploits the attraction of a given molecule for a polar or nonpolar environment, and in the case of proteins, this tendency is controlled by the hydrophobicity or hydrophilicity of the residues on the outer surface of the protein that are exposed. Thus, proteins are fractionated according to their different degrees of attraction to a hydrophobic matrix, typically an inert support with an alkyl linker of 2-18 carbon chain length. The stationary phase consists of small nonpolar groups (butyl, octyl, or phenyl) attached to a hydrophilic polymer backbone (e.g., cross-linked Sepharose TM , dextran, or agarose). Thus, HIC columns are typically Butyl SEPHAROSE TM columns or Phenyl SEPHAROSE TM columns, and most typically Phenyl SEPHAROSE TM columns.

[0346] In some embodiments, hydrophobic interaction chromatography includes the use of Phenyl SEPHAROSE TM High Performance, Phenyl SEPHAROSE TM 6 Fast Flow (low substitution) or Phenyl SEPHAROSE TM 6 Fast Flow (high substitution) in one or more of the phenyl chromatography methods.

[0347] In some embodiments, subjecting an arylsulfatase A sample to hydrophobic interaction chromatography includes: loading the arylsulfatase A sample onto an HIC column, washing the HIC column, and eluting the arylsulfatase A from the column. The loading, washing, and elution in HIC generally follow the same principles as the above ion exchange chromatography, but usually employ conditions that are almost the opposite of those used in ion exchange chromatography. Thus, the HIC process involves using a high-salt loading buffer that can unfold the protein to expose hydrophobic sites. The protein is retained on the column by the hydrophobic ligand and is exposed to a gradient buffer containing a decreasing salt concentration. As the salt concentration decreases, the protein resumes its native conformation and is finally eluted from the column. Alternatively, the protein can be eluted with PEG.

[0348] In some embodiments, loading the arylsulfatase A sample onto the HIC column is carried out with a loading buffer. In one embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration in the loading buffer is about 0.5 M to about 2.5 M, such as about 1 M or about 1.5 M. In another embodiment, the loading buffer contains sodium phosphate. For example, the sodium phosphate concentration in the loading buffer is about 10 mM to about 100 mM, such as about 25 mM, about 50 mM, or about 75 mM. In some embodiments, loading the arylsulfatase A sample onto the HIC column is carried out at a pH of about 5 to about 7, such as about 5.5 to about 6.5, such as about 5.5, about 6.0, or about 6.5. In some embodiments, the arylsulfatase A sample is loaded onto the HIC column with a binding capacity of about 12 AU / L resin or lower, such as about 10 AU / L resin or lower, about 9 AU / L resin or lower, about 7 AU / L resin or lower, or about 5 AU / L resin or lower, such as 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.

[0349] Using Phenyl SEPHAROSE TMThe solid phase in HIC is typical in the present disclosure. Similarly, it is obvious that there are many different possibilities when it comes to the exact conditions and the buffers and combinations of buffers used for the loading, washing, and elution processes. In a typical embodiment, the column can be equilibrated in a buffer containing 0.05 M NaPO4, 1 M NaCl (pH 5.5). For convenience, the sample can be loaded in the buffer from the previous step of the purification process, or the sample can be loaded using a loading buffer.

[0350] In some embodiments, the HIC column is washed with one or more wash buffers. For example, washing the HIC column can include two or more (e.g., first and second) washing steps, each using a different wash buffer. In some embodiments, the wash buffer contains sodium chloride. For example, the sodium chloride concentration in the wash buffer is from about 100 mM to about 1.5 M, such as from about 250 mM to about 1 M, such as about 250 mM, about 500 mM, about 750 mM, or about 1 M. In another embodiment, the wash buffer contains sodium phosphate. For example, the sodium phosphate concentration in the loading buffer is from about 10 mM to about 100 mM, such as about 25 mM, about 50 mM, or about 75 mM. In some embodiments, washing the HIC column is carried out at a pH of about 5 to about 7, such as about 5.5 to about 6.5, such as about 5.5, about 6.0, or about 6.5. For example, the washing can be performed by washing the column 1 - 2 times with the equilibration buffer, and then with 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 buffer, loading buffer, and wash buffer described herein for HIC.

[0351] In some embodiments, elution of arylsulfatase A from the HIC column is carried out with an elution buffer. In some embodiments, the elution buffer contains sodium chloride. For example, the sodium chloride concentration in the elution buffer is from about 30 mM to about 100 mM, such as from about 45 mM to about 85 mM, such as about 50 mM, about 60 mM, about 70 mM, or about 80 mM. In some embodiments, elution of arylsulfatase A from the HIC column is carried out at a pH of about 5 to about 9, such as about 6 to about 8, such as 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.

[0352] In some embodiments, eluting arylsulfatase A from the HIC column includes one or more elution peak collection steps. For example, elution peak collection starts at about 50 mAU on the rising side and ends at about 50 mAU on the falling side, such as starting at about 100 mAU on the rising side and ending at about 50 mAU on the falling side, starting at about 200 mAU on the rising side and ending at about 50 mAU on the falling side, starting at about 50 mAU on the rising side and ending at about 100 mAU on the falling side, starting at about 50 mAU on the rising side and ending at about 200 mAU on the falling side, or starting at about 100 mAU on the rising side and ending at about 100 mAU on the falling side, for example, determined by spectrophotometry, such as at 280 nM.

[0353] In some embodiments, purifying arylsulfatase A by HIC is successfully purified by ion exchange chromatography (e.g., anion exchange chromatography) and / or mixed mode chromatography. However, it is contemplated that these steps can be performed in the reverse order.

[0354] The loading buffer, wash 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, such as 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, such as about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0355] The yield after HIC can vary. In some embodiments, the activity yield of arylsulfatase A is at least about 60%, such as at least about 70%, such as between about 70% and about 100%. In some embodiments, the protein yield (AU or absorbance units), determined by spectrophotometry, such as at 280 nm, is about 45% to 100%, such as about 50% to about 95%, or about 55% to about 90%.

[0356] The purity is greatly improved after HIC. In some embodiments, the specific activity of the purified arylsulfatase A is at least about 50 U / mg to about 140 U / mg, such as at least about 70 U / mg, at least about 90 U / mg, at least about 100 U / mg, or at least about 120 U / mg, as determined by the method described herein, for example.

[0357] In some embodiments, arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8% or at least about 99.9%. The purity of arylsulfatase A can be measured by, for example, one or more of the following: host cell protein (HCP) Western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse-phase HPLC, and size-exclusion HPLC. In some embodiments, the log reduction value (LRV) of host cell protein (HCP) is between about 0.6 and about 1.2, such as between about 0.7 and 0.95.

[0358] Ultrafiltration / diafiltration

[0359] The purification methods described herein may include one or more steps of downstream ultrafiltration and / or diafiltration. In some embodiments, the method further includes concentrating and / or filtering the arylsulfatase A sample, for example, by ultrafiltration and / or diafiltration, such as by tangential flow ultrafiltration.

[0360] Ultrafiltration refers to a membrane separation process driven by a pressure gradient, in which the components of a liquid are fractionated by the membrane according to their solvated size and structure. Diafiltration is a special type of ultrafiltration process in which the retentate is diluted with water and ultrafiltered again to reduce the concentration of soluble permeate components and further increase the concentration of the retained components. Ultrafiltration and diafiltration are typically combined into an ultrafiltration / diafiltration (UFDF) purification step.

[0361] Embodiments of the invention utilize at least one, at least two, at least three or more downstream UFDF purification steps. Diafiltration may occur one or more times within a UFDF step (such as UFDFDF). In some embodiments, the protein yield (AU or absorbance units) after downstream UFDF, as measured by spectrophotometry, for example, at 280 nm, relative to the amount from a previous purification step, is from about 90% to 105%, such as from about 95% to about 100%, such as from about 97% to about 99%. In some embodiments, there is substantially no protein loss during UFDF.

[0362] In some embodiments of the present invention, the purity of rASA produced by downstream UFDF, as determined by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and / or reverse phase-high performance liquid chromatography (RP-HPLC), is at least about 95%, at least about 97%, at least about 98%, at least about 99% or higher. In some embodiments, arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8% or at least about 99.9%. The purity of arylsulfatase A can be measured by, for example, one or more of the following: host cell protein (HCP) Western blot, Coomassie staining of SDS-PAGE, silver staining of SDS-PAGE, reverse phase HPLC and size exclusion HPLC. The specific activity of rASA, as determined by, for example, the sulfatase release assay described below, is at least about 60 U / mg to about 100 U / mg, such as at least about 65 U / mg, at least about 90 U / mg, at least about 70 U / mg or at least about 90 U / mg.

[0363] In some embodiments, arylsulfatase A is purified by tangential flow filtration to separate arylsulfatase A from contaminants based on the size of the contaminants in an acidic environment. Arylsulfatase A forms an octamer at low pH with a theoretical molecular weight of 480 kDa and will thus be retained by a relatively open membrane, while most contaminants will pass through the membrane (Sommerlade et al., (1994) Biochem. J., 297; 123-130; Schmidt et al., (1995) Cell, 82 271-278; Lukatela et al., (1998) Biochemistry, 37, 3654-3664).

[0364] In a typical embodiment, the diafiltration buffer contains 0.01 M sodium citrate phosphate, 0.137 M NaCl (pH 6.0).

[0365] In some embodiments, since the starting material of the process is a suspension of arylsulfatase A eluted from a chromatography column in the previous step of the process, the pH in the suspension is adjusted to 4 - 5 by adding 0.2 - 1 M sodium acetate at pH 4.5. Then, diafiltration is carried out with 1 - 10 buffer volumes of sodium acetate at pH 4.0 - 5.5 in a manner well-known to those skilled in the art. Filtration can be carried out using several different filter types with nominal weight cut-offs in the range of 20 - 450 kDa, but filters with cut-offs in the range of 100 - 300 kDa are generally used. To further process the solution containing arylsulfatase A, the pH is adjusted to a value in the range of 7 - 8 by adding Tris-base to a final concentration of approximately 20 - 50 mM.

[0366] As an alternative to the above acidic tangential flow filtration, essentially the same conditions and buffer composition can be used to separate ASA from contaminants by acidic gel filtration. Filtration is carried out through a gel filtration column at low pH, which has been equilibrated in a solution at low pH (e.g., 0.2 - 0.9 M sodium acetate solution 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 gel filtration. The degree of concentration can vary widely such that arylsulfatase A can be concentrated from about 0.1 mg / ml to about 50 mg / ml, preferably to about 5 mg / ml.

[0367] In some embodiments, the sample pool is concentrated using a 30 kDa Biomax A screen. Diafiltration is carried out by performing 3 - 5 column washes with 20 mM sodium acetate (pH 5.4 - 5.7).

[0368] In an embodiment, a surfactant such as polysorbate - 20 (P20) is added to the composition containing the purified ASA protein prior to refrigeration. In an embodiment, the composition contains a surfactant such as P20 at 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).

[0369] Characterization of the purified ASA protein

[0370] Various methods can be used to characterize the purified recombinant ASA protein.

