Method for purifying aryl sulfatase A

Abstract

The present invention provides, inter alia, improved methods for purifying arylsulfatase A (ASA) proteins that are recombinantly produced for enzyme replacement therapy. The present invention is based, in part, on the surprising discovery that recombinant ASA proteins can be purified from untreated biological materials (e.g., ASA-containing cell culture media) using processes that include only four chromatographic columns and only one step of post-chromatographic column ultrafiltration / diafiltration.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of Chinese invention patent application No. 201480003307.5, filed on January 9, 2014, and entitled “Methods for Purifying Arylsulfatase A.” The original application is a national phase application with International Application No. PCT / US2014 / 010856, which claims priority to U.S. Provisional Patent Application No. 61 / 750,693, filed on January 9, 2013. The aforementioned application is hereby incorporated by reference in its entirety. background

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

[0004] The most characteristic clinical feature of the disorder is central nervous system (CNS) degeneration, which leads to cognitive impairment (such as mental retardation, confusion, and blindness, among others).

[0005] MLD can manifest itself in young children (late infantile form), where infected children typically begin to show symptoms just after their first birthday (e.g., around 15-24 months), and they typically do not survive past the age of 5. MLD can manifest itself in children (juvenile form), where infected children approximately 3-10 years of age typically show cognitive impairment, with a variable lifespan (e.g., ranging from 10-15 years after symptom onset). MLD can manifest itself in adults (adult-onset form), and can occur in individuals of any age (e.g., typically 16 years of age and older), and the progression of the disease can vary widely.

[0006] Enzyme replacement therapy (ERT) is an improved therapy for treating MLD that involves administering exogenous replacement ASA enzyme, particularly recombinant arylsulfatase A (rASA) (eg, recombinant human arylsulfatase A (rhASA)), to patients with MLD. Overview

[0007] The present invention, among other things, provides improved methods for purifying recombinantly produced ASA proteins for use in enzyme replacement therapy. The present invention is based, in part, on the surprising discovery that recombinant ASA proteins can be purified from untreated biological material (e.g., cell culture medium containing ASA) using a process comprising only a single step, ultrafiltration / diafiltration after a chromatography column, to yield a pharmaceutically acceptable drug substance that can be directly incorporated into the final formulation buffer. As described in the Examples below, this single-step UF / DF process is accomplished by simply combining the eluates from the chromatography steps and adjusting the pH of the combined eluate to approximately 6.0. Prior to the present invention, processes for purifying recombinantly produced rASA proteins included at least two steps, ultrafiltration / diafiltration (UF / DF) after a chromatography column. As described in the Examples section, recombinant ASA proteins purified using a one-step UF / DF process according to the present invention have comparable purity, activity, and yield compared to recombinant ASA proteins purified using multiple UF / DF steps. For example, the recombinant ASA enzyme purified according to the present invention has only 100 pg / mg of host cell DNA and maintains a high specific activity (e.g., approximately 50-140 U / mg). Other distinguishing features include improved bioavailability and / or lysosomal targeting of the recombinant ASA protein. Thus, this simplified process is faster, less expensive, and equally effective in purifying recombinant ASA protein. This process is particularly useful when combined with a high-loading capacity chromatography step, thereby facilitating large-scale production of recombinant ASA protein.

[0008] Thus, in one aspect, the present invention provides a method for purifying a recombinant arylsulfatase A (ASA) protein, comprising the steps of purifying the recombinant arylsulfatase A (ASA) protein from an impure preparation by performing one or more chromatography steps; combining the eluates from the one or more chromatography steps; adjusting the pH of the combined eluates to about 5.8, 5.9 or 6.0 or greater than about 5.8, 5.9 or 6.0; and subjecting the pH-adjusted eluate to ultrafiltration and / or diafiltration. In some embodiments, the pH is adjusted to about 5.8-7.0 (e.g., about 5.8-6.8, about 5.8-6.6, about 5.8-6.4, about 5.8-6.2, about 5.8-6.1, about 5.8-6.0, about 5.9-7.0, about 5.9-6.8, about 5.9-6.6, about 5.9-6.4, about 5.9-6.2, about 5.9-6.1, about 5.9-6.0, about 5.95-6.20, about 5.95-6.15, about 5.95-6.10, or about 5.95-6.05). In some embodiments, the pH is adjusted to about 6.0.

[0009] In some embodiments, the pH is adjusted using a buffer comprising sodium phosphate, sodium chloride, and sodium citrate at pH 7.0. In some embodiments, the buffer comprises about 0.1-0.5 M (e.g., about 0.1-0.4 M, 0.1-0.3 M, 0.2-0.4 M, or 0.2-0.3 M) sodium phosphate, about 0.5-2.5 M (e.g., about 0.5-2.0 M, 0.5-1.5 M, 0.75-2.5 M, 0.75-2.0 M, 0.75-1.5 M, 1.0 In some embodiments, the buffer comprises about 0.25 M sodium phosphate, about 1.33 M sodium chloride, and about 0.34 M sodium citrate, and the pH of the buffer is about 7.0.

[0010] In some embodiments, virus filtration is performed before the ultrafiltration and diafiltration steps. In some embodiments, a single step of ultrafiltration and / or diafiltration is performed. In some embodiments, the single step of ultrafiltration and / or diafiltration includes only one diafiltration. In various embodiments, the ultrafiltration is tangential flow ultrafiltration.

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

[0012] In some embodiments, the one or more chromatography steps include affinity chromatography. In some embodiments, the affinity chromatography is the first chromatography step. In some embodiments, the affinity chromatography is the last chromatography step and the eluents from the affinity chromatography are combined before adjusting the pH. In some embodiments, one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed before performing the affinity chromatography. In some embodiments, one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed after performing the affinity chromatography.

[0013] In some embodiments, the anion exchange chromatography is Q chromatography. In some embodiments, the anion exchange chromatography comprises a TMAE resin (e.g., TMAE). In some embodiments, once the impure product is loaded, the TMAE resin is washed with a first wash buffer comprising MES-Tris at a pH of about 7.0. In certain embodiments, the first wash buffer comprises about 20-75 mM MES-Tris (e.g., about 30-60 mM, 40-70 mM, or 40-60 mM). In certain embodiments, the first wash buffer comprises about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, or 75 mM MES-Tris.

[0014] In some embodiments, the TMAE resin is washed using a second wash buffer comprising MES-Tris and NaCl at a pH of about 7.0. In some embodiments, the second wash buffer comprises about 5-75 mM MES-Tris (e.g., about 5-70 mM, 5-60 mM, 5-50 mM, 5-40 mM, 5-30 mM, 10-60 mM, 10-50 mM, 10-40 mM, 10-30 mM, or 15-25 mM) and about 50-150 mM NaCl (e.g., about 50-140 mM, 50-130 mM, 50-120 mM, 50-110 mM, 75-150 mM, 75-140 mM, 75-130 mM, 75-120 mM, or 75-110 mM), and the pH of the second wash buffer is about 7.0. In some embodiments, the second wash buffer comprises about 5mM, 10mM, 20mM, 30MM, 40mM, 50mM, 60mM or 75mM MES-Tris and about 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM NaCl, and the pH of the second wash buffer is about 7.0. In some embodiments, the second wash buffer comprises about 20mM MES-Tris and about 100mM NaCl, and the pH of the second wash buffer is about 7.0.

[0015] In some embodiments, once the impure preparation is loaded, the TMAE resin is eluted using an elution buffer comprising MES-Tris and NaCl at a pH of about 7.0. In some embodiments, the elution buffer comprises about 5-75 mM MES-Tris (e.g., about 5-70 mM, 5-60 mM, 5-50 mM, 5-40 mM, about 5-30 mM, 10-75 mM, 10-60 mM, 10-50 mM, 10-40 mM, or 10-30 mM) and about 150-300 mM NaCl (e.g., about 150-250 mM, about 180-260 mM, about 200-280 mM, 200-260 mM, 200-240 mM, 210-300 mM, 210-280 mM, 210-260 mM, or 210-240 mM), and the pH of the elution buffer is about 7.0. In some embodiments, the elution buffer comprises about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or 75 mM MES-Tris and about 150 mM, 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM NaCl, and the pH of the elution buffer is about 7.0. In some embodiments, the elution buffer comprises about 20 mM MES-Tris and about 220 mM NaCl, and the pH of the elution buffer is about 7.0.

[0016] In some embodiments, the anion exchange chromatography utilizes a chromatography column selected from the group consisting of: QSEPHAROSE TM Fast Flow Column, Q SEPHAROSE TM High-performance columns, Q SEPHAROSE TM XL, CAPTO TM Q, DEAE, TOYOPEARL Q. TMAE, ESHMUNO TM Q. NUVIA TM Q or UNOSPHERE TM Q.

[0017] In some embodiments, a suitable mixed-mode chromatography for use in the present invention is hydroxyapatite (HA) chromatography.

[0018] In some embodiments, a suitable hydrophobic interaction chromatography for use in the present invention is phenyl chromatography.

[0019] In some embodiments, anion exchange chromatography (e.g., using TMAE resin), mixed mode chromatography (e.g., HA chromatography), hydrophobic interaction chromatography (e.g., phenyl chromatography), and cation exchange chromatography (e.g., SP chromatography) are performed in the following order.

[0020] In some embodiments, suitable affinity chromatography for use with the present invention utilizes an anti-arylsulfatase A antibody (eg, an anti-human arylsulfatase A antibody).

[0021] In some embodiments, suitable anion exchange chromatography uses a column with a loading capacity greater than about 4.5 g / L (e.g., greater than about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L). In some embodiments, suitable anion exchange chromatography uses a column with a loading capacity ranging between about 4.5-20 g / L (e.g., ranging between about 5-20 g / L, 5-19 g / L, 5-18 g / L, 5-17 g / L, 5-16 g / L, 5-15 g / L, 7.5-20 g / L, 7.5-19 g / L, 7.5-18 g / L, 7.5-17 g / L, 7.5-16 g / L, 7.5-15 g / L, 10-20 g / L, 10-19 g / L, 10-18 g / L, 10-17 g / L, 10-16 g / L, or 10-15 g / L). In specific embodiments, the suitable loading capacity of the column is about 10-15 g / L.

[0022] In some embodiments, ultrafiltration of the impure product is performed prior to the one or more chromatography steps. In some embodiments, an ultrafiltration step suitable for the present invention is tangential flow ultrafiltration. In certain embodiments, suitable ultrafiltration utilizes a membrane filter comprising a pore size having a molecular weight cutoff of at least about 10 kDA, at least about 20 kDA, at least about 30 kDA, at least about 40 kDA, or at least about 50 kDA. In certain embodiments, at least 75% of the recombinant ASA is retained in the impure product. In certain embodiments, at least 75% of the recombinant ASA permeates through the filter. In certain embodiments, suitable ultrafiltration steps utilize polyethersulfone or cellulose membranes.

[0023] In some embodiments, clarification of the impure product is performed prior to ultrafiltration of the impure product. In some embodiments, a clarification step suitable for the present invention is filtration using one or more depth filters. In certain embodiments, a series of depth filters is used to filter the impure product. In certain embodiments, a suitable depth filter series utilizes a membrane comprising cellulose, diatomaceous earth, polyethersulfone, or a combination thereof.

[0024] In some embodiments, the ultrafiltration is followed by one or more steps of depth filtration and / or viral inactivation. In some embodiments, the one or more steps of depth filtration and / or viral inactivation are followed by one or more chromatography steps. In some embodiments, the viral inactivation step comprises adding a detergent to the impure product.

[0025] In some embodiments, the methods according to the present invention can be used to purify recombinant ASA protein produced by mammalian cells cultured in suspension. In some embodiments, the mammalian cells are cultured in a bioreactor. In some embodiments, the culture medium is serum-based. In some embodiments, the culture medium is serum-free. In some embodiments, the serum-free culture medium is a chemically defined culture medium.

[0026] In some embodiments, the impure product is a feed stream from a perfusion bioreactor. In some embodiments, the impure product is prepared from a culture medium (e.g., serum-based or serum-free) containing recombinant ASA protein secreted by mammalian cells. In some embodiments, the impure product is thawed from a frozen culture medium product.

[0027] In some embodiments, the ultrafiltration and / or diafiltration steps comprise exchanging the purified recombinant ASA protein into a pharmaceutical formulation buffer.

[0028] In some embodiments, the recombinant ASA protein has an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the recombinant ASA protein has an amino acid sequence that is identical to SEQ ID NO: 1.

[0029] In some embodiments, the purified recombinant ASA protein according to the present invention contains less than about 150 ng / mg, 140 ng / mg, 130 ng / mg, 120 ng / mg, 110 ng / mg, 100 ng / mg, 90 ng / mg, 80 ng / mg, 70 ng / mg, 60 ng / mg, 50 ng / mg, 40 ng / mg, 30 ng / mg, 20 ng / mg, or 10 ng / mg of Host Cell Protein (HCP).

[0030] In some embodiments, the purified recombinant ASA protein has no new bands with an intensity greater than 1.0% of the assay control when subjected to SDS-PAGE using Coomassie Blue staining.

[0031] In some embodiments, the purified recombinant ASA protein contains less than about 150 pg / mg, 140 pg / mg, 130 pg / mg, 120 pg / mg, 110 pg / mg, 100 pg / mg, 90 pg / mg, 80 pg / mg, 70 pg / mg, 60 pg / mg, 50 pg / mg, 40 pg / mg, 30 pg / mg, 20 pg / mg, or 10 pg / mg of host cell DNA.

[0032] In some embodiments, the present invention provides a method for purifying a recombinant arylsulfatase A (ASA) protein, comprising the steps of purifying the recombinant arylsulfatase A (ASA) protein from an impure preparation by performing one or more chromatography steps, wherein the first chromatography step uses about 4.5 g protein / L of resin (e.g., greater than about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, etc.). L or 20 g / L) or greater than about 4.5 g protein / L resin (e.g., greater than about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, or 20 g / L); combining the eluates from the one or more chromatography steps; adjusting the pH of the combined eluates to a pH of about 6.0 or greater; and subjecting the pH adjusted eluate to ultrafiltration and / or diafiltration.

[0033] In some embodiments, the step wherein performing one or more chromatography steps comprises performing in the following order: a first ion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and cation exchange chromatography.

[0034] In some embodiments, the one or more chromatography steps comprise affinity chromatography. In certain embodiments, the affinity chromatography is performed before the first ion exchange chromatography step. In certain embodiments, the affinity chromatography is performed after the second ion exchange chromatography step. In some embodiments, the affinity chromatography is performed between the first ion exchange chromatography step and the second ion exchange chromatography step.

[0035] In some embodiments, the step of performing one or more chromatography steps comprises performing in the following order: anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and cation exchange chromatography.

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

[0037] In some embodiments, the step of performing one or more chromatography steps comprises performing in the following order: anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, cation exchange chromatography, and affinity chromatography.

[0038] In some embodiments, the anion exchange chromatography uses a post with TMAE resin. In certain embodiments, the TMAE post has a loading capacity of about 5-20g protein / L resin (for example, about 5-19g / L, 5-18g / L, 5-17g / L, 5-16g / L, 5-15g / L, 7.5-20g / L, 7.5-19g / L, 7.5-18g / L, 7.5-17g / L, 7.5-16g / L, 7.5-15g / L, 10-20g / L, 10-19g / L, 10-18g / L, 10-17g / L, 10-16g / L or 10-15g / L).

[0039] In some embodiments, the single step of ultrafiltration and / or diafiltration is performed after the one or more chromatography steps.

[0040] In addition, the present invention provides recombinant arylsulfatase A (ASA) proteins purified according to the methods of the present invention described herein and pharmaceutical compositions comprising the same.

[0041] In some embodiments, the present invention provides a composition comprising purified recombinant arylsulfatase A (ASA) having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, wherein the purified recombinant ASA has a specific activity of at least about 50 U / mg, and further wherein the purified recombinant ASA contains less than 150 ng / mg host cell protein (HCP) and / or 150 pg / mg host cell DNA (HCD). In some embodiments, the purified recombinant ASA has an amino acid sequence that is identical to SEQ ID NO: 1. In some embodiments, the purified recombinant ASA contains less than about 140 ng / mg, 130 ng / mg, 120 ng / mg, 110 ng / mg, 100 ng / mg, 90 ng / mg, 80 ng / mg, 70 ng / mg, 60 ng / mg, 50 ng / mg, 40 ng / mg, 30 ng / mg, 20 ng / mg, or 10 ng / mg of host cell protein (HCP). In some embodiments, the purified recombinant ASA contains less than about 140 pg / mg, 130 pg / mg, 120 pg / mg, 110 pg / mg, 100 pg / mg, 90 pg / mg, 80 pg / mg, 70 pg / mg, 60 pg / mg, 50 pg / mg, 40 pg / mg, 30 pg / mg, 20 pg / mg, or 10 pg / mg of host cell DNA.

[0042] In some embodiments, the purified recombinant ASA has a specific activity of at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg. In some embodiments, the purified recombinant ASA has a concentration in the range of about 50-200 U / mg (e.g., about 50-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-140 U / mg, 60-130 U / mg, 60-120 U / mg, 60-110 U / mg, 60-100 U / mg, 70-140 U / mg, 70-130 U / mg, 70-120 U / mg, 70-110 U / mg). or 130 U / mg).

[0043] In some embodiments, the present invention provides a composition comprising a purified recombinant arylsulfatase A (ASA) having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, wherein the purified ASA is characterized by a glycan profile comprising one or more peak groups selected from peak groups indicative of neutral (peak group 1), monosialylation (peak group 2), blocked mannose-6-phosphorylation (peak group 3), disialylation (peak group 4), monomannose-6-phosphorylation (peak group 5), hybrid (peak group 6), and dimannose-6-phosphorylation (peak group 7). In some embodiments, the purified ASA is characterized by a glycan profile comprising at least two or more, three or more, four or more, five or more, six or more, or seven or more of peak groups 1-7. In some embodiments, the purified recombinant ASA has an amino acid sequence identical to SEQ ID NO: 1.