[0371] purity

[0372] The purity of a purified recombinant ASA protein is typically measured by the levels of various impurities (e.g., host cell proteins or host cell DNA) present in the final product.

[0373] For example, the level of host cell protein (HCP) can be measured by ELISA or SDS-PAGE. In some embodiments, the purified recombinant ASA protein contains less than 150 ng HCP / mg ASA protein (e.g., less than 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 30, 20, 10 ng HCP / mg ASA protein). In embodiments, the purified recombinant ASA protein contains less than about 100, about 80, or about 60 ng HCP / mg ASA.

[0374] In some embodiments, the purified recombinant ASA protein contains less than about 150 pg / mg, 140 pg / mg, 130 pg / mg, 120 pg / mg, 110 pg / mg, 100 pg / mg, 90 pg / mg, 80 pg / mg, 70 pg / mg, 60 pg / mg, 50 pg / mg, 40 pg / mg, 30 pg / mg, 20 pg / mg, or 10 pg / mg host cell DNA (HCD). In embodiments, the purified recombinant ASA protein contains less than about 50, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 pg HCD / mg ASA protein.

[0375] In some embodiments, the purified recombinant ASA protein has no new bands with an intensity greater than 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% of the assay control when subjected to Coomassie Brilliant Blue stained SDS-PAGE.

[0376] In some embodiments, the purified recombinant ASA protein has no band with an intensity greater than the 15 kDa HCP band assay control when subjected to Western blot SDS-PAGE for HCP, and no new band with an 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.

[0377] In some embodiments, the purified recombinant ASA protein has no new bands with an intensity greater than 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% of the assay control when subjected to silver stain SDS-PAGE.

[0378] In some embodiments, the log reduction value (LRV) of host cell protein (HCP) is between about 0.3 to about 0.6, such as between about 0.4 to 0.5. Various assay controls can be used, particularly those acceptable to regulatory agencies such as the FDA.

[0379] The purity of the purified recombinant ASA protein can 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 chromatography (RP-HPLC) (e.g., using an octadecyl (C18) bonded silica column and TFA as a counterion at acidic pH). In some embodiments of the present invention, the main peak in the chromatogram is ASA. Parameters that can be changed or optimized to improve separation include gradient conditions, organic modifiers, counterions, temperature, column pore size and particle size, solvent composition, and flow rate. As known to those skilled in the art, the level of purity can be identified by the percentage of the main peak. For example, the purity can be determined by integrating the observed main peak and side peaks and calculating the percentage of the main peak to the total area.

[0380] In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and 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 more). In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and 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 more).

[0381] In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and determined by the main peak percentage of RP-HPLC is greater than or equal to 97% (i.e., about 97%, about 98%, about 99% or more). In some embodiments of the invention, the purity of ASA purified by the methods disclosed herein and determined by the main peak percentage of RP-HPLC is greater than or equal to 98% (i.e., about 98%, about 99% or more).

[0382] Specific activity

[0383] Purified recombinant ASA protein can also be characterized by evaluating function and / or biological activity. Methods known in the art can be used to determine the enzymatic activity of recombinant ASA compositions. Typically, these methods include detecting the removal of sulfate from a synthetic substrate, which is referred to as a sulfate release assay. An example of enzyme activity assay involves the use of ion chromatography. The method quantifies the amount of sulfate ions enzymatically released from a substrate by recombinant ASA. The substrate can be a natural substrate or a synthetic substrate. In some cases, the substrate is heparan sulfate, dermatan sulfate, or their functional equivalents. Typically, the sulfate ions released are analyzed by ion chromatography with a conductivity detector. In this example, the result can be expressed as U / mg protein, where 1 unit is defined as the amount of enzyme required to release 1 μmol sulfate ions from a substrate per hour. In some embodiments, the specific activity of purified recombinant ASA is at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg. In some embodiments, the specific activity of purified recombinant ASA is between about 50-200 U / mg (e.g., about 50-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-200 U / mg). / mg、60-190U / mg、60-180U / mg、60-170U / mg、60-160U / mg、60-150U / mg、60-140U / mg、60-130U / mg、60-120U / mg、60-110U / mg、60-100U / mg、70-200U / mg、70-190U / mg、70-180U / mg、70-170U / mg, 70-160U / mg, 70-150U / mg, 70-140U / mg, 70-130U / mg, 70-120U / mg, 70-110U / mg, 70-100U / mg, 80-200U / mg, 80-190U / mg, 80-180U / mg, 80-170U / mg, 80-160U / mg, 80-150U / mg, 80-140U / m g, 80-130U / mg, 80-120U / mg, 80-110U / mg, 80-100U / mg, 90-200U / mg, 90-190U / mg, 90-180U / mg , 90-170U / mg, 90-160U / mg, 90-150U / mg, 90-140U / mg, 90-130U / mg, 90-120U / mg, 90-110U / mg,90-100U / mg, 100-200U / mg, 100-190U / mg, 100-180U / mg, 100-170U / mg, 100-160U / mg, 100-150U / mg, 100-140U / mg, 100-130U / mg , 100-120U / mg, 100-110U / mg, 110-200U / mg, 110-190U / mg, 110-180U / mg, 110-170U / mg, 110-160U / mg, 110-150U / mg, 110-140U / In embodiments, the specific activity of the purified recombinant ASA is about 50 to about 130 U / mg, about 70 to about 100 U / mg, about 80 to about 90 U / mg, or about 75 to about 95 U / mg. ,

[0384] In another example, the enzyme activity of the recombinant ASA composition can be determined by measuring the removal of sulfate from a 4-methylumbelliferyl-sulfate (4-MUF-sulfate) substrate to form fluorescent methylumbelliferone. In this example, the fluorescent signal generated by the test sample can be used to calculate the enzyme activity (in mU / mL) using a 4-MUF standard. One milliunit of activity is defined as the amount of enzyme required to convert 1 nmol of 4-MUF sulfate to 4-MUF in 1 minute at 37°C. The specific activity can then be calculated by dividing the enzyme activity by the protein concentration.

[0385] In some embodiments, activity is determined by hydrolysis of a synthetic chromogenic substrate, p-nitrocatechol sulfate (pNCS), whose final product, p-nitrocatechol (pNC), absorbs light at 515 nm. The following equation can be used to calculate enzyme activity in μmol pNCS hydrolyzed / min×ml (=units / ml):

[0386] Vtotal (ml) x ΔA = units / ml (1)

[0387] εM / 1000 x Vsample (ml) x incubation time (min), where:

[0388] ΔA = absorbance of sample – absorbance of blank

[0389] Vtotal (ml) = total reaction volume, in ml (0.15 ml in this case)

[0390] Vsample (ml) = volume of added sample, in ml (0.05 ml in this case)

[0391] εM = molar extinction coefficient of product pNC, 12,400 M-1 cm-1 in this case.

[0392] Equation 1 can be simplified to:[[]]

[0393] ΔA x (0.15 / (12,400 / 1000 x 0.05 x 30)) = X μmol / (min x ml) (= units / ml) (1)

[0394] To calculate the specific activity (in μmol of pNC consumed / (min x mg) (= units / mg)), divide Equation 1 by the protein concentration of the sample:

[0395] Equation 1 / protein concentration (mg / ml) = Y μmol / (min x mg) = units / mg (2)

[0396] In any instance, the protein concentration of the recombinant ASA composition can be determined by any suitable method known in the art for determining protein concentration. In some cases, the protein concentration is determined by ultraviolet light absorption assay. Such absorption assays are typically performed at a wavelength of about 280 nm (A280).

[0397] In some embodiments, the specific activity of the purified recombinant ASA against the 4-methylumbelliferyl substrate is from about 1.0 x 10 3 mU / mg to 100.0 x 10 3 mU / mg, from about 1.0 x 10 3 mU / mg to 50.0 x 10 3 mU / mg, from about 1.0 x 10 3 mU / mg to 40.0 x 10 3 mU / mg, from about 1.0 x 10 3 mU / mg to 30.0 x 10 3 mU / mg, from about 1.0 x 10 3 mU / mg to 20.0 x 10 3 mU / mg, from about 1.0 x 10 3 mU / mg to 10.0 x 10 3 mU / mg, from about 4.0 x 10 3mU / mg to 8.0x 10 3 mU / mg, about 4.0x 10 3 mU / mg to 10.0x 10 3 mU / mg, about 4.5x 10 3 mU / mg to 10.0x 10 3 mU / mg, about 5.0x 10 3 mU / mg to 10.0x 10 3 mU / mg, about 5.5x 10 3 mU / mg to 15.0x 10 3 mU / mg or about 4.0x 10 3 mU / mg to 20.0x 10 3 mU / mg. In some embodiments, the specific activity of the purified recombinant ASA against the 4-methylumbelliferone substrate is about 1.0x 10 3 mU / mg, 2.0x10 3 mU / mg, 3.0x 10 3 mU / mg, 4.0x 10 3 mU / mg, 5.0x 10 3 mU / mg, 10.0x 10 3 mU / mg, 15.0x 10 3 mU / mg, 20.0x 10 3 mU / mg, 25.0x 10 3 mU / mg, 30.0x 10 3 mU / mg, 35.0x 10 3 mU / mg, 40.0x 10 3 mU / mg, 45.0x 10 3 mU / mg, 50.0x 10 3 mU / mg or greater.

[0398] Charge distribution

[0399] The purified recombinant ASA can be characterized by the charge distribution associated with the protein. Generally, the protein charge distribution reflects the pattern of side-chain charges of residues that are typically present on the protein surface. The charge distribution can be determined by performing ion-exchange (IEX) chromatography (e.g., HPLC) assays on the protein. In some embodiments, "charge distribution" refers to a set of values that represent the amount of protein eluted from a column at a certain time point after adding a mobile phase containing exchange ions to the ion-exchange column.

[0400] Typically, a suitable ion exchange column is an anion exchange column. For example, a high performance liquid chromatography (HPLC) system can be used to determine the charge distribution by strong anion exchange (SAX) chromatography. Generally speaking, recombinant ASA adsorbs to the fixed positive charges of a strong anion exchange column, and a gradient of increasing ionic strength with a predetermined flow rate of the mobile phase elutes the recombinant ASA species from the column in proportion to their strength of ionic interaction with the positively charged column. The ASA species with more negative charge (higher acidity) elute later than the ASA species with less negative charge (lower acidity). The concentration of protein in the eluate is detected by ultraviolet light absorption (at 280 nm).

[0401] In some embodiments, recombinant ASA adsorbs to the fixed positive charges of a Mini Q PE column in 20 mM TRIS-HCl at about pH 8.0, and a gradient of increasing ionic strength with a flow rate of 0.8 ml / min of a mobile phase consisting of 20 mM TRIS-HCL, 1 M sodium chloride (pH 8.0) elutes the recombinant ASA species from the column in proportion to their strength of ionic interaction with the positively charged column.

[0402] In some embodiments, the charge distribution can be depicted by a chromatogram of absorbance units versus time after elution from an HPLC column. The chromatogram can include a set of one or more peaks, and each peak in the set identifies a subpopulation of recombinant ASA having a composition with similar surface charge.