[0044] In some embodiments, the present invention provides a formulation comprising a composition described herein and a physiologically acceptable carrier. In some embodiments, the formulation is suitable for intravenous administration. In some embodiments, the formulation is suitable for intrathecal administration. In some embodiments, the formulation is suitable for subcutaneous administration.

[0045] In addition, the present invention provides a method for treating metachromatic leukodystrophy, comprising administering to a subject in need of treatment a purified recombinant ASA protein, a pharmaceutical composition, or a formulation described herein.

[0046] As used herein, unless expressly stated otherwise, the terms "ASA protein," "ASA," "ASA enzyme," or grammatical equivalents refer to preparations of recombinant ASA protein molecules.

[0047] As used in this application, the terms "about" and "approximately" are used equivalently. Any numerical value used in this application with or without about / approximately is intended to cover any normal fluctuations understood by a person of ordinary skill in the relevant art.

[0048] Other features, objects and advantages of the present invention will become apparent in the detailed description that follows. However, it should be understood that while the detailed description illustrates embodiments of the present invention, it is provided by way of illustration only and not limitation. Various changes and modifications within the scope of the present invention will be readily apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The following figures, which together form part of the accompanying drawings, are for illustration purposes only and are not limiting.

[0050] Figure 1 An overall flow diagram of an exemplary purification process is shown.

[0051] Figure 2 An overall flow diagram of an exemplary purification process including the steps of combining eluates from a cation exchange column and adjusting the pH of the combined eluates is shown.

[0052] Figure 3 Exemplary results illustrating the relationship between feed flow rate, transmembrane pressure (TMP), and permeate flux for each membrane are shown.

[0053] Figure 4 An exemplary SDS-PAGE (silver) gel is shown showing similar band profiles for Fractogel and Q FF throughout the process.

[0054] Figure 5 Exemplary results are shown showing that feasibility runs and control runs including a single UFDF or UFDFDF step exhibited similar banding patterns to the reference standard (lane 3), indicating that the drug substance from all runs exhibited comparable low levels of HCPs.

[0055] Figure 6 Exemplary results are shown demonstrating comparability between experimental (feasibility) and control runs and reference standards, with no additional banding patterns detected by SDS-PAGE (Coomassie).

[0056] definition

[0057] In order to make the present invention more easily understood, some terms are first defined as follows. The following terms and other terms are further defined throughout the specification.

[0058] About or approximately: As used herein, the term "about" or "approximately" as applied to one or more values ​​of interest refers to a value similar to a specified reference value. In certain embodiments, unless otherwise indicated or apparent from the context, the term "about" or "approximately" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a specified reference value in either direction (greater than or less than), except where such values ​​exceed 100% of the possible values.

[0059] Biological activity: As used herein, the term "biological activity" refers to the property of any agent that is active in a biological system, particularly an organism. For example, an agent that has a biological effect on an organism when administered to that organism is considered biologically active. In specific embodiments, where a protein or polypeptide is biologically active, the portion of the protein or polypeptide that shares at least one biological activity of that protein or polypeptide is generally referred to as the "biologically active" portion.

[0060] 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 a mannose-6-phosphate (M6P) tag on acid hydrolase precursors in the Golgi apparatus destined for transport to lysosomes. In addition to mannose-6-phosphate, CI-MPR also binds other proteins including IGF-II. CI-MPR is also known as "M6P / IGF-II receptor," "CI-MPR / IGF-II receptor," "IGF-II receptor," or "IGF2 receptor." These terms and their abbreviations are used interchangeably herein.

[0061] Chromatography: As used herein, the term "chromatography" refers to a technique for separating mixtures. Typically, the mixture is dissolved in a fluid called the "mobile phase," which carries it through a structure containing another material called the "stationary phase." Column chromatography is a separation technique using a fixed bed within a tube (i.e., a column).

[0062] Diluent: As used herein, the term "diluent" refers to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) diluent substance used to prepare a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0063] Elution: As used herein, the term "elution" refers to the process of extracting one material from another by washing with a solvent. For example, in ion exchange chromatography, elution is the process of washing a loaded resin to remove trapped ions.

[0064] Eluent: As used herein, the term "eluent" refers to the combination of the mobile phase "support" and the analyte material that emerges from the chromatography, typically as a result of elution.

[0065] Enzyme replacement therapy (ERT): As used herein, the term "enzyme replacement therapy (ERT)" refers to any therapeutic strategy that corrects an enzyme deficiency by providing the missing enzyme. Once administered, the enzyme is taken up by the cell and transported to the lysosome, where it is used to eliminate the material that has accumulated in the lysosome due to the enzyme deficiency. Typically, for effective lysosomal enzyme replacement therapy, the therapeutic enzyme is delivered to the lysosomes in appropriate cells of the target tissue that exhibits the storage defect. The purification process described herein can be used to purify and formulate recombinant arylsulfatase A as an ERT pharmaceutical for MLD.

[0066] Equilibrate or equilibrate: As used herein, the terms "equilibrate" or "equilibrate" in relation to chromatography refer to bringing a first fluid (e.g., a buffer) into equilibrium with another fluid, typically to a point where the components of the fluid (e.g., buffer) are stabilized and evenly distributed. For example, in some embodiments, a chromatography column can be equilibrated by passing one or more column volumes of a desired fluid (e.g., a buffer) through the column.

[0067] Improve, increase, or decrease: As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, indicate a value relative to a baseline measurement (e.g., a value measured in the same individual before initiation of a treatment described herein or a value measured in a control individual (or multiple control individuals) in the absence of a treatment described herein). A "control individual" is an individual who has the same form of lysosomal storage disease as the individual being treated and is of similar age to the individual being treated (to ensure that the disease stage is comparable in the individual being treated and the control individuals).

[0068] Impurity: As used herein, the term "impurity" refers to an internal, finite amount of liquid, gas, or solid matter that is chemically different from the target material or compound. Impurities are also called contaminants.

[0069] Loading: As used herein, the term "loading" refers to adding a liquid or solid containing a sample to a column in a chromatographic column. In some embodiments, as the loaded sample passes through the column, the sample-specific components loaded on the column are captured. In some embodiments, as the loaded sample passes through the column, the sample-specific components loaded on the column are not captured by the column or do not "flow through" the column.

[0070] Polypeptide: As used herein, generally, a "polypeptide" is a string of at least two amino acids linked to one another by peptide bonds. In some embodiments, a polypeptide may include at least 3-5 amino acids, each linked to the other amino acids by means of at least one peptide bond. Those skilled in the art understand that polypeptides sometimes include "unnatural" amino acids or other entities, but these can still be optionally incorporated into the polypeptide chain.

[0071] Pool: As used herein, the term "pool" in relation to chromatography refers to combining one or more fractions of fluid that have passed through a column. For example, in some embodiments, one or more fractions (e.g., "peak fractions") containing the desired components of a sample separated by chromatography can be pooled together to produce a single "pooled" fraction.

[0072] Alternative enzyme: As used herein, the term "alternative enzyme" refers to any enzyme that can be used as at least a partial replacement for a deficient or missing enzyme in a disease to be treated. In some embodiments, the term "alternative enzyme" refers to any enzyme that can be used as at least a partial replacement for a deficient or missing lysosomal enzyme in a lysosomal storage disease to be treated. In some embodiments, an alternative enzyme (e.g., rASA) can reduce the amount of material that accumulates in mammalian lysosomes or can rescue or alleviate one or more lysosomal storage disease (e.g., MLD) symptoms. Alternative enzymes suitable for the present invention include wild-type or modified lysosomal enzymes and can be produced using recombinant and synthetic methods or purified from natural sources. The alternative enzyme can be a recombinant enzyme, a synthetase, a genetically activated enzyme, or a natural enzyme.

[0073] 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 into which the therapeutic agent is administered and transported to the target site of action is sufficient to allow delivery of a therapeutically effective amount of the therapeutic agent to the site of action targeting. Multiple factors can affect the solubility of the therapeutic agent. For example, relevant factors that can affect protein solubility include ionic strength, amino acid sequence, and the presence of other auxiliary solubilizers or salts (e.g., calcium salts). In some embodiments, the therapeutic agent according to the present invention is soluble in its corresponding pharmaceutical composition.

[0074] Stability: As used herein, the term "stable" refers to the ability of a therapeutic agent (e.g., a recombinant enzyme) to maintain its therapeutic efficacy (e.g., all or most of its intended biological activity and / or physiochemical integrity) over an extended period of time. The stability of therapeutic agents and the ability of pharmaceutical compositions to maintain the stability of these therapeutic agents can be estimated over an extended period of time (e.g., at least 1, 3, 6, 12, 18, 24, 30, 36 months or longer). In the context of a formulation, a stable formulation is one in which the physical and / or chemical integrity and biological activity of the therapeutic agent are substantially maintained during storage and processing (e.g., freeze / thaw, mechanical mixing, and lyophilization). For protein stability, it can be measured by changes in high molecular weight (HMW) aggregate formation, loss of enzyme activity, peptide fragment generation, and charge profile.

[0075] Virus treatment: As used herein, the term "virus treatment" refers to "virus removal," where viruses are simply removed from a sample (e.g., virus filtration), or "virus inactivation," where viruses remain in the sample, but in a non-infectious form. In some embodiments, virus removal can utilize nanofiltration and / or chromatography techniques, among others. In some embodiments, virus inactivation can utilize solvent inactivation, detergent inactivation, pasteurization, acidic pH inactivation, and / or ultraviolet inactivation, among others. Details

[0076] In addition, the present invention provides improved methods for purifying ASA proteins that are produced recombinantly for use in enzyme replacement therapy. In some embodiments, the present invention provides methods for purifying recombinant ASA proteins from impure products (e.g., untreated biological materials, such as cell culture media containing ASA) using a process comprising only one step of ultrafiltration / diafiltration after the chromatography column. In some embodiments, the single-step UF / DF process is accomplished by combining the eluates from the chromatography steps and adjusting the pH of the combined eluates to about 6.0 or greater. In some embodiments, this simplified process is combined with a high loading capacity chromatography step to facilitate large-scale production of recombinant ASA proteins.

[0077] Various aspects of the present invention are further described in detail in the following subsections. The use of subsections is not intended to limit the present invention. Each subsection is applicable to any aspect of the present invention. In this application, unless otherwise indicated, the use of "or" means "and / or."

[0078] Arylsulfatase A

[0079] Arylsulfatase A (ASA, ARSA or cerebroside-sulfatase) is an enzyme that breaks down cerebroside-3-sulfate (or sulfatide) into cerebroside and sulfate. Specifically, galactosylsulfatide is normally metabolized 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 saponin B to hydrolyze the 3-O-sulfate bond to form galactocerebroside. Deficiency of arylsulfatase A is present in all tissues of patients with metachromatic leukodystrophy (MLD) in late infancy, juvenile and adult forms. As used herein, arylsulfatase A protein will be referred to as "ASA" or "ARSA", and saponin B will be referred to as "Sap-B".

[0080] Arylsulfatase A is an acidic glycoprotein with a low isoelectric point. At pH 6.5, the enzyme exists as a monomer with a molecular weight of approximately 100 kDa. Under acidic conditions (pH≤about 5.0), ASA exists as a 480 kDa octamer and decomposes into dimers at neutral pH levels. In human urine, the enzyme is composed of two non-identical subunits of 63 and 54 kDA (Laidler PM et al., Biochim Biophys Acta. 1985, 827, 73-83). Arylsulfatase A purified from human liver, placenta, and fibroblasts is also composed of two subunits of slightly different sizes, varying between 55 and 64 kDA (Draper RK et al., Arch Biochemica Biophys. 1976, 177, 525-538; Waheed A et al., Hoppe Seylers Z Physiol Chem. 1982, 363, 425-430; Fujii T et al., Biochim Biophys Acta. 1992, 15 1122, 93-98). Like other lysosomal enzymes, arylsulfatase A is synthesized as a glycosylated precursor on membrane-bound ribosomes. This glycosylated precursor then passes through the endoplasmic reticulum and Golgi apparatus, where the N-linked oligosaccharides of the glycosylated precursor are processed to form phosphorylated and sulfated complex oligosaccharides (Waheed A et al., Biochim Biophys Acta. 1985, 847, 53-61; Braulke T et al., Biochem Biophys Res Commun. 1987, 143, 178-185). In normal cultured fibroblasts, a 62 kDa precursor polypeptide is generated, which is translocated to the acidic prolysosome by binding to the mannose-6-phosphate receptor (Braulke T et al., J Biol Chem. 1990, 265, 6650-6655) (Kelly BM et al., Eur J Cell Biol. 1989, 48, 71-78).

[0081] The methods described herein can be used to purify arylsulfatase A from any source, such as from tissue or cultured cells (e.g., human cells (e.g., fibroblasts) that recombinantly produce arylsulfatase A). Arylsulfatase A from any source, including but not limited to humans and other animals, can be produced by the methods described herein.

[0082] 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. Therefore, for the deduced human ASA cDNA (EMBL gene bank accession numbers J04593 and X521151), the cleavage of the signal peptide should be carried out after residue number 18 (Ala) in all cells to produce the mature form of human arylsulfatase A. As used herein, recombinant arylsulfatase A will be abbreviated as "rASA". Mature forms of arylsulfatase A (including mature forms of human arylsulfatase A) will be referred to as "mASA", and mature recombinant human ASA will be referred to as "mrhASA".

[0083] Various forms of arylsulfatase A have been detected in cells from human urine (Luijten J A F M et al., J Mol Med. 1978, 3, 213), leukocytes (Dubois et al., Biomedicine. 1975, 23, 116-119; Manowitz P et al., Biochem Med Metab Biol. 1988, 39, 117-120), platelets (Poretz et al., Biochem J. 1992, 287, 979-983), cultured fibroblasts (Waheed A et al., Hoppe Seylers Z Physiol Chem. 1982, 363, 425-430; Stevens R L et al., Biochim Biophys Acta. 1976, 445, 661-671; Farrell DF et al., Neurology. 1979, 29, 16-20) and liver (Stevens R L et al., Biochim Biophys Acta. 1989, 446, 661-671). The results were confirmed by electrophoresis and isoelectric focusing of enzyme preparations of arylsulfatase A (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 reduced the molecular size and the complexity of the electrophoretic pattern, indicating that the large charge heterogeneity of arylsulfatase A is due to variations in the carbohydrate content of the enzyme.

[0084] The active site of arylsulfatase A contains an essential histidine residue (Lee GD and Van Etten RL, Arch Biochem Biophys. 1975, 171, 424-434) and two or more arginine residues (James GT, Arch Biochem Biophys. 1979, 97, 57-62). Many anions are inhibitors of the enzyme at millimolar concentrations or lower.

[0085] The human arylsulfatase A gene structure has been described. As used herein, the gene will be referred to as "ARSA". However, in some cases, "ARSA" may also be referred to as arylsulfatase A protein. The ARSA gene is located near the end of the long arm of chromosome 22 (22q13.31-qter), spanning 3.2 kb (Kreysing et al., Eur J Biochem. 1990, 191, 627-631) and consisting of eight exons that define 507 amino acid enzyme units (Stein et al., J Biol Chem. 1989, 264, 1252-1259). Messenger RNAs of 2.1, 3.7, and 4.8 kb have been detected in fibroblasts, wherein the 2.1-kb message is apparently derived from a large amount of active arylsulfatase A produced by the cell (Kreysing et al., Eur J Biochem. 1990, 191, 627-631). The ARSA sequence has been deposited in the EMBL gene bank with accession number X521150. The difference between the coding portion of disclosed cDNA and ARSA is described by the people such as Kreysing (Eur J Biochem. 1990, 191, 627-631). The cDNA sequence described by the people such as Stein (J Biol Chem. 1989, 264, 1252-1259) and the cDNA sequence described by the people such as Kreysing (Eur J Biochem. 1990, 191, 627-631) are deposited in the EMBL gene bank with the following accession numbers J04593 and X521151 respectively.

[0086] Several polymorphisms and more than 40 disease-associated mutations have been identified in the ARSA gene (Gieselmann et al., Hum Mutat. 1994, 4, 233-242; Barth et al., Hum Mutat. 1995, 6, 170-176; Draghia et al., Hum Mutat. 1997, 9, 234-242). Disease-associated mutations in the ARSA gene can be divided into two major groups that are closely related to the clinical phenotype of MLD. One group (I) does not produce active enzymes, no immunoreactive proteins, and does not express ASA activity when introduced into cultured animal cell lines. The other group (A) produces a small amount of cross-reactive substances and low levels of functional enzymes in cultured cells. Individuals with purified mutations from group (I) or individuals with two different mutations from this group express MLD in late infancy. Most individuals with one (I)-type and one (A)-type mutation develop the juvenile-onset form, while those with two (A)-type mutations typically develop MLD in adulthood. Some mutations have been found to occur quite frequently, while others are only detected in a single family. Specific mutations can be traced through many family members, but universal carrier screening is not yet feasible.

[0087] In addition to the mutation related to the above-mentioned disease, multiple polymorphisms have been identified in the ARSA gene. In some clinically normal parents of MLD patients and also in the general population, extremely low ASA activity is found. This so-called pseudo-lack of ASA is associated with the common polymorphism of the ARSA gene (Gieselmann et al., Dev Neurosci. 1991, 13, 222-227).

[0088] Recombinant ASA protein

[0089] As used herein, the term "recombinant ASA protein" refers to any molecule or portion of a molecule that can replace at least a portion of the activity of a naturally occurring arylsulfatase A (ASA) protein or rescue one or more phenotypes or symptoms associated with ASA deficiency. As used herein, the terms "recombinant ASA enzyme" and "recombinant ASA protein" and grammatical equivalents are used interchangeably. In some embodiments, the present invention is used to purify a recombinant ASA protein, which is a polypeptide having an amino acid sequence substantially similar to or identical to that of a mature human ASA protein.