[0403] Glycan mapping

[0404] In some embodiments, the purified recombinant ASA protein can be characterized by proteoglycan composition (commonly referred to as glycan mapping). Without wishing to be bound by any theory, it is believed that the shape and complexity of the glycan linkages as well as the branching structure can affect in vivo clearance, lysosomal targeting, bioavailability, and / or efficacy.

[0405] Typically, the glycan profile can be determined by enzymatic digestion and subsequent chromatographic analysis. Various enzymes can be used for enzymatic digestion, including but not limited to suitable glycosidases, peptidases (e.g., endopeptidases, exopeptidases), proteases, and phosphatases. In some embodiments, a suitable enzyme is alkaline phosphatase. In some embodiments, a suitable enzyme is neuraminidase. Glycans (e.g., phosphoglycans) can be detected by chromatographic analysis. For example, phosphoglycans can be detected by high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) or size exclusion high performance liquid chromatography (HPLC). The amount of glycan (e.g., phosphoglycan) represented by each peak on the glycan profile can be calculated using a standard curve of the glycan (e.g., phosphoglycan) according to methods known in the art and disclosed herein.

[0406] In an embodiment, the purified recombinant ASA protein exists in the form of the following species: neutral recombinant ASA protein (Group A), sialylated recombinant ASA protein (Group B), mannose-6-phosphorylated recombinant ASA protein (Group C), N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (Group E), or mixed recombinant ASA protein (Group E), or any combination thereof. In an embodiment, the purified recombinant ASA protein exists in the form of: neutral recombinant ASA protein (Group A), sialylated recombinant ASA protein (Group B), mannose-6-phosphorylated recombinant ASA protein (Group C), N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (Group E), and mixed recombinant ASA protein (Group E).

[0407] In some embodiments, the purified recombinant ASA protein according to the present invention is characterized by a glycan profile that comprises at least seven peak groups that respectively indicate neutral ASA protein (peak group 1), monosialylated ASA protein (peak group 2), capped mannose-6-phosphorylated ASA protein (peak group 3), disialylated ASA protein (peak group 4), monomannose-6-phosphorylated ASA protein (peak group 5), mixed ASA protein (peak group 6), and dimannose-6-phosphorylated ASA protein (peak group 7).

[0408] The relative amount of the glycan corresponding to a group (e.g., any of groups A-E or peak groups 1-7) can be determined based on the peak group area relative to the peak group area of the corresponding peak group in a predetermined reference standard.

[0409] In an embodiment, the peak group area of Group A (neutral recombinant ASA protein) relative to the peak group area of the corresponding peak group in the reference standard can be in the range of about 15% to about 25% (e.g., about 16% to about 22% or about 20% to about 25%). In an embodiment, the peak group area of the neutral recombinant ASA protein is characteristic of the range of any exemplary formulation in Example 3, with a variation of about 1% to about 10% in either direction for each endpoint of the exemplified range (e.g., with a variation of about 1% to about 5% for each endpoint of the exemplified range).

[0410] In an embodiment, the peak group area of Group B (sialylated recombinant ASA protein) relative to the total peak group area of the sialylated recombinant ASA protein substance of any corresponding peak group in the reference standard can be in the range of about 35% to about 45% (e.g., about 35% to about 42% or about 37% to about 42%). In an embodiment, the total peak group area of the sialylated recombinant ASA protein is characteristic of the range of any exemplary formulation in Example 3, with a variation of about 1% to about 10% in either direction for each endpoint of the exemplified range (e.g., with a variation of about 1% to about 5% for each endpoint of the exemplified range).

[0411] In an embodiment, the total peak group area of the mannose-6-phosphorylated recombinant ASA protein in group C relative to any corresponding peak group area in the reference standard can be in the range of about 20% to about 30% (e.g., about 20% to about 26%, about 25% to about 28%, or about 27% to about 32%). In an embodiment, the total peak group area of the mannose-6-phosphorylated ASA protein is a range characteristic of any exemplary formulation in Example 3, and for each endpoint of the exemplified range, there is a variation of about 1% to about 10% in either direction (e.g., for each endpoint of the exemplified range, there is a variation of about 1% to about 5%).

[0412] In an embodiment, the total peak group area of the N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein in group D relative to any corresponding peak group area in the reference standard can be in the range of about 1% to about 10% (e.g., about 3% to about 5%, about 4% to about 6%, or about 4.5% to about 5.5%). In an embodiment, the total peak group area of the N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein is a range characteristic of any exemplary formulation in Example 3, and for each endpoint of the exemplified range, there is a variation of about 1% to about 10% in either direction (e.g., for each endpoint of the exemplified range, there is a variation of about 1% to about 5%).

[0413] In an embodiment, the peak group area of the mixed recombinant ASA protein in group E relative to any corresponding peak group area in the reference standard can be in the range of about 5% to about 15% (e.g., about 7% to about 10%, about 7% to about 9%, or about 7.5% to about 8.5%). In an embodiment, the total peak group area of the mixed recombinant ASA protein is a range characteristic of any exemplary formulation in Example 3, and for each endpoint of the exemplified range, there is a variation of about 1% to about 10% in either direction (e.g., for each endpoint of the exemplified range, there is a variation of about 1% to about 5%).

[0414] Various reference standards for glycan mapping are known in the art and can be used to practice the present invention.

[0415] It is expected that the glycosylation pattern of the purified recombinant ASA protein (e.g., a composition comprising the purified recombinant ASA protein having a threshold population of mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein)) can affect the bioavailability, targeting, or efficacy of the protein.

[0416] Peptide mapping

[0417] In some embodiments, peptide mapping can be used to characterize amino acid composition, post-translational modifications, and / or cellular processing, such as cleavage of signal peptides and / or glycosylation. Typically, a recombinant protein can be fragmented into discrete peptide fragments by controlled or random cleavage to generate a pattern or peptide profile. In some cases, the purified ASA protein can be subjected to enzymatic digestion prior to analysis. Prior to analysis, digestion can be performed using peptidases, glycoside hydrolases, phosphatases, lipases, or proteases and / or combinations thereof. Methods well known in the art can be used to determine the structural composition of the peptides. Exemplary methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR), or HPLC.

[0418] Metal analysis

[0419] In some embodiments, the purified recombinant ASA protein can be characterized by metal analysis. Various methods for analyzing trace metals in purified drug substances are known in the art and can be used to practice the present invention.

[0420] In some embodiments, residual phosphorus 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 the pH of the drug substance. In some embodiments of the present invention, the residual phosphorus 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 of the recombinant ASA purified according to the methods disclosed herein ranges 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).

[0421] In some embodiments, the recombinant ASA purified according to the methods disclosed herein contains calcium. Without wishing to be bound by any particular theory, it is hypothesized that the calcium ions present in the active site of ASA may be essential for enzyme activity. In some embodiments of the present invention, calcium is present at a level 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).

[0422] Examples

[0423] Example 1. Exemplary Process

[0424] A purified recombinant ASA protein is prepared according to the exemplary processes described herein. Table 3 specifies certain particular features of the exemplary processes for preparing the purified recombinant ASA protein. In an embodiment, the process for purifying the recombinant ASA protein includes one or more of the features described in Table 3.

[0425] Table 3

[0426]

[0427]

[0428] Example 2. Exemplary Formulations

[0429] A composition comprising a purified ASA protein is prepared as described herein. Table 4 provides the characteristics of a composition comprising a purified ASA protein prepared according to the exemplary processes described herein. In an embodiment, the composition comprising the purified recombinant ASA protein is characterized by one or more of the features described in Table 4. In an embodiment, where a numerical range is provided, each boundary of the exemplified range may vary by about 1% to about 20% (e.g., about 1% to about 15%, about 1% to about 10%, about 1% to about 7.5%, about 1% to about 5%, or about 1% to about 2.5%).

[0430] Table 4. Exemplary Compositions Comprising Recombinant ASA Drug Substance (DS)

[0431]

[0432] Example 3: Comparability Study

[0433] A non-clinical comparability program was conducted to evaluate the activity, safety, and pharmacokinetics of Process B rhASA compared to Process A rhASA in the non-clinical comparability program (Table 5). All doses were administered intrathecally. Immunohistochemical staining of lysosome-associated membrane protein 1 (LAMP-1) was used to evaluate pharmacodynamic comparability in immunotolerant MLD mice. Pharmacokinetic comparability was evaluated by administering a single dose of 6.0 mg (equivalent to 100 mg / kg brain weight) of Process A or Process B rhASA to juvenile cynomolgus monkeys. Biodistribution was compared by quantitative whole-body autoradiography in rats. The potential toxicity of Process B rhASA was evaluated by repeated administration of rhASA at a dose of 18.6 mg to juvenile cynomolgus monkeys.

[0434] Table 5.

[0435]

[0436] AUC, area under the concentration-time curve; CSF, cerebrospinal fluid; M6P, mannose-6-phosphate; MLD, metachromatic leukodystrophy; QWBA, quantitative whole body autoradiography; rhASA, recombinant human arylsulfatase A.

[0437] Pharmacodynamic comparability

[0438] Immunotolerant MLD mice are ASA knockout mice (ASA− / −) with a stably integrated transgene encoding an inactive variant of human ASA, rendering them immunotolerant to injected rhASA. Animals were assigned to six groups: untreated group (n = 6); vehicle group (154 mM NaCl, 0.005% polysorbate-20, pH 6.0, n = 4); process A rhASA groups at 0.04 mg (n = 9) and 0.21 mg (n = 10); and process B rhASA groups at 0.04 mg (n = 10) and 0.21 mg (n = 10). Animals were dosed intrathecally on days 1, 8, 15, and 21 or 22. A group of seven untreated C57 / B16 mice was used as wild-type controls. Animals were sacrificed 24 hours after the final dose of rhASA.

[0439] Histological analysis involved immunohistochemical staining for lysosome-associated membrane protein 1 (LAMP-1), a lysosomal protein marker used to detect lysosomal storage diseases and as an indicator of disease status. Rabbit polyclonal anti-LAMP-1 antibody (Abcam, Cat# ab24170, Lot# ER127402-2, 1:400, Cambridge, MA, USA) and Bond TMThe PolymerRefine Detection Kit (Leica, Cat#DS9800, Buffalo Grove, IL, USA) was used for LAMP-1 immunostaining of mouse brain and spinal cord in 5-μm paraffin sections. LAMP-1 signals were obtained by staining with 3,3-diaminobenzidine and then counterstaining with hematoxylin. The primary antibody was replaced with isotype IgG of the negative control. Digital images of LAMP-1-stained slides were created and analyzed using an Aperio scanner and ImageScope software (Leica Microsystems Inc., Buffalo Grove, IL, USA). The relative staining positivity rate was obtained using the following formula: positivity rate (%) = (number of LAMP-1-positive pixels / total number of pixels) × 100. Statistical comparisons were made between the vehicle and untreated animals for each tissue using the Mann–Whitney test. Based on this assessment (inter-group p > 0.01 for all tissues), the data were combined into one group (hereinafter referred to as the control). The data were normalized to the control group, and statistical comparisons were made using one-way ANOVA and Tukey's multiple comparison test. Comparison results with p < 0.01 were considered statistically significant.