[0090] Typically, human ASA is produced as a precursor molecule that is processed into a mature form. This process typically occurs by removing an 18-amino acid signal peptide. Typically, the precursor form, also referred to as the full-length precursor or full-length ASA protein, contains 507 amino acids. The N-terminal 18 amino acids are cleaved to produce a mature form of 489 amino acids in length. Therefore, it is estimated that the N-terminal 18 amino acids are generally not required for ASA protein activity. The amino acid sequences of the mature form (SEQ ID NO: 1) and the full-length precursor (SEQ ID NO: 2) of a typical wild-type or naturally occurring human ASA protein are shown in Table 1.

[0091] Table 1. Human arylsulfatase A

[0092]

[0093] Thus, in some embodiments, the recombinant ASA protein purified by the embodiments of the present invention is a mature human ASA protein (SEQ ID NO: 1). In some embodiments, the recombinant ASA protein purified by the embodiments of the present invention may be a homolog or analog of a mature human ASA protein. For example, a homolog or analog of a mature human ASA protein may be a modified mature human ASA protein containing one or more amino acid substitutions, deletions, and / or insertions as compared to a wild-type or naturally occurring ASA protein (e.g., SEQ ID NO: 1), but still substantially retaining ASA protein activity. Thus, in some embodiments, the recombinant ASA protein purified by the embodiments of the present invention is substantially homologous to a mature human ASA protein (SEQ ID NO: 1). In some embodiments, the recombinant ASA protein purified by 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 embodiments of the present invention is substantially identical to mature human ASA protein (SEQ ID NO: 1). In some embodiments, the recombinant ASA protein purified by 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 embodiments of the present invention contains fragments or portions of mature human ASA protein.

[0094] Alternatively, the recombinant ASA protein purified by the embodiments of the present invention is a full-length ASA protein. In some embodiments, the recombinant ASA protein may be a homolog or analog of the full-length human ASA protein. For example, a homolog or analog of the full-length human ASA protein may be a modified full-length human ASA protein containing one or more amino acid substitutions, deletions, and / or insertions as compared to the wild-type or naturally occurring full-length ASA protein (e.g., SEQ ID NO: 2), but still substantially retains ASA protein activity. Thus, in some embodiments, the recombinant ASA protein purified by the embodiments 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 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 greater homologous to SEQ ID NO: 2. In some embodiments, the recombinant ASA protein purified by embodiments of the present invention is substantially identical to SEQ ID NO: 2. In some embodiments, the recombinant ASA protein purified by 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: 2. In some embodiments, the recombinant ASA protein purified by embodiments of the present invention contains a fragment or portion of a full-length human ASA protein. As used herein, a full-length ASA protein typically contains a signal peptide sequence.

[0095] Homologs or analogs of human ASA proteins can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences (e.g., methods found in references compiling 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, "conservative amino acid substitutions" are substitutions that do not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.

[0096] In some embodiments, the recombinant ASA protein may include a moiety that binds to a receptor on the surface of the target cell to facilitate cellular uptake and / or lysosomal targeting. For example, these receptors may be the cation-independent mannose-6-phosphate receptor (CI-MPR) that binds to mannose-6-phosphate (M6P) residues. Furthermore, CI-MPR also binds to other proteins, including IGF-II. In some embodiments, the recombinant ASA protein contains M6P residues on the protein surface. In particular, the recombinant ASA protein may contain diphosphorylated oligosaccharides with a high binding affinity for CI-MPR. In some embodiments, suitable enzymes contain an average of about at least 20% diphosphorylated oligosaccharides per enzyme. In other embodiments, suitable enzymes may contain about 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% diphosphorylated oligosaccharides per enzyme.

[0097] 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 the target cell. Suitable lysosomal targeting moieties can be IGF-I, IGF-II, RAP, p97, and variants, homologs, or fragments thereof (e.g., peptides comprising sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95% identical to wild-type mature IGF-I, IGF-II, RAP, or 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.

[0098] Production of recombinant ASA protein

[0099] The present invention is useful for purifying recombinant ASA proteins produced by various methods. For example, ASA proteins can be recombinantly produced using host cell systems engineered to express nucleic acids encoding ASA. Alternatively, ASA proteins can be produced by activating endogenous ASA genes. It is contemplated that the present invention can be used to purify recombinant ASA proteins produced using various expression systems. Suitable expression systems include, for example, eggs, baculovirus, plants, yeast, or mammalian cells.

[0100] In some embodiments, the ASA enzyme is produced in mammalian cells. Non-limiting examples of mammalian cells that can be used according to the present invention include a BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); a monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); a human embryonic kidney line (HEK293 or 293 cells grown in suspension culture and subcloned; Graham et al., J. Gen Virol., 36:59, 1977); a human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (e.g., Sertoli cells). cell) (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0101] In some embodiments, the methods of the invention according to the present invention are used to purify recombinant ASA enzymes produced by human cells (e.g., HT1080). In some embodiments, the methods of the invention according to the present invention are used to purify recombinant ASA enzymes produced by CHO cells.

[0102] Typically, cells engineered to express recombinant ASA can include a transgene encoding a recombinant ASA protein as described herein. It should be understood that nucleic acids encoding recombinant ASA can contain regulatory sequences, gene control sequences, promoters, non-coding sequences, and / or other suitable sequences for expressing recombinant ASA. Typically, the coding region can be operably linked to one or more of these nucleic acid components.

[0103] "Regulatory sequence" generally refers to a nucleotide sequence that is located upstream (5' non-coding sequence), within or downstream (3' non-coding sequence) of a coding sequence and that affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. Sometimes, "regulatory sequence" is also referred to as a "gene control sequence."

[0104] " Promoter " generally refers to the nucleotide sequence that can control coding sequence or functional RNA expression. Usually, coding sequence is located at 3 ' place of promoter sequence. Promoter sequence is made up of proximal and more distal upstream elements, and latter element is usually called enhancer. Therefore, " enhancer " is the nucleotide sequence that can stimulate promoter activity and can be the innate element of promoter or insert to strengthen the level of promoter or tissue-specific heterologous element. Promoter can all originate from natural gene or be made up of the different elements that derive from the different promoters found in nature, or even comprise synthetic nucleotide fragment. It is understood by those skilled in the art that different promoters can guide the expression of genes in different tissues or cell types or at different developmental stages or in response to different environmental conditions.

[0105] "3' non-coding sequences" generally refer to nucleotide sequences located downstream of the coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is generally characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0106] "Translation leader sequence" or "5' non-coding sequence" generally refers to the nucleotide sequence located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence can affect the processing of the primary transcript into mRNA, mRNA stability, or translation efficiency.

[0107] Generally, the terms "operably linked" or "operably linked" refer to the association of two or more nucleic acid segments on a single nucleic acid segment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either sense or antisense orientation.

[0108] The coding region of a transgene can include one or more silent mutations to optimize codon usage for a specific cell type. For example, the codon usage of an ASA transgene can be optimized for expression in vertebrate cells. In some embodiments, the codon usage of an ASA transgene can be optimized for expression in mammalian cells. In some embodiments, the codon usage of an ASA transgene can be optimized for expression in human cells.

[0109] Optionally, the construct may comprise additional components, such as one or more of the following: splice sites, enhancer sequences, a selectable marker gene under the control of an appropriate promoter, an amplifiable marker gene under the control of an appropriate promoter, and a matrix attachment region (MAR) or other elements known in the art that enhance expression of the region into which it is inserted.

[0110] Once transfected or transduced into a host cell, appropriate vectors may be expressed extrachromosomally (episomally) or integrated into the genome of the host cell.

[0111] Cell culture media and conditions

[0112] Various cell culture media and conditions can be used to produce recombinant ASA protein. For example, recombinant ASA protein can be produced in serum-containing or serum-free medium. In some embodiments, recombinant ASA protein is produced in serum-free medium. In some embodiments, recombinant ASA protein is produced in animal-free medium (i.e., medium without components of animal origin). In some embodiments, recombinant ASA protein is produced in a chemically defined medium. As used herein, the term "chemically defined nutrient medium" refers to a medium in which substantially all chemical components are known. In some embodiments, the chemically defined nutrient medium does not have components of animal origin, such as serum, serum-derived proteins (e.g., albumin or fetuin) and other components. In some cases, the chemically defined medium comprises one or more proteins (e.g., protein growth factors or cytokines). In some cases, the chemically defined nutrient medium comprises one or more protein hydrolysates. In other cases, the chemically defined nutrient medium is a protein-free medium, i.e., a serum-free medium that does not comprise a hydrolysate or component of an unknown composition.

[0113] In some embodiments, the chemically defined culture medium may be supplemented with one or more animal-derived components. These animal-derived components include, but are not limited to, fetal bovine serum, horse serum, goat serum, donkey serum, human serum, and serum-derived proteins such as albumin (e.g., bovine serum albumin or human serum albumin). Although the addition of serum is desirable because it contains components such as vitamins, amino acids, growth factors, and hormones, it also consists of concentrated sources of exogenous proteins that can hinder the purification of recombinant proteins. For example, fetuin is a family of related serum proteins that are secreted by cells, primarily hepatocytes, and have three characteristic domains (Brown WM, et al., Eur J Biochem. 1992 Apr 1; ​​205(1): 321-31). Fetuin is abundant in the fetal period and, therefore, is typically abundant in commercially available fetal-derived serum, and fetuin constitutes the primary protein contaminant in purification. Therefore, in some embodiments, a suitable culture medium is a xeno-free culture medium, for example, a culture medium that does not contain any bovine serum or bovine serum-derived components. For example, the xeno-free culture medium may contain one or more human serum albumin, human transferrin, human insulin, and human lipids. In some embodiments, the suitable culture medium contains fetuin-depleted serum. Fetuin can be depleted from the serum using various methods known in the art. For example, fetuin can be depleted from the serum by antibody affinity chromatography. (See, e.g., Toroian D and Price PA, Calcif Tissue Int (2008) 82: 116–126). In some embodiments, the suitable culture medium is fetuin-free.