[0440] The specific activities of process A and process B rhASA were 89 U / mg and 106 U / mg, respectively, both within the target range of the assay. The comparison results of the two substances showed that the changes in M6P and sialic acid contents were the largest, and the specific activities were similar.

[0441] As Figures 1A - 1F shown, LAMP-1 staining of immunotolerant MLD mice was performed in the spinal cord, cerebellar white matter, fimbria of the hippocampus, cerebral peduncle, cerebral cortex, and striatum of the mice. Regions showing effects of treatment with process A substances were selected from previous studies for evaluation. No statistically significant differences in LAMP-1 staining were observed in the spinal cord, cerebellar white matter, and fimbria of the hippocampus between animals administered process A and process B rhASA. Similarly, no statistical differences were observed in the cerebral peduncle between process A and process B rhASA, but differences were observed in the cerebral cortex and striatum. These differences were considered sporadic and not biologically relevant because only one of the two tested doses reached statistical significance.

[0442] Compared with control group animals, in the spinal cord (0.21 mg dose group, p < 0.0001; Figure 1A ), cerebellar white matter (0.04 mg and 0.21 mg dose groups, p < 0.01; Figure 1B ), cerebral peduncle (0.04 mg and 0.21 mg dose groups; Figure 1D ) and fimbria of the hippocampus (0.04 mg dose group, p < 0.01; Figure 1C) In it, there was a statistically significant decrease in LAMP-1 staining of process A and process B rhASA. In the cerebral cortex (0.04 mg and 0.21 mg dose groups, p ≤ 0.0001; Figure 1E ) and striatum (0.04 mg dose group, p = 0.0005; Figure 1F ) In the striatum, there was also a statistically significant decrease in LAMP-1 staining of process B rhASA.

[0443] Pharmacokinetic comparability

[0444] In phase 1, animals were randomly divided into a process A rhASA 6.0 mg group (n = 6 [3 males and 3 females]) or a process B rhASA 6.0 mg group (n = 6 [3 males and 3 females]) based on body weight. After pretreatment with subcutaneous atropine sulfate (0.01 mg / kg), an intrathecal lumbar catheter was surgically implanted, and then the animals were anesthetized with intramuscular ketamine hydrochloride (8 mg / kg) and approximately 1 L / min of oxygen and 2% isoflurane. The rhASA dose was administered in a 1 mL dose volume through the implanted intrathecal lumbar catheter. When normalized to a 60 g brain weight in cynomolgus monkeys, the 6.0 mg dose was equivalent to 100 mg / kg brain weight. This dose was based on the highest dose of rhASA administered in a phase 1 / 2 clinical study in late infantile MLD children. In this clinical study, the highest dose administered was 100 mg, which was equivalent to 100 mg / kg brain weight when normalized to 1 kg brain weight (determined for the age group of the study participants).

[0445] After a washout period of at least 8 days, phase 2 was initiated. In this phase, if the animals had been administered process A rhASA in phase 1, they were administered process B rhASA; conversely, if the animals had been administered process B rhASA in phase 1, they were administered process A rhASA. Twenty-four hours after the last dose of rhASA, the animals were humanely euthanized.

[0446] Body weight, food consumption, and clinical parameters were monitored, and blood and CSF samples were collected at pre-specified time intervals before and after dosing. The concentration of rhASA in serum and CSF was measured using an enzyme-linked immunosorbent assay. The levels of rhASA in serum and CSF of juvenile cynomolgus monkeys were quantified using a GLP-validated ELISA assay. Microtiter plates were coated with a rabbit polyclonal antibody against rhASA (SH040). After incubation with samples and standards, the microtiter plates were washed, and a horseradish peroxidase-conjugated anti-ASA mouse monoclonal antibody (clone 19-16-3) was added to all wells. The lower limit of quantification for monkey serum was 39.1 ng / mL, and for CSF was 19.5 ng / mL. The upper limit of quantification for each method was 1250 ng / mL.

[0447] When using Process A and Process B rhASA, after a single intrathecal dose of 6.0 mg was administered to juvenile cynomolgus monkeys, the concentration-time curves of rhASA in serum ( Figure 2A ) and CSF ( Figure 2B ) were similar. Process A and Process B rhASA were similar with respect to pharmacokinetic parameters, including maximum plasma concentration (Cmax), exposure (area under the concentration-time curve from time 0 to the last measurement [AUClast]), and clearance (Table 6).

[0448] Table 6. Mean cerebrospinal fluid and serum pharmacokinetic parameters in juvenile cynomolgus monkeys after intrathecal lumbar administration of 6.0 mg of rhASA manufactured using Process A or Process B.

[0449]

[0450]

[0451] λz, t 1 1 / 2, Vz, CL, and MRTinf could not be determined in serum samples.

[0452] AUCinf, area under the concentration-time curve from time 0 to infinity; AUClast, area under the concentration-time curve from time 0 to the last measurement; CL, clearance; Cmax, maximum plasma concentration; λz, terminal rate constant; h, hour; MRTinf, mean residence time to infinity; n, number; ND, not detected; t 1 1 / 2, terminal elimination phase half-life; rhASA, recombinant human arylsulfatase A; SD, standard deviation; Tmax, time to reach maximum plasma concentration; Vz, volume of distribution.

[0453] Biodistribution comparability

[0454] This study used quantitative whole body autoradiography (QWBA) and gamma counting after a single intrathecal dose of 125 I]-rhASA. To evaluate tissue distribution, 28 rats implanted with intrathecal ports were randomly grouped such that each rat received 0.62 mg of 125I]-rhASA dose. At each of seven time points after administration (approximately 1, 4, 12, 24, 48, 96, and 168 hours after administration), two rats in each dose group were euthanized and subjected to QWBA, and the radioactivity concentration was determined for the selected tissues. Before euthanasia, blood samples were collected from the animals anesthetized with isoflurane by cardiac puncture. To evaluate the pharmacokinetic parameters, gamma counting was used to analyze the total radioactivity of aliquots of whole blood, plasma, and tissues. To evaluate the excretion pattern of rhASA, six male Sprague Dawley rats were assigned to a group that received 0.62 mg of 125 I]-rhASA dose. At 168 hours after administration 125 I]-rhASA, the rats were housed in individual metabolic cages to collect urine and feces separately.

[0455] Trichloroacetic acid precipitation of plasma samples indicated that approximately 33% of 125 I was not bound to rhASA. Representative whole-body autoradiographs showed the distribution of Figure 3A ) and process B ( Figure 3B ) 125 I]-rhASA in male Sprague Dawley rats 4 hours after a single intrathecal dose of 0.62 mg. The Cmax and AUClast of process A and process B rhASA and the ratio of process B rhASA to process A rhASA in different tissues are shown in Table 6. 125 The highest calculated concentration (Cmax) of 125 I]-rhASA was found in the thyroid, which may be due to the uptake of unbound iodine. This was followed by the pituitary gland, spinal cord, and liver, all of which had similar levels (Table 7). 125 The lowest concentration of

[0456] Table 7. Pharmacokinetic parameters of rhASA equivalents in male Sprague Dawley rats after a single intrathecal dose of 0.62 mg of process A or process B 125 I]-rhASA.

[0457]

[0458] AUClast, area under the concentration-time curve from time 0 to the last measurement; Cmax, maximum plasma concentration; rhASA, recombinant human arylsulfatase A.

[0459] For the brain, spinal cord, and lung, the Cmax values of Process B rhASA were approximately 1.5-fold higher (highest ratio) than those of Process A 125 I]-rhASA, and for the kidney, adrenal gland, stomach, and pancreas, the Cmax values of Process B rhASA were approximately 0.2–0.6-fold lower (lowest ratio; Table 5) than those of Process A 125 I]-rhASA. For all tissues except skin and adrenal gland, the exposure differences (AUClast) between Process B and Process A 125 I]-rhASA were less than two-fold, with ratios of 2.27 and 2.19 for skin and adrenal gland, respectively (Table 5). The terminal elimination-phase half-life of Process A 125 I]-rhASA in plasma was 37.2 hours, while that of Process B was 42.5 hours.

[0460] As Figure 4 shown, the excretion curves in urine and feces were evaluated. The excretion patterns of Process A and Process B 125 I]-rhASA were similar. Most of the Process A and Process B products were excreted in urine (76% and 71%, respectively), indicating that rhASA was systemically cleared mainly by renal elimination, and approximately 6% of both products were excreted in feces. The proportion of the administered dose recovered in the carcass was 6% for the Process A product and 8% for the Process B product, with most located in the thyroid, CSF, and meninges. For animals administered Process A or Process B 125 I]-rhASA, the total recoveries (sum of all excreta and carcass) were 90% and 83%, respectively.

[0461] Toxicology

[0462] After pretreatment with meloxicam (0.2 mg / kg) followed by ketamine hydrochloride (5 mg / kg) and medetomidine (0.06 mg / kg), intrathecal port catheters were surgically implanted. The rhASA dose was administered via slow bolus delivery using an intrathecal port catheter system. Juvenile cynomolgus monkeys (n = 8 [4 males and 4 females]) were dosed every 2 weeks with 18.6 mg of Process B rhASA in a dose volume of 0.6 mL or vehicle control (NaCl 154 mM, 0.005% polysorbate-20, pH 6.0; n = 8 [4 males and 4 females]) for 11 weeks (a total of six doses). The maximum feasible dose of 18.6 mg of rhASA was used in this study, based on the maximum tolerable volume (0.6 mL) in juvenile cynomolgus monkeys and the concentration of the Process B rhASA formulation (31.0 mg / mL).

[0463] Evaluations included clinical examinations (body weight assessment, food consumption assessment, physical assessment, cardiovascular assessment (electrocardiogram and blood pressure), neurological assessment, and ophthalmic assessment) and pathological examinations (serum chemistry, hematology, coagulation, urine analysis, CSF cell count, and CSF chemistry). The antigenicity of Process B rhASA was evaluated in serum and CSF. Two physical and neurological examinations were performed on unanesthetized animals during the pre-dose phase (before surgery and ≥5 days after surgery), and one physical and neurological examination was performed at weeks 1, 5, and 9 during the dosing phase (approximately 24 hours after dosing), and one physical and neurological examination was performed before necropsy. Electrocardiograms were performed on unanesthetized, temporarily restrained animals during the pre-dose phase (after surgery) and during week 11 of the dosing phase (near dosing and 3 hours after dosing). Twenty-four hours after the final dose, all animals were humanely euthanized. A complete set of tissues was collected, but only the target tissues (i.e., spinal cord and dorsal root ganglia) identified using Process A in the initial study were evaluated histopathologically.

[0464] Juvenile cynomolgus monkeys tolerated intrathecal administration of Process B rhASA well, with no drug-related effects on clinical, neurological, or pathological examinations.

[0465] No levels of side effects were observed following intrathecal administration of 18.6 mg of B rhASA to juvenile cynomolgus monkeys every 2 weeks for 11 weeks (a total of six doses). Administration of Process B rhASA was associated with eosinophilic infiltration in the following areas: the leptomeningeal areas around the brain and spinal cord; the perivascular areas in the brain and spinal cord; the peritubular (adjacent to the central canal) area in the spinal cord; and the perineurium and epineurium areas around the spinal nerve roots. The severity of these Process B rhASA-related changes was not reasonably expected to alter nervous system function, and based solely on morphology, these Process B rhASA-related changes were not considered side effects. The presence of infiltration in the spinal cord was consistent with what was expected in animals receiving intrathecal administration of protein.

[0466] On day 2, the mean concentration of process B rhASA in serum was 544 ng / mL, while that in CSF was 2185 ng / mL. At week 11, the mean concentration in serum was 37 ng / mL, while that in CSF was 171 ng / mL (Figure 2). Anti-drug antibodies were generated in all animals administered rhASA by week 10, and the anti-drug antibody levels observed in serum were higher than those in CSF (mean values: 391,462 ng / mL and 7,383 ng / mL, respectively); immunohistochemistry and tissue ELISA confirmed that the anti-drug antibodies did not affect rhASA exposure to the target tissue (data not shown).

[0467] Example 4: Pharmacokinetic / Pharmacodynamic Characteristics of Recombinant Human Arylsulfatase A in Patients with MLD

[0468] The pharmacokinetic / pharmacodynamic (PK / PD) characteristics of rhASA were studied after intrathecal administration in patients with MLD. The clinical efficacy was evaluated and the optimal dosing regimen was determined using the effect of rhASA on sulfatide levels in cerebrospinal fluid (CSF) and the change in the total score of the Gross Motor Function Measure 88 (GMFM-88) in MLD patients receiving rhASA.

[0469] All patients received rhASA every other week via an intrathecal drug delivery device (IDDD) for up to 338 weeks. In the initial dose-escalation study, patients in groups 1, 2, and 3 were less than 12 years old at enrollment and received 10, 30, and 100 mg rhASA, respectively. Patients in group 4 were less than 8 years old at enrollment and received 100 mg rhASA manufactured using an improved process. All patients received a drug product formulated with 30 mg / ml rhASA in an isotonic aqueous solution containing 154 mM sodium chloride and 0.005% polysorbate-20 (pH 6.0), which was delivered via IT administration through the in vivo IDDD. The results of multiple efficacy measures indicated that rhASA might be associated with a slowed disease progression.

[0470] Patients who complete the dose-escalation study may continue to receive rhASA every other week via IDDD and enter the extension study. Serum rhASA concentration is evaluated after the initial rhASA dose and at Week 38 of treatment. Patients in Group 4 receive rhASA manufactured using an improved process. Blood samples are collected ≤1 hour before IT injection and at 0.5, 1, 2, 4, 8, 12, 24, and 48 hours after IT injection. During the dose-escalation study, the rhASA concentration in CSF is evaluated every 2 or 4 weeks, and in the extension study, quarterly from Week 52 to Week 104 and semi-annually from Week 104 to Week 338. In the dose-escalation study, the GMFM-88 observations are evaluated at Weeks 0, 16, 24, and 40, and in the extension study, quarterly from Week 52 to Week 104 and semi-annually from Week 104 to Week 338.

[0471] Evaluations include adverse events (AEs) that occur during treatment; serum chemistry; 12-lead electrocardiogram (ECG) results; vital signs; physical examinations; cell counts; glucose and protein levels in cerebrospinal fluid (CSF); and anti-rhASA antibodies in CSF and serum. AE is defined as all adverse events that occur between the first dose of the study product or the device implantation surgery (whichever occurs earlier) and the date of the last follow-up visit of the study. Follow-up evaluations are conducted 4 weeks after the last rhASA injection or 2 weeks after removal of the last IDDD (whichever is later) for patients who do not participate in the extension study. For individuals who participate in the extension study, follow-up evaluations are conducted at the end-of-study visit (Week 40).

[0472] Anti-drug antibodies (ADA) in serum and CSF samples are first screened using an electrochemiluminescence bridging immunoassay (Meso Scale Diagnostics, LLC, Rockville, MD), which utilizes biotinylated and thio-labeled rhASA for capture and detection, respectively. ADA-positive samples are confirmed by ligand competition binding using the same bridging immunoassay, and then antibody titers and neutralizing activities are evaluated using an arylsulfatase A activity assay with 4-nitrocatechol sulfate as the substrate.

[0473] Evaluate the pharmacokinetics of rhASA in CSF and serum after administration, and the change in motor function relative to baseline. Serum and CSF samples were collected from patients at week 0 (first dose) and week 38 (last dose). The rhASA concentration was determined using a validated enzyme-linked immunosorbent assay method. The change in motor function relative to baseline was evaluated using the GMFM-88 total score (0-100%). Other endpoints included: levels of sulfatide and lysosulfatide in CSF evaluated by liquid chromatography-tandem mass spectrometry; N-acetylaspartate (NAA) / creatine and choline / creatine levels in deep brain white and gray matter evaluated by proton magnetic resonance spectroscopy (MRS); change in the total MLD severity score based on brain magnetic resonance imaging (MRI) relative to baseline; and peripheral nerve function evaluated by electromyography. Based on the highest concentrations observed in samples from 60 children without leukodystrophy who had undergone lumbar puncture (and thus might have other psychiatric disorders), the upper limits of normal for CSF sulfatide and lysosulfatide were defined as 0.113 μg / mL and 0.0277 ng / mL, respectively. Each patient received serial brain MRI measurements. Based on an MRI-based visual scoring method (where a higher score indicates more severe brain symptoms), the total MLD severity score (range: 0-34) for each individual was calculated at each time point. Peripheral nerve function was also evaluated by electromyography. If overall means and standard deviations (SDs) could be obtained within the appropriate age range, nerve conduction velocity, amplitude, distal latency, and F-wave latency were measured and converted to z-scores.

[0474] Treatment with rhASA was well tolerated, with no deaths, no discontinuation due to adverse events (AEs), and no life-threatening AEs reported. IT rhASA did not appear to have a high immunogenic potential, although treatment-induced antibodies were produced in the sera of 10 patients, and there was no correlation between serum antibody response and AEs.

[0475] After IT administration, the concentration of rhASA in serum increased slowly (mean t max in the range of 5.1 - 18.2 hours), and the observed maximum serum concentration (C max ) and the area under the serum concentration-time curve increased with increasing dose. In CSF, the rhASA concentration generally increased in a dose-dependent manner, and measurable concentrations were detected at each pre-dose visit, indicating that rhASA was present in CSF throughout the 2-week period between doses. The mean CSF concentrations of rhASA by dose cohort and treatment week are presented in Figure 14 The pharmacokinetic results were similar for patients receiving 100 mg of rhASA manufactured using Process A or Process B.

[0476] After rhASA treatment, a decrease in the mean GMFM-88 total score relative to baseline was observed in each of the four groups ( Figure 15 ). The mean (SD) decrease in the GMFM-88 total score at week 40 was similar in the two 100 mg groups (Groups 3 and 4) compared to baseline (–19.0 [16.12] and –22.6 [25.48], respectively). However, these decreases were not as pronounced as those observed in the 10 mg and 30 mg groups (Groups 1 and 2; –29.2 [24.45] and –27.7 [17.76], respectively). The changes in the GMFM-88 total score over time relative to baseline are summarized in Table 8. A decline in gross motor function was observed in all patients, indicating a trend towards a smaller decline with increasing rhASA dose levels. The MLD severity score remained stable in patients receiving 100 mg rhASA (Group 3; Procedure A).

[0477] Table 8. Changes in GMFM-88 total score relative to baseline

[0478]

[0479] CSF sulfatide levels were assessed as an indicator of drug activity in the CNS. As Figure 16A shown, at the end of the study, the mean CSF sulfatide levels were below the upper limit of normal in Groups 3 and 4 (0.07 [0.03] and 0.08 [0.03], respectively). A decrease in the mean CSF sulfatide levels over time was also observed in all groups ( Figure 16B ). Starting from week 2 in Group 3, week 4 in Group 4, and week 12 in Group 2, the mean CSF sulfatide levels were consistently below the upper limit of normal. This data indicates that rhASA is active in the CNS.

[0480] NAA can be used as a surrogate measure of the number and / or density of mature neurons and axons, and in neurological disorders, a decrease in NAA concentration is associated with damage and loss of neurons and axons. Changes in the NAA / creatine ratio were evaluated as an indicator of the severity of MLD in patients ( Figure 17A ). In the right frontal white matter, the NAA / creatine ratio decreased from baseline to the end of the study, but this was not as pronounced in patients treated with 30 mg or 100 mg rhASA (Groups 2–4) as in those treated with 10 mg rhASA (Group 1) ( Figure 17A ). Similar results were observed in the right fronto-parietal white matter and the right parieto-occipital white matter ( Figure 18A and Figure 19A ). The NAA / creatine ratio in gray matter and rhASA dose are shown in Figure 20A .

[0481] The ratio of choline / creatine was also evaluated in all groups. It has been shown that choline levels are elevated in patients with MLD, possibly due to increased membrane turnover associated with demyelination. In the right frontal white matter, right fronto-parietal white matter, and right parieto-occipital white matter, the choline / creatine ratio consistently increased in patients treated with the lowest dose (10 mg; group 1) compared to baseline, but there was no clear trend in patients treated with 30 mg or 100 mg (groups 2 - 4; Figure 17B , Figure 18B and Figure 19B ). In the middle occipital gray matter, the choline / creatine ratio was variable in groups 1 and 2, but decreased compared to baseline in group 3 and remained stable in group 4 ( Figure 20B ).

[0482] Over time, the mean total MLD severity score increased in patients treated with 10 mg, 30 mg, and 100 mg (B) rhASA (groups 1, 2, and 4), indicating disease progression ( Figure 21 ). The greatest increase relative to baseline was in patients receiving 10 mg rhASA (group 1; mean change of 11.0). In contrast, in patients treated with 100 mg (A) rhASA (group 3), the mean total MLD severity score remained stable, which may indicate disease stability in the CNS ( Figure 21 ).

[0483] In all groups, a significant deterioration in the nerve conduction ability of the median motor abductor pollicis was observed at baseline (Table 9). Similar results were also found in sensory nerves (data not shown). At subsequent time points, the nerve function of the four groups did not appear to deteriorate.

[0484] Table 9. Z-scores of the median motor abductor pollicis obtained by electroneurography visits.

[0485]

[0486]

[0487]

[0488] Multiple studies have also reported changes in metabolite levels in the CNS of patients with neurological disorders, including MLD. For example, reduced NAA concentration is associated with damage and loss of neurons and axons, while elevated choline levels have been shown in patients with MLD. As expected, we found that the ratio of NAA / creatine in white matter generally decreased over time. However, this was less pronounced for the 100 mg dose than for the 10 mg or 30 mg doses, which may indicate less neuronal damage at the highest dose. Additionally, in patients treated with the lowest dose (10 mg; group 1), the ratio of choline / creatine in white matter consistently increased compared to baseline, but there was greater variability in the results in patients treated with higher doses. In our study, we did not observe any clear trends in the levels of other metabolites, including lactate, inositol, and glutamine + glutamate. Analysis of metabolite levels may need to be performed in more patients and different brain regions to clearly determine the impact of rhASA treatment.