[0114] Various cell culture conditions can be used for large-scale production of recombinant ASA protein, including but not limited to roller bottle culture, batch culture in a bioreactor, and fed-batch culture in a bioreactor. In some embodiments, recombinant ASA protein is produced by cells cultured in suspension. In some embodiments, recombinant ASA protein is produced by adherent cells.

[0115] Purification of recombinant arylsulfatase A

[0116] Embodiments of the present invention include purification processes for producing arylsulfatase A ("ASA") (particularly recombinant human ASA ("rhASA")) pharmaceutical substances. Various techniques can be optionally combined, in whole or in part, with the modifications described herein to produce purified ASA pharmaceutical substances. For example, Figure 1An overall flow diagram of an embodiment of the present invention is shown. The exemplary purification process begins with thawing and pooling of the rhASA unpurified bulk (UPB), which is then captured and filtered by ultrafiltration and diafiltration ("UFDF"). Following filtration, the captured material can be virus-inactivated (not shown) prior to chromatographic purification. In the specifically shown embodiment, the next four purification process steps utilize four chromatographic columns in succession: an anion exchange column (e.g., a Q Sepharose Fast Flow ("Q-FF") column), a ceramic hydroxyapatite type I (HA) column, a phenyl column, and a cation exchange (e.g., SP) column. Following the fourth and final chromatographic step, the SP eluate is concentrated and diafiltered using tangential flow ultrafiltration. The resulting filtrate is then virus filtered (e.g., by 20N virus reduction filter), followed by a second concentration and diafiltration step to obtain the final target protein concentration. Figure 2 Another exemplary embodiment of the present invention is shown. Figure 1 The chromatographic step is performed using one or more different chromatography resins (e.g., TMAE anion exchange resin). However, after the fourth or last chromatography step, the SP eluates are combined and the pH of the combined solution is adjusted. In some embodiments, the pH is adjusted to between 5.5 and 6.5 (e.g., about 6.0). The resulting pH-adjusted cation exchange eluate is then virus filtered and then passed through a concentration and diafiltration step to obtain the final target protein concentration. Optional refinement of these processes and other embodiments is described herein.

[0117] As used herein, "contaminants" are materials other than the desired polypeptide product (e.g., arylsulfatase A (ASA)). Contaminants can be variants of the desired polypeptide (e.g., deamidated variants or amino-aspartic acid variants of the desired polypeptide) or other molecules, such as polypeptides, nucleic acids, and endotoxins.

[0118] As used herein, "purifying" a polypeptide from a composition or sample comprising the polypeptide and one or more contaminants refers to increasing the purity of the polypeptide in the composition or sample by removing (completely or partially) at least one contaminant from the composition or sample. A "purification step" can be part of a total purification process to obtain a composition comprising at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% by weight of the polypeptide of interest, based on the total weight of the composition. The purity of arylsulfatase A can be determined, for example, by one or more of the following methods: host cell protein (HCP) Western blotting, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reversed-phase HPLC, and size exclusion HPLC. In some embodiments, the specific activity of the purified arylsulfatase A is at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg, e.g., as determined by the methods described herein. In some embodiments, the purified recombinant ASA has a range of about 50-200 U / mg (e.g., about 50-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-140 U / mg, 60-130 U / mg, 60-120 U / mg, 60-110 U / mg, 60-100 U / mg, 70-140 U / mg, 70-130 U / mg, 70-120 U / mg, 70-110 U / mg, 70-100 U / mg).

[00155] In some embodiments, the specific activity of the present invention may be an activity of at least one of the following: a) 80-140 U / mg, 90-130 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), e.g., as determined by the methods described herein.

[0119] The raw material for the purification process is any impure product. For example, the impure product can be an untreated cell culture medium containing recombinant ASA protein secreted by cells (e.g., mammalian cells) that produce the ASA protein or a raw cell lysate containing the ASA protein. In some embodiments, the impure product can be a partially treated cell culture medium or cell lysate. For example, the cell culture medium or cell lysate can be concentrated, diluted, or treated with virus inactivation, virus treatment, or virus removal. In some embodiments, virus removal can utilize nanofiltration and / or chromatography techniques, etc. In some embodiments, virus inactivation can utilize 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 proteases, DNA enzymes, and / or RNA enzymes to reduce the level of host cell proteins and / or nucleic acids (e.g., DNA or RNA). In some embodiments, untreated or partially treated 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, impure preparations are also referred to as starting materials or loading materials.

[0120] The purification methods described herein may include, but are not limited to, one or more of the following steps: depth filtration, viral inactivation, ion exchange chromatography (e.g., ion exchange chromatography and / or cation exchange chromatography), mixed mode chromatography, hydrophobic interaction chromatography, ultrafiltration / diafiltration, and viral removal filtration. In some embodiments, the purification methods described herein further include affinity chromatography.

[0121] In the chromatography step, when resin is filled into chromatographic column, the appropriate volume of resin used is fed back by column size (that is, the diameter of column and the height of resin), and depends on the amount of protein in the solution of application and the binding capacity of the resin used to change. However, increasing the scale of production technology and purification process to obtain the production and purification of ASA on an industrial scale, this is also within the scope of the present disclosure. Therefore, parameters (such as column size, diameter and flow velocity) can be increased to adapt to the speed and efficiency of this large-scale production. In some embodiments, the scope of column diameter is about 50-100mm; Volume range is about 100-300ml; and flow velocity is between about 40-400cm / hour (for example, between about 100cm / hour and 150cm / hour) or about 5 to 100ml.

[0122] Ultrafiltration – Capture

[0123] 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 the perfusion bioreactor. As used herein, ultrafiltration refers to filtration using membranes with filter pore sizes of 0.001 to 0.1 μm, which can be used to concentrate and desalt dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), exchange buffers, and generally fractionate. Ultrafiltration methods used in embodiments of the present invention include tangential flow ultrafiltration or cross-flow filtration.

[0124] As used herein, tangential flow filtration and ultrafiltration refer to an arrangement in which the feed stream passes parallel to the membrane surface, i.e., when a portion of the feed stream passes through the membrane (permeate), the remaining portion (retentate) is simultaneously circulated back to the feed storage tank. In some embodiments, the pore size of the tangential flow ultrafiltration filter is selected so that recombinant ASA can permeate through the filter. In other embodiments, the pore size is selected so that substantially all of the ASA is retained in the feed passing through the filter. As described elsewhere, under acidic conditions (pH≤about 5.0), ASA exists as a 480 kDa octamer and decomposes into dimers at neutral pH levels. Therefore, the pH of the feed can be adjusted in combination with the selection of an appropriate pore size to retain ASA on the filter membrane or to allow it to pass as permeate.

[0125] Can select to have the pore size of the molecular weight cut-off of at least 10kDa, at least 20kDa, at least 30kDa, at least 40kDa, at least 50kDa, at least 60kDa, at least 70kDa, at least 80kDa, at least 90kDa, at least 100kDa, at least 300kDa, at least 400kDa or at least 500kDa.For example, the filter with at least 10kDa pore size will retain most of protein with about 11kDa or larger molecular weight in feed.As another example, the filter with at least 400kDa pore size will retain most of protein higher than 400kDa molecular weight.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, can select the pore size of specific size permeate separation.For example, the filter with at least 500kDa pore size will make most of be less than the protein of 500kDa molecular weight penetrate through. In some embodiments, the permeability is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more.

[0126] The filtration area or capacity can also be optimized for the process disclosed herein. Considerations affecting the selection of the filtration area include robustness, cost, material flow rate (i.e., cross-flow velocity), transmembrane pressure, permeate flux rate, plant fit, and throughput. In some embodiments, the permeate flux is about 50–100 liters / square meter / hour ("LMH"). In some embodiments, the feed stream is about 250–600 LMH in a bracketed manner. In some embodiments, the feed stream is about 250–350 LMH in a bracketed manner. In some embodiments, the feed stream is about 175-245 LMH in a bracketed manner. In some embodiments, the feed stream is about 170–230 LMH in a bracketed manner. In some embodiments, the feed stream is about 120–160 LMH in a bracketed manner. In some embodiments, the feed stream is about 15–30 LMH in a bracketed manner. In some embodiments, the feed stream is about 11-21 LMH in a bracketed manner. In some embodiments, the filtration area is about 0.02 m 2 , about 0.14m 2 , about 0.7 or about 3.5m 2 In certain embodiments, the transmembrane pressure is about 55-60 psi. In some embodiments, the transmembrane pressure is about 15-25 psi. In some embodiments, the transmembrane pressure is about 10-20 psi. In some embodiments, the transmembrane pressure is about 5-15 psi.