[0489] Overall, these findings suggest that IT rhASA treatment may be associated with disease stabilization in the CNS, particularly at the 100 mg EOW dose. However, the small sample size means that our results should be interpreted with caution. In particular, the number of patients for whom data were available at each time point varied with some parameters, and further studies are needed over a longer time period and in more patients to determine the long-term efficacy of rhASA in patients with MLD. Additionally, there was some difference in the age of inclusion between patients and groups, which may have an impact on comparing treatment responses between different dose groups. Similarly, metabolite level data obtained by MRS were not standardized to account for patient age.

[0490] Despite these limitations, our findings suggest that children with MLD generally tolerate IT rhASA well. Although motor function generally declines over time, there is evidence that disease progression is reduced in patients receiving rhASA, particularly at the highest dose. Extension studies are ongoing to evaluate the long-term safety, immunogenicity, and clinical outcomes associated with rhASA treatment, and subsequent clinical phase studies with higher doses of rhASA will soon start recruiting patients. It is hoped that the results of these studies will help to guide dosing and determine whether rhASA can be considered a potential treatment for patients with MLD in the future.

[0491] Example 5: Modeling and Simulation of Recombinant Human Arylsulfatase A in Patients with MLD

[0492] Data from the clinical study described in Example 4 were used for pharmacokinetic (PK) and pharmacodynamic (PD) modeling and simulation of recombinant human arylsulfatase A in patients with MLD. As Figure 5As described, for PK analysis, all patients received at least one dose of rhASA, and the concentration of rhASA in serum or CSF was measured at least once. For PK / PD analysis: Estimates of PK parameters for all patients were obtained from the PK analysis, and the sulfatide in CSF or the total GMFM-88 score was measured at least once. A population PK model based on the serum and CSF concentrations of rhASA was developed, as Figure 6 shown. The elimination of rhASA was represented by a two-compartment model in the central nervous system (CNS), which had distribution volumes VCNS and VCSF and an inter-compartment clearance QCSF. A hypothetical transport compartment was used to characterize the clearance of rhASA from the CSF and its delayed appearance in serum.

[0493] The PK / PD relationship was modeled, including the effect of rhASA on the sulfatide concentration measured in the CSF and the exposure-response (ER) relationship between rhASA and the total GMFM-88 score. The PK drivers of the PK / PD relationship were the patient-specific time-course PK profiles in the CSF and CNS. The PD effects were modeled sequentially, and all PK-related parameters were fixed during the estimation of PD parameters. An indirect response PK / PD model was used to characterize the time course of CSF sulfatide measurements, in which rhASA was modeled as enhancing the elimination of sulfatide. The concentration of rhASA in the CNS compartment predicted by the population PK model was used as the driver of the PD response. The E-R relationship between rhASA exposure and the total GMFM-88 score was evaluated using a beta regression model constrained by a logit link function to limit the model predictions to the range of 0 - 100%.

[0494] Population PK, PK / PD, and E-R models were used to simulate the expected rhASA concentration-time curves and total GMFM-88 scores. The profiles of 750 patients were simulated; 250 were randomly sampled evenly from each age group. All data combinations, plots, and summary tables were prepared using R (v3.3 or higher; Lucent Technologies, Swindon, UK; http: / / www.rproject.org). Population PK, PK / PD, and E-R modeling analyses and simulations were performed using NONMEM software (v7.3 or higher; ICON Development Solutions, Ellicott City, MD, USA).

[0495] As Figure 8As shown, four different dosing regimens were used. Regimen 1 included 100 mg every other week; Regimen 2 included 100 mg weekly for 12 weeks (initially once weekly), then 100 mg every other week; Regimen 3 included 150 mg weekly for 12 weeks (initially once weekly), then 150 mg every other week; and Regimen 4 included age-adjusted dosing weekly for 12 weeks (initially once weekly), then once every other week (Regimen 4: 80 mg, <8 months; 120 mg, 8–<30 months; 150 mg, ≥30 months). All data combinations, plots, and summary tables were prepared using R (v3.3 or higher; Lucent Technologies, Swindon, UK; http: / / www.rproject.org). Profiles of 750 patients were simulated; 250 were randomly sampled evenly from each age group. Population PK, PK / PD, and E-R modeling analyses and simulations were performed using NONMEM software (v7.3 or higher; ICON Development Solutions, Ellicott City, MD, USA).

[0496] Patients (n = 24) were evenly distributed among four groups. At baseline, the mean patient age was 44.9 (standard deviation [SD], 22.8; range, 19.0 - 107.0) months, and the mean weight was 15.4 (SD, 4.14; range, 10.5 - 24.8) kg.

[0497] Fixed effects were estimated with higher precision except for the proportionality coefficients of VCSF (76.1% RSE), VCNS (121% RSE), and Ktrans (40.2% RSE). The precision of the estimated inter-individual variability was poor. The model showed that rhASA rapidly distributed into the brain tissue and systemic circulation: the median distribution half-life of rhASA in the CNS was 1.02 (range 0.394 - 1.66) hours, and the median distribution half-life from CSF to serum was 1.19 (range 0.555–2.09) hours. However, the median terminal half-life of rhASA in the CNS was approximately 20 days (477 [range 256 - 1010] hours) (Table 10). The median transport rate constant (expressed as transport half-life) was 1.19 (range 0.555 - 2.09) hours, consistent with CSF physiological turnover (approximately 6 hours).

[0498] Table 10. Summary statistics of the estimated values of each PK parameter

[0499]

[0500] CNS, central nervous system; CSF, cerebrospinal fluid; PK, pharmacokinetics; 0 CSF; Intercompartmental clearance in the CNS; rhASA, recombinant human arylsulfatase A; SD, standard deviation

[0501] A concentration-dependent decrease in sulfatide concentration in CSF was observed (model-estimated EC50: 184 ng / mL rhASA, with a steep Hill slope of 3.59), and the concentration-dependence of gross motor function loss was inhibited (model-estimated IC50: 120 ng / mL rhASA, in brain tissue, with a shallow Hill slope of 0.554). Goodness-of-fit plots for predicted rhASA concentration in CSF, sulfatide concentration in CSF, and total GMFM-88 scores for the population and individuals are shown in Figure 7A 、 Figure 7B and Figure 7C respectively. All three models showed a reasonable characterization of the observed rhASA concentration; the precision of the models varied, likely due to the small number of patients included in the study. For the four simulated dosing regimens, median pre-dose trough rhASA concentrations in CSF and CNS are shown in Figure 8 .

[0502] In the case of dosing 100 mg every other week (Regimen 1), it took approximately 16 weeks to reach PK steady state, which was consistent with a median rhASA elimination half-life of 477 hours. In the case of dosing 100 mg or 150 mg weekly for 12 weeks and then every other week (Regimens 2 and 3), higher rhASA trough concentrations were achieved in CSF and CNS compared to Regimen 1. The age-based dosing regimen (Regimen 4) showed more consistent rhASA steady-state trough concentrations over time for the three age groups. However, patients less than 8 months old did not reach the same high rhASA trough concentrations as in Regimens 2 and 3 during the weekly dosing phase.

[0503] Simulation of total GMFM-88 scores showed some patients stabilizing over time as expected ( Figure 9 ), which was consistent with the observed clinical study results. Regimens 1 - 3 predicted that some patients in all groups would show an early response to treatment. Regimen 3 predicted the largest number of patients with total GMFM-88 scores higher than 35 or 50 at 12 months and 24 months, respectively. The age-based dosing regimen (Regimen 4) predicted that fewer patients in the <8 months and 8–<30 months groups would respond to treatment compared to those in the >30 months group.

[0504] Delivery of rhASA via IDDD reduces sulfatide levels in the CSF of patients with MLD and slows the rate of motor function loss in a dose- and exposure-dependent manner. Pharmacometric modeling approaches were used to predict population PK, PK / PD, and E–R relationships with the GMFM-88 total score. In all cases, the models provided good characterization of the measurements observed in patients receiving rhASA. Given the small number of patients, the precision between models varied, with variability in rhASA concentrations observed in the CSF and CSF concentration variability only observed at pre-dose time points. However, the models indicated rapid distribution of rhASA into the brain tissue and systemic circulation, but a slower terminal elimination half-life in the CNS.

[0505] Example 6: Analysis of responders and non-responders to intrathecal delivery of recombinant human arylsulfatase A in children with late infantile metachromatic leukodystrophy.

[0506] As Figure 10 shown, during weeks 0 to 38, patients received rhASA every other week via an intrathecal drug delivery device (IDDD). Group 1 (n = 6) received 10 mg rhASA, group 2 (n = 6) received 30 mg rhASA, group 3 (n = 6) received 100 mg rhASA, and group 4 (n = 6) received 100 mg rhASA manufactured using an improved process.

[0507] Eligibility criteria for groups 1 - 4 included confirmed MLD (evaluated based on ASA deficiency by elevated sulfatide concentrations in leukocytes and urine); first manifestation of MLD symptoms at 30 months of age or earlier; presence of neurological symptoms of MLD at screening examination; able to walk at screening (able to walk 10 steps forward with support of one hand). Patients included in groups 1 - 3 at screening were also less than 12 years old. Additional eligibility criteria for group 4 patients included a Gross Motor Function Measure-88 (GMFM-88) total score of at least 40 and at least 35 at baseline, and less than 8 years old at screening.

[0508] Prior to receiving the first dose of rhASA, each patient had an IDDD surgically implanted ( II [Smiths Medical ASD, Inc., St Paul, MN, USA] or Mini S [Sophysa, Orsay, France]). The position of the IDDD was confirmed after surgery. The treatment response of patients in groups 3 and 4 was retrospectively evaluated using the GMFM-88 total score (0–100%) (which provides an indication of motor function) (Bjornson KF et al., Pediatr Phys Ther 1998;10:43–7).

[0509] Responders were defined as individuals with a baseline GMFM-88 total score ≥ 35 and whose GMFM-88 total score at the end of the study remained unchanged (GMFM-88 total score decrease ≤ 10) or increased relative to baseline. Non-responders were defined as individuals who did not meet this criterion. Other key outcomes evaluated included changes in sulfatide concentration in cerebrospinal fluid (CSF), changes in the severity of MLD (measured by brain magnetic resonance imaging (MRI) using a scoring method similar to that of Eichler et al. (Giugliani R et al., JIEMS 2017; 5:314:A699) to obtain an MLD-MRI severity score), and changes in N-acetylaspartate (NAA) levels in white matter of the brain (evaluated by magnetic resonance spectroscopy (MRS)). Figure 22 Shown are the GMFM-88 total scores by age in months of individual sibling patients from the rhASA clinical study. Biomarkers and motor response appear to improve together. Additionally, CSF sulfatide and MRS NAA levels appear to improve together. Motor response was associated with a dose-dependent decrease in sulfatide accumulated in the CSF.

[0510] Of the 12 patients who were enrolled in NCT01510028 and received 100 mg rhASA, four were identified as responders: two from cohort 3 and two from cohort 4. The remaining eight patients in cohorts 3 and 4 were identified as non-responders. Figure 11 Shown are the GMFM-88 total scores of the 12 patients over time. The demographics and baseline characteristics of the four responders are shown in Table 11.