[0127] Ultrafiltration filters used in embodiments of the present invention may include membrane materials known to those skilled in the art, including but not limited to polyethersulfone and stabilized cellulose. An exemplary filter cartridge used in embodiments of the present invention is a Sartorius Another exemplary filter cartridge for use in embodiments of the present invention is the Sartorius 30kD standard membrane.

[0128] Depth Filtering

[0129] Purification process as herein described can comprise one or more steps of depth filtration. Depth filter is to use porous filter medium to make particles pass through medium rather than only various filters at the surface of the medium. They comprise filter medium with graded density, which makes it possible for larger particles to be trapped near the filter surface, while smaller particles penetrate through the larger open area at the filter surface and are only trapped at the smaller openings closer to the center of the filter. Certain embodiments only adopt the depth filtration step in the upstream recovery stage (that is, before the subsequent chromatographic purification step), but other embodiments adopt the depth filter in one or more other purification stages. In some embodiments of the present invention, Cuno Zeta Plus depth filter is used.

[0130] Depth filtration may optionally be followed by 0.45 micron (± 2 μm) filtration to remove particulates and reduce bioburden in the product for downstream processing.

[0131] Virus inactivation

[0132] The purification methods described herein may include one or more steps of virus inactivation. In some embodiments, virus inactivation includes a solvent and / or a detergent. The solvent or detergent may include, for example, polysorbate 80, tributyl phosphate (TnBP), or both. Virus inactivation may include incubation in the solvent or detergent for 3-24 hours. In another embodiment, virus inactivation includes virus filtration, for example, by using a Planova TM Filter.

[0133] It is understood that these methods are expected to result in the preparation of enzymes that are substantially free of infectious viruses and can therefore be labeled as "virus-safe products." Furthermore, it is expected that the various methods can be used independently or in combination.

[0134] Viral inactivation can be achieved by adding one or more "viral inactivating agents" to the enzyme-containing solution. In some embodiments, the viral inactivation step is performed before the chromatographic purification step (i.e., before loading the impure product onto the first chromatography column) to ensure that the agent is not present in any amount or concentration in the final product when used as a pharmaceutical or when the product is used to prepare a pharmaceutical; other embodiments employ depth filters in one or more additional purification stages. For example, in some embodiments, the inventive methods according to the present invention further include a viral removal step after the final chromatography column.

[0135] The term "viral inactivating agent" is intended to mean an agent (e.g., a detergent) or method that can be used to inactivate lipid-encapsulated viruses as well as non-lipid-encapsulated viruses. The term "viral inactivating agent" is understood to include combinations of these agents and / or methods, as well as only one type of these agents or methods, whenever appropriate.

[0136] Typical viral inactivators are detergents and / or solvents, most common detergent-solvent mixtures. It is understood that the viral inactivator is optionally a mixture of one or more detergents and one or more solvents. A wide range of detergents and solvents can be used for viral inactivation. The detergent can be selected from the group consisting of nonionic and ionic detergents and is selected from substantially non-denaturing detergents. Typically, nonionic detergents are used because they facilitate subsequent 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 commercially available under the trademarks Triton X-100 and Tween 20 or Tween 80. Preferred solvents for use in viral inactivators are di- or trialkyl phosphates as described, for example, by Neurath and Horowitz in U.S. Patent No. 4,764,369. A typical solvent is tributyl phosphate (TnBP). A particularly preferred virus-inactivating agent in the practice of the present invention is Tween 80, but other agents or combinations of agents may alternatively be used. Typical agents are added so that the concentration of Tween 80 in the ASA-containing solution is in the range of approximately 0.5-4.0% by weight (preferably a concentration of approximately 1% by weight). TnBP may then be added to a final concentration of 0.3% calculated based on the new volume of the ASA-containing sample.

[0137] The virus-inactivation step is performed under conditions that inactivate the encapsulated virus to obtain a solution containing substantially virus-safe rhASA. Typically, these conditions include a temperature of 4-37°C (e.g., 19-28°C, 23-27°C, typically about 25°C) and an incubation time that has been shown to be effective by validation studies. Generally, an incubation time of 1-24 hours is sufficient, preferably 10-18 hours, for example, about 14 hours, to ensure sufficient viral inactivation. However, suitable conditions (temperature and incubation time) depend on the viral inactivator employed, the pH and protein concentration of the solution, and the lipid content.

[0138] It is contemplated that other methods of removing or inactivating viruses may also be employed to produce a virus-safe product, for example, the addition of methylene blue followed by inactivation by irradiation using ultraviolet light.

[0139] Purification process as herein described may include one or more steps of virus removal filtration. Typically, virus filtration is performed after enzyme purification by one or more steps of chromatography. In some embodiments, the virus filtration step is performed by passing an ASA solution (which is the result of the purification step) through a sterile filter and subsequently passing the sterile-filtered solution through a nanofilter. So-called "sterile filter" refers to a filter that substantially removes all microorganisms that can breed and / or cause infection. While conventional filters have a pore size of approximately 0.1 micron, the range of pore size can be between approximately 0.05 and 0.3 microns. When carrying out purification process, it is feasible to replace the virus filtration of the sample with contacting the sample with a detergent or to merge the virus filtration of the sample with contacting the sample with a detergent.

[0140] Some embodiments of the present invention include at least two steps of virus inactivation and / or filtration. For example, virus inactivation before column chromatography can be combined with virus removal after all chromatography steps are complete. Post-chromatographic column virus removal can be performed before or after one or more steps of ultrafiltration / diafiltration (UFDF) (e.g., tangential flow ultrafiltration). In a specific example, the eluate is obtained from the last step of chromatographic purification (e.g., cation exchange (SP) chromatography) and the pH of the combined eluate is adjusted to about 5.5, about 6.0, about 6.5, or about 7.0, followed by virus filtration. Thus, in certain embodiments, a single step of UFDF is performed before virus filtration (e.g., using Planova TM In other examples, the eluate obtained from the final step of chromatographic purification (e.g., cation exchange (SP) chromatography) is subjected to the first step of UFDF without pH adjustment, and then to the second step of virus filtration and UFDF.

[0141] In certain embodiments, the pH-adjusted cation exchange combined eluate is virus-filtered on a Planova 20N filter. In some embodiments, after virus filtration of the pH-adjusted cation exchange eluate, the yield relative to the input, as measured by A280 absorbance, is between about 90–100%, i.e., about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater. Thus, in some embodiments, substantially no recombinant ASA is lost during virus filtration. The virus filtration yield is important because it demonstrates that adjusting the pH to approximately 6.0 allows the octamer (which has a diameter of approximately 20 nm) of ASA to dissociate into dimeric forms. Therefore, the pore size of the virus filter can be selected to ensure that only the dimeric form is filtered (i.e., the octamer is retained by the filter or causes clogging of the virus filter). For example, a virus filter with a 20 nm pore size will retain the octamer of ASA, but not the dimeric form.

[0142] Affinity chromatography

[0143] The purification methods described herein can include one or more steps of affinity chromatography (eg, immunoaffinity chromatography, immobilized metal ion affinity chromatography, and / or immobilized ligand affinity chromatography).

[0144] In short, affinity chromatography is a chromatographic technique that depends on high interaction (for example, between receptor and ligand, antigen and antibody or enzyme and substrate). As known to those skilled in the art, the selective molecule adopted in the affinity chromatography step of purification method as herein described can be based on the various characteristics (for example, three-dimensional structure, glycosylation, etc.) of ASA produced by the available recombinant production of selective molecule. Exemplary selective molecules (or capture agents) available in the affinity chromatography step include protein A, protein G, antibodies, metal ions (for example, nickel), specific substrates, ligands or antigens. In some embodiments, the suitable selective molecule for the affinity chromatography step of the present invention utilizes anti-arylsulfatase A antibodies (for example, anti-human arylsulfatase A antibodies). Suitable anti-arylsulfatase A antibodies are commercially available or obtained by immobilization of non-human animals (for example, mice, rats, rabbits, chickens, goats, sheep, horses or other suitable animals thereof for producing antibodies against human proteins).

[0145] In general, the molecule of interest (e.g., recombinant ASA) is captured on a solid or stationary phase or medium by interaction with the selective molecule, while other undesirable molecules are not captured because they are not bound by the selective molecule. The solid medium can then be removed from the mixture, optionally washed, and the molecule of interest is then released from the capture by elution. In some embodiments, the affinity column can be eluted by changing the ionic strength by a gradient. For example, salt concentration, pH, pI, and ionic strength can be used to separate or form a separation gradient.

[0146] In some embodiments, recombinant ASA proteins can be produced with tags to facilitate purification by affinity chromatography. As 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 within a sample. For example, certain lectins specifically bind to specific sugar molecules and can be used to separate glycoproteins from non-glycosylated proteins, or to separate one glycoform from another.

[0147] Ion exchange chromatography

[0148] The purification methods described herein can include one or more steps of ion exchange chromatography (eg, ion exchange chromatography and / or cation exchange chromatography).