[0511] Table 11. Demographics and baseline characteristics of individuals identified as responders

[0512]

[0513]

[0514] CSF, cerebrospinal fluid; GMFM-88, Gross Motor Function Measure-88; MLD, metachromatic leukodystrophy; MRS, magnetic resonance spectroscopy; NAA, N-acetylaspartate

[0515] One month later, the CSF sulfatide concentrations of responders 1, 2, and 3 were below the upper limit of normal (0.113 μg / mL), and there was no follow-up information on the CSF sulfatide concentration of responder 4. At the end of the study (week 40), the CSF sulfatide concentrations of responders 1, 2, and 3 were reported as 0.0294 μg / mL, 0.0489 μg / mL, and 0.0653 μg / mL, respectively.

[0516] One month later, the CSF sulfatide concentration of one patient among the eight non-responders was below the upper limit of normal. In the remaining patients, after 8–36 weeks, the CSF sulfatide concentration dropped below the upper limit of normal. One patient in Group 1 also met the post hoc responder criteria, while the CSF sulfatide level did not reach the normal level at the end of the study.

[0517] By week 40, the MLD-MRI severity score relative to baseline decreased in two responders and increased in one responder ( Figure 12 ). Information on the MLD-MRI severity score of responder 4 at the end of the study was not available. Two of the four responders had an NAA / creatine ratio ≥1.0 in the fronto-parietal white matter at baseline. None of the eight non-responders with available baseline data had an NAA / creatine ratio ≥1.0 at baseline.

[0518] The NAA / creatine ratio in the fronto-parietal white matter of one responder increased compared to baseline at week 40, while in the other two responders for whom data were available at baseline and week 40, the ratio remained stable ( Figure 13 ).

[0519] Four of the twelve patients receiving 100 mg rhASA were identified as responders to treatment; the responders differed in age and baseline characteristics. These findings suggest a trend towards disease stabilization in the subgroup of patients receiving the highest dose and provide support for intrathecal treatment of LI-MLD patients with rhASA. Factors influencing treatment response may include CSF rhASA concentration, baseline NAA / creatine ratio, stabilization of the NAA / creatine ratio, disease duration, age, and the time required for normalization of the CSF sulfatide level.

[0520] Example 7: Intrathecal administration of recombinant human arylsulfatase A in patients with late infantile metachromatic leukodystrophy

[0521] The lack of the lysosomal enzyme arylsulfatase A in MLD results in the accumulation of sulfated glycosphingolipids (collectively called sulfatides). These substances accumulate in the cells that maintain the central and peripheral myelin insulating sheaths of axons and are toxic to these cells. Intrathecal (IT) administration of recombinant human arylsulfatase A may be sufficient to hydrolyze the sulfatides that accumulate in the cells of the nervous system and may slow or prevent further accumulation, which would translate into motor system benefits. Direct CNS administration, such as by IT delivery, can be used to effectively treat MLD patients. This example illustrates a multicenter open-label matched historical control clinical study that was designed to evaluate the efficacy of weekly intrathecal administration of 150 mg rhASA in patients with late infantile MLD. The study will investigate the potential of 150 mg IT-administered rhASA to stabilize or slow the progression of motor dysfunction in pediatric subjects with late infantile MLD.

[0522] Approximately 35 patients will be recruited to evaluate the safety and tolerability of rhASA and its delivery via the SOPH-A-PORT Mini S intrathecal drug delivery device. The rate and severity of disease progression are well documented in late infantile MLD. A notable feature of the definition of late infantile MLD is the early age of onset of disease symptoms, with most late infantile MLD patients first showing motor dysfunction before 18 months of age. Based on the age at enrollment and the degree of motor dysfunction, four treatment groups will be included:

[0523] ● Group A - Early symptoms (18 - 48 months; Gross Motor Function Classification for MLD [GMFC-MLD] level 1 - 2; n = 16),

[0524] ● Group B - Intermediate symptoms (18 - 72 months; GMFC-MLD level 3; n ≤ 8),

[0525] ● Group C - Late symptoms (18 - 72 months; GMFC-MLD level 4; n ≤ 8), and

[0526] ● Group D - Presymptomatic or mildest symptoms (<18 months; participants have a presymptomatic sibling with the same ASA allele constitution; n = 3).

[0527] In Groups A - C, the onset of symptoms must be before 30 months. All subjects will receive a weekly dose of 150 mg of IT rhASA for 105 weeks. The study will use the GMFC-MLD and GMFM-88 total scores to measure disease progression and thus evaluate the safety and efficacy of the treatment regimen on gross motor function. The Alberta Infant Motor Scale (Piper and Darrah, 1994) will be used to evaluate subjects in Group D. The primary endpoint will be the proportion of children in Group A whose GMFC-MLD level has increased by no more than 2 (indicating a decline in motor function) relative to baseline by 2 years. The secondary endpoint will be the proportion of children in Group A with a GMFM-88 total score > 40 after 2 years.

[0528] Other secondary endpoints will include the effect of IT-administered rhASA on the time course of decline in gross motor function using the GMFC-MLD; the effect of IT-administered rhASA on the time course of decline in gross motor function using the GMFM-88; the effect of IT-administered rhASA on expressive language using the Expressive Language Function Classification (ELFC-MLD); the effect of IT-administered rhASA on cerebrospinal fluid (CSF) biomarkers (i.e., sulfatide levels); and the effect of IT-administered rhASA on brain proton magnetic resonance spectroscopy (MRS), specifically N-acetylaspartate / creatine (NAA / Cr) in white matter. Results will be compared to historical control data matched by propensity score in MLD children.

[0529] The safety and tolerability of IT rhASA will be based on the occurrence of treatment-emergent adverse events (TEAEs), clinical laboratory tests (serum chemistry, hematology, and urinalysis), vital signs, physical examinations (including documentation of MLD signs and symptoms), 12-lead electrocardiograms (ECGs), CSF laboratory parameters (chemistry and cell count), the presence and activity of anti-rhASA antibodies in CSF and serum, and in MLD subjects Mini S device.

[0530] Other evaluations of the effects of IT-administered rhASA may be based on serum and urine biomarkers (i.e., sulfatide levels), severity scores measured by brain magnetic resonance imaging (MRI), volumetric analysis based on brain MRI, swallowing ability evaluated by fiberoptic endoscopic evaluation of swallowing (FEES), nerve conduction evaluated by electroneurography (ENG), and communication and cognition evaluated using the Battelle Developmental Inventory-2 New Update (BDI-2NU). The impact of caregiver burden and the subject's health-related quality of life (HRQOL) in MLD children will be explored by evaluating caregiver burden (assessed by the Caregiver Impact Questionnaire (CIQ)) and HRQOL (assessed by the Infant Toddler Quality of Life Questionnaire-97 items (ITQOL-97)).

[0531] The primary efficacy endpoint was defined as the response of Group A to maintain gross motor function at 2 years (Week 106), evaluated as a change in GMFC-MLD of no more than 2 levels relative to baseline. The key secondary efficacy endpoint in Group A was to maintain gross motor function at 2 years (Week 106), evaluated as a GMFM-88 total score > 40. The efficacy of rhASA will be evaluated by comparing the enrolled subjects in Group A with matched historical control subjects. Data for these untreated MLD subjects (i.e., subjects who have not received any study product or therapy) will be from the ongoing Global Leukodystrophy Initiative - MLD (GLIA-MLD) natural history study. Other matched historical control data from MLD subjects exposed to rhASA in previous studies may also be used. Matched historical controls from GLIA-MLD and previous Shire studies must have at least baseline data and 1 post-baseline gross motor function assessment data. The inclusion / exclusion criteria for these controls are very similar to those of the enrolled subjects in this study, and comparable efficacy assessments are performed at similar time points.

[0532] Although several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision numerous other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications of the teachings of this invention. Those skilled in the art will recognize, or be able to determine using only routine experimentation, many equivalents to the specific inventive embodiments described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that the embodiments of this invention may be practiced otherwise than as specifically described and claimed within the scope of the appended claims and their equivalents. The inventive embodiments of this disclosure relate to each and every separate feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention of this disclosure if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

Claims

1. A method for treating metachromatic leukodystrophy (MLD), the method comprising intrathecal administration to a subject in need thereof of a therapeutically effective dose of a preparation comprising purified recombinant human arylsulfatase A (rhASA) protein, wherein the purified rhASA protein is characterized by a proteoglycan profile that comprises one or more peaks corresponding to a neutral recombinant ASA protein (neutral ASA protein), a sialylated recombinant ASA protein (sialic acid ASA protein), a mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), an N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and a mixed recombinant ASA protein (mixed ASA protein), and wherein administration of the preparation results in stabilization or reduction of the progression of at least one symptom of MLD.

2. The method according to claim 1, wherein the therapeutically effective dose is 100 mg.

3. The method according to claim 1, wherein the therapeutically effective dose is 150 mg.

4. The method according to claim 1, wherein the preparation is administered once a week.

5. The method according to claim 1, wherein the subject has late infantile MLD.

6. The method according to any one of the preceding claims, wherein stabilization or reduction of the progression of MLD is measured using changes in the Gross Motor Function Classification (GMFC-MLD).

7. The method according to any one of the preceding claims, wherein administration of the preparation results in a change in GMFC-MLD level relative to baseline of ≤4, ≤3, or ≤2 levels after 2 years of treatment in the subject.

8. The method according to any one of the preceding claims, wherein administration of the preparation results in maintenance of the GMFC-MLD score after two years of treatment.

9. The method according to claim 7 or 8, wherein the maintenance of the GMFC-MLD score is a change in GMFC-MLD relative to baseline of no more than 2 levels after two years of treatment.

10. The method according to any one of claims 7-9, wherein the baseline is the evaluated score of GMFC-MLD before the first administration of the preparation.

11. The method according to any one of the preceding claims, wherein stabilization or reduction of the progression of MLD is measured using changes in the Gross Motor Function Measure (GMFM-MLD).

12. The method according to claim 11, wherein administration of the preparation results in maintenance of the GMFM-MLD score after two years of treatment.

13. The method according to claim 11 or 12, wherein the GMFM-MLD score is maintained at >40.

14. The method according to any one of the preceding claims, wherein stabilization or reduction of the progression of MLD is measured by the level of sulfatide in cerebrospinal fluid.

15. The method according to any one of the preceding claims, wherein stabilization or reduction of the progression of MLD is measured by the brain N-acetylaspartate / creatine ratio (NAA / cr).

16. The method according to any one of the preceding claims, wherein the stabilization or reduction of the progression of MLD is measured using Expressive Language Function Classification - MLD Level (ELFC-MLD).

17. The method according to any one of claims 5-16, wherein the baseline GMFC-MLD level of the subject is 1-2.

18. The method according to any one of claims 5-16, wherein the baseline GMFC-MLD level of the subject is 3.

19. The method according to any one of claims 5-16, wherein the baseline GMFC-MLD level of the subject is 4.

20. The method according to any one of claims 5-16, wherein the subject is pre-symptomatic.

21. The method according to claim 20, wherein the subject is less than 18 months of age.

22. The method according to claim 21, wherein the subject is evaluated using the Alberta Infant Motor Scale.

23. The method according to any one of the preceding claims, wherein the rhASA protein has an amino acid sequence that is at least 70% identical to SEQ ID NO:

1.

24. A method of treating late infantile MLD, the method comprising: administering to a subject a preparation comprising a purified recombinant human arylsulfatase A (rhASA) protein having an amino acid sequence that is at least 70% identical to SEQ ID NO:1, wherein the purified rhASA protein is characterized by a proteoglycan profile comprising one or more peaks corresponding to neutral recombinant ASA protein (neutral ASA protein), sialylated recombinant ASA protein (sialic ASA protein), mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and mixed recombinant ASA protein (mixed ASA protein), and wherein the preparation is administered intrathecally at a dose of 150 mg at intervals of once a week.

25. The method according to any one of the preceding claims, wherein the preparation contains less than about 150 ng / mg of host cell protein (HCP).

26. The method according to any one of the preceding claims, wherein at least about 23% of the purified recombinant ASA protein, which accounts for the total purified recombinant ASA protein, corresponds to mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein).

27. The method according to any one of the preceding claims, wherein the proteoglycan profile of the purified recombinant ASA protein comprises: about 15% to about 25% of neutral recombinant ASA protein (neutral ASA protein), about 35% to about 45% of sialylated recombinant ASA protein (sialic ASA protein), about 23% to about 33% of mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), about 1% to about 10% of N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and About 5% to about 15% of a mixed recombinant ASA protein (mixed ASA protein).

28. The method according to claim 27, wherein the proteoglycan profile of the purified recombinant ASA protein comprises: About 18% to about 22% of a neutral ASA protein, About 37% to about 41% of a sialic acid ASA protein, About 26% to about 29% of an M6P ASA protein, About 4% to about 6% of a capped M6P ASA protein, and About 7% to about 9% of a mixed ASA protein.

29. A composition comprising a purified recombinant human arylsulfatase A (rhASA) protein having an amino acid sequence that is at least 70% identical to SEQ ID NO:1, wherein the purified rhASA protein is characterized by one or more proteoglycan species selected from: neutral recombinant ASA protein (neutral ASA protein), sialylated recombinant ASA protein (sialic acid ASA protein), mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), or mixed recombinant ASA protein (mixed ASA protein).

30. The composition according to claim 29, wherein at least about 23% of the total purified recombinant ASA protein corresponds to mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein).

31. A composition comprising a purified recombinant arylsulfatase A (rhASA) protein having an amino acid sequence that is at least 70% identical to SEQ ID NO:1, wherein at least about 23% of the total purified recombinant ASA protein corresponds to mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein) characterized by a proteoglycan profile; and the composition contains less than about 150 ng / mg of host cell protein (HCP).

32. The composition according to claim 30 or 31, wherein the M6P ASA protein is present in an amount of at least about 26% of the total purified rhASA protein content.

33. The composition according to claim 30 or 31, wherein the M6P ASA protein is present in an amount of at least about 28% of the total purified rhASA protein content.

34. The composition according to any one of the preceding claims, wherein the M6P ASA protein is present in an amount of about 20% to about 33% of the total purified rhASA protein content.

35. The composition according to any one of claims 29-34, wherein the total purified rhASA protein comprises neutral recombinant ASA protein (neutral ASA protein), sialylated recombinant ASA protein (sialic acid ASA protein), N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), or mixed recombinant ASA protein (mixed ASA protein) or any combination thereof.

36. The composition according to claim 30 or 31, wherein the total purified rhASA protein comprises: From about 23% to about 33% of the mannose-6-phosphorylated recombinant ASA protein (M6P ASA protein), from about 15% to about 25% of the neutral recombinant ASA protein (neutral ASA protein), from about 35% to about 45% of the sialylated recombinant ASA protein (sialic acid ASA protein), from about 1% to about 10% of the N-acetylglucosamine mannose-6-phosphorylated recombinant ASA protein (capped M6P ASA protein), and from about 5% to about 15% of the mixed recombinant ASA protein (mixed ASA protein).

37. The composition according to any one of claims 29-36, wherein the neutral ASA protein is present in an amount of about 16% to about 22% of the total purified rhASA protein.

38. The composition according to any one of claims 29-37, wherein the neutral ASA protein is present in an amount of about 20% to about 25% of the total purified rhASA protein.

39. The composition according to any one of claims 29-38, wherein the sialic acid ASA protein is present in an amount of about 35% to about 40% of the total purified rhASA protein.

40. The composition according to any one of claims 29-39, wherein the sialic acid ASA protein is present in an amount of about 37% to about 42% of the total purified rhASA protein.

41. The composition according to any one of claims 29-40, wherein the capped M6P ASA protein is present in an amount of about 3% to about 5% of the total purified rhASA protein.

42. The composition according to any one of claims 29-41, wherein the capped M6P ASA protein is present in an amount of about 4% to about 6% of the total purified rhASA protein.

43. The composition according to any one of claims 29-42, wherein the capped M6P ASA protein is present in an amount of about 4.5% to about 5.5% of the total purified rhASA protein.

44. The composition according to any one of claims 29-43, wherein the mixed ASA protein is present in an amount of about 7% to about 10% of the total purified rhASA protein.

45. The composition according to any one of claims 29-44, wherein the mixed ASA protein is present in an amount of about 7.5% to about 8.5% of the total purified rhASA protein.

46. The composition according to claim 30 or 31, wherein the total purified rhASA protein comprises: from about 16% to about 23% of the neutral ASA protein, from about 37% to about 42% of the sialic acid ASA protein, from about 23% to about 27% of the M6P ASA protein, from about 4% to about 8% of the capped M6P ASA protein, and from about 7% to about 10% of the mixed ASA protein.

47. The composition according to claim 30 or 31, wherein the total purified rhASA protein comprises: from about 20% to about 23% of the neutral ASA protein, from about 35% to about 39% of the sialic acid ASA protein, from about 26% to about 32% of the M6P ASA protein, From about 3% to about 5% of the capped M6P ASA protein, and from about 7% to about 9% of the mixed ASA protein.

48. The composition according to claim 30 or 31, wherein the total purified rhASA protein comprises: From about 18% to about 22% of the neutral ASA protein, from about 37% to about 41% of the sialic acid ASA protein, from about 26% to about 29% of the M6P ASA protein, from about 4% to about 6% of the capped M6P ASA protein, and from about 7% to about 9% of the mixed ASA protein.

49. The composition according to any one of claims 29-48, wherein the total purified rhASA protein is present at a concentration of about 20 mg / mL to about 45 mg / mL.

50. The composition according to claim 49, wherein the total purified rhASA protein is present at a concentration of about 25 mg / mL to about 34 mg / mL or about 28 mg / mL to about 32 mg / mL.

51. The composition according to claim 49, wherein the total purified rhASA protein is present at a concentration of about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL or about 34 mg / mL.

52. The composition according to any one of claims 29-51, wherein the purified rhASA protein has a specific activity of about 50 to about 130 U / mL.

53. The composition according to claim 52, wherein the purified rhASA protein has a specific activity of about 70 to about 100 U / mg.

54. The composition according to claim 52, wherein the purified rhASA protein has a specific activity of about 80 to about 90 U / mg.

55. The composition according to claim 52, wherein the purified rhASA protein has a specific activity of about 75 to about 95 U / mg.

56. The composition according to any one of claims 29-55, wherein the purified rhASA protein contains less than about 140 ng / mg of host cell protein (HCP).

57. The composition according to claim 56, wherein the purified rhASA protein contains less than about 100 ng / mg of HCP.

58. The composition according to claim 56, wherein the purified rhASA protein contains less than about 80 ng / mg of HCP.

59. The composition according to claim 56, wherein the purified rhASA protein contains less than about 60 ng / mg of HCP.

60. The composition according to any one of claims 29-59, wherein the purified rhASA protein contains less than about 100 pg / mg of host cell DNA (HCD).

61. The composition according to claim 60, wherein the purified rhASA protein contains less than about 50 pg / mg of HCD.

62. The composition according to claim 60, wherein the purified rhASA protein contains less than about 10 pg / mg of HCD.

63. The composition according to claim 60, wherein the purified rhASA protein contains less than about 5 pg / mg of HCD, less than about 4 pg / mg of HCD, less than about 3 pg / mg of HCD, less than about 2 pg / mg of HCD, or less than about 1 pg / mg of HCD.

64. The composition according to any one of claims 29-63, wherein the purified rhASA protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:

1.

65. The composition according to claim 64, wherein the purified rhASA protein has an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

66. The composition according to claim 64, wherein the purified rhASA protein has an amino acid sequence that is at least 95% identical to SEQ ID NO:

1.

67. The composition according to claim 64, wherein the purified rhASA protein has the same amino acid sequence as SEQ ID NO:

1.

68. The composition according to any one of claims 29-67, the composition comprising from about 0.001% to about 0.01% of polysorbate-20 (P20).

69. A formulation comprising the composition according to any one of claims 29-68 and a physiologically acceptable carrier.

70. The formulation according to claim 69, wherein the formulation is suitable for intravenous administration.

71. The formulation according to claim 69, wherein the formulation is suitable for intrathecal administration.

72. The formulation according to claim 69, wherein the formulation is suitable for subcutaneous administration.

73. A method for purifying a recombinant arylsulfatase A (ASA) protein, the method comprising: purifying a recombinant arylsulfatase A (ASA) protein from an impure preparation by performing one or more chromatographic steps; combining the eluates from the one or more chromatographic steps; optionally adjusting the pH of the combined eluate to a pH of about 6.0 to about 8.0; optionally subjecting the combined eluate or the pH-adjusted eluate to ultrafiltration and / or diafiltration; obtaining an eluate comprising the purified recombinant arylsulfatase A (ASA) protein; and adding a surfactant to the eluate comprising the purified recombinant arylsulfatase A (ASA) protein.

74. The method according to claim 73, comprising the step of adjusting the pH of the combined eluate to a pH of about 6.0 to about 8.

0.

75. The method according to claim 74, comprising the step of subjecting the pH-adjusted eluate to ultrafiltration and / or diafiltration.

76. The method according to any one of claims 73-75, wherein the addition of the surfactant is performed before refrigerating the eluate comprising the purified recombinant ASA protein.

77. The method according to any one of claims 73 - 75, wherein the surfactant is present at a concentration of from about 0.0001% (v / v) to about 0.01% (v / v).

78. The method according to claim 77, wherein the surfactant is present at a concentration of from about 0.001% (v / v) to about 0.01% (v / v).

79. The method according to any one of claims 73 - 78, wherein the surfactant is polysorbate - 20 (P20).

80. The method according to any one of claims 73 - 79, wherein the step of performing one or more chromatographic steps comprises sequentially performing anion - exchange chromatography, mixed - mode chromatography, hydrophobic interaction chromatography i.e. phenyl chromatography, and cation - exchange chromatography.

81. The method according to claim 80, wherein the anion - exchange chromatography uses a column with a TMAE resin.

Images