[0149] As 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 the attached charged groups. For example, the following matrices can be used, wherein the mentioned materials are more or less cross-linkable: agarose based (e.g. SEPHAROSE TM CL-6B, SEPHAROSE TM Fast Stream and SEPHAROSE TM High performance), cellulose-based (such as DEAE ), glucanyl (e.g. ), silica-based and synthetic polymer-based.

[0150] Ion exchange resins can be prepared according to known methods. Typically, an equilibration buffer, which allows the resin to bind to its counter ions, is passed over the ion exchange resin before a sample or composition comprising a polypeptide and one or more contaminants is loaded onto the resin. Conveniently, the equilibration buffer may also be the loading buffer, but this is not required.

[0151] In an optional embodiment of the present invention, after the polypeptide is eluted, the ion exchange resin can be regenerated using a regeneration buffer so that the column can be reused. Generally, the salt concentration and / or pH of the regeneration buffer is such that substantially all of the contaminants and the polypeptide of interest are eluted from the ion exchange resin. Generally, the regeneration buffer has a very high salt concentration for eluting contaminants and polypeptides from the ion exchange resin.

[0152] Anion exchange chromatography

[0153] 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 (e.g., anion exchange chromatography as described herein). For anion exchange resins, the charged groups covalently attached to the matrix can be, for example, diethylaminoethyl (DEAE), quaternary ammoniumethyl (QAE) and / or quaternary ammonium (Q). In some embodiments, the anion exchange resin employed is a Q Sepharose column. Using, for example, a QSEPHAROSE TM Fast Flow Column, Q SEPHAROSE TM High-performance columns, Q SEPHAROSE TM XL, CAPTO TM Q, DEAE, TOYOPEARL Q. TMAE (trimethylaminoethyl, quaternary ammonium resin), ESHMUNO TM Q. NUVIA TM Q or UNOSPHERE TMQ can be used for anion exchange chromatography. Other anion exchangers can be used within the scope of the present invention, including but not limited to quaternary ammonium resins or "Q-resins" (e.g., CAPTO™-Q, Q- 、QAE ); diethylaminoethane (DEAE) resin (e.g., DEAE- 、DEAE Benzoylated naphthol DEAE, diethylaminoethyl );AMBER Resin; Resin; Resins (e.g., AMBE IRA-67 Strong alkalinity, Weakly alkaline); cholestyramine resin; Resins (e.g. SAX-10, WAX-10 WCX–10);TSK- resins (e.g., TSKgel DEAE–NPR, TSKgel DEAE–5PW); and In some embodiments, the sample of arylsulfatase A is subjected to anion exchange chromatography at about 23°C or below. 、 The reaction is carried out at a temperature of about 18°C ​​or less or about 16°C or less (e.g., about 23°C, about 20°C, about 18°C, or about 16°C).

[0154] In certain embodiments, 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, mobile phase is included in the separation process at any given time between about 1% to about 100%, about 5% to about 95%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70% or about 40% to about 60% polar solution.

[0155] In certain embodiments, rASA is loaded at a binding capacity of about 23 AU / L resin or less, for example, about 19 AU / L resin or less, about 15 AU / L resin or less, or about 12 AU / L resin or less; for example, 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, a sample of arylsulfatase A is loaded onto an anion exchange chromatography column at a binding capacity of at least about 4.5 g / L resin (for example, 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, a sample of arylsulfatase A is loaded onto an anion exchange chromatography column at a binding capacity ranging from about 4.5-20 g / L resin (e.g., ranging from 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).

[0156] The aqueous solution comprising ASA and pollutants can be loaded onto the anion resin as mobile phase using a loading buffer having a salt concentration and / or pH that allows the polypeptide and pollutants to bind to the anion exchange resin. The resin can then be washed with one or more column volumes of loading buffer, followed by one or more column volumes of wash buffer in which the salt concentration increases. Finally, the ASA can be eluted with an elution buffer increased by salt concentration. Optionally, the elution of the enzyme can also be mediated by gradually or progressively reducing the pH. The active fraction containing ASA can be collected and merged for further purification.

[0157] In some embodiments, arylsulfatase A sample is loaded onto an anion exchange chromatography column using loading buffer. In one embodiment, loading buffer does not contain sodium chloride. In another embodiment, loading buffer contains sodium chloride. For example, the sodium chloride concentration of loading buffer is about 1mM to about 25mM, for example, about 1mM to about 10mM, about 1mM to about 5mM or about 5mM to about 10mM. In some embodiments, salt concentration is gradient (for example, linear or nonlinear gradient) in mobile phase. In some embodiments, salt concentration is constant in mobile phase. In some embodiments, salt concentration can progressively increase or decrease in mobile phase. In some embodiments, arylsulfatase A sample is loaded onto an anion exchange chromatography column at a pH of about 5 to about 9 (for example, about 6 to about 8, for example, about 7).

[0158] In some embodiments, one or more wash buffers are used to carry out the washing of anion exchange chromatography column. For example, washing anion exchange column may include two or more (for example, first and second) washing steps, and each step uses different wash buffers. In one embodiment, wash buffer does not contain sodium chloride. In another embodiment, wash buffer contains sodium chloride. For example, the sodium chloride concentration of wash buffer is about 50mM to about 200mM, for example, about 50mM to about 150mM, about 100mM to about 200mM or about 100mM to about 150mM; for example, about 80mM, about 100mM, about 120mM or about 140mM. In some embodiments, the washing of anion exchange chromatography column is carried out at a pH of about 5 to about 9 (for example, about 6 to about 8, for example, about 7).

[0159] In one embodiment, the elution buffer contains sodium phosphate. For example, the sodium phosphate concentration of the elution buffer is from about 20mM to about 50mM, for example, from about 25mM to about 45mM; for example, from about 30mM, about 35mM or about 40mM. In another embodiment, the elution buffer does not contain sodium chloride. In yet another embodiment, the elution buffer contains sodium chloride. For example, the sodium chloride concentration of the elution buffer is from about 200mM to about 300mM, for example, from about 240mM to about 280mM. In some embodiments, arylsulfatase A is eluted by an anion exchange chromatography column at a pH of about 5 to about 9 (for example, from about 6 to about 8, for example, about 7).

[0160] In some embodiments, elution of arylsulfatase A from an anion exchange chromatography column includes one or more steps of elution peak collection. For example, as determined by spectrophotometry (e.g., at 280 nM), elution peak collection starts from about 50 mAU on the rising side to about 50 mAU on the falling side, for example, about 100 mAU on the rising side to about 50 mAU on the falling side; about 200 mAU on the rising side to about 50 mAU on the falling side; about 50 mAU on the rising side to about 100 mAU on the falling side; about 50 mAU on the rising side to about 200 mAU on the falling side or about 100 mAU on the rising side to about 100 mAU on the falling side.

[0161] It will be apparent to those skilled in the art that a variety of different buffers can be used for the loading, washing, and elution steps. However, typically, the column can be equilibrated using 1–10 column washes of a buffer comprising 0.05M MES-Tris, pH 7.0. For convenience, the sample can be loaded in the buffer of the previous step of the purification process, or the sample can be loaded using a loading buffer. The column can be washed using 1–10 column volumes of a buffer for equilibration, followed by a wash buffer comprising 0.02MES-Tris, 0.12M NaCl, pH 7.0. Alternatively, the column can be equilibrated, loaded, and washed using any other equilibration, loading, and washing buffer described herein for anion exchange chromatography. The sample can be eluted in a buffer comprising 0.02MES-Tris, 0.26M NaCl, pH 7.0. Alternatively, the sample can be eluted in any other elution buffer described herein for anion exchange chromatography.

[0162] The loading buffer, wash buffer, and elution buffer described herein may include one or more buffers. For example, the buffer may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffer is between about 1 mM and about 500 mM, for example, 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; for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0163] In some embodiments, the arylsulfatase A activity productive rate is at least about 75%, for example, at least about 85%, for example, between about 85% to about 99% or between about 90% to about 99%. In some embodiments, for example, as determined by spectrophotometry (for example, at 280nM), the protein yield (AU or absorbance unit) is about 10% to 50%, for example, about 20% to about 35% or about 25% to about 30%. In some embodiments (e.g., those using a TMAE column as described below), the eluted pooled protein activity yield (AU or absorbance units) is, for example, about 70% to 400%, for example, about 80% to about 390%, or about 90% to about 350%, or about 100% to 150%, greater than at least 95%, as determined spectrophotometrically (e.g., at 280 nM). In some embodiments (e.g., those using a TMAE column as described below), the host cell protein (HCP) log reduction value (LRV) is between about 0.5 and about 1.1, for example, between about 0.6 and 0.9, or between about 0.7 and 0.8. In some embodiments (e.g., those using a TMAE column as described below), the purity is at least 75%, for example, at least 80%, at least 85%, at least 90%, or more, as determined by, for example, capillary electrophoresis-SDS PAGE. In preferred embodiments, the activity yield, HCP after anion exchange chromatography, The LRV and purity (as determined by capillary electrophoresis-SDS PAGE) are at least about 90%, at least about 0.6, and at least about 80%, respectively.

[0164] In a preferred embodiment of the present invention, use anion exchange column with high loading capacity.In certain embodiments of the present invention, post is characterised in that scope is between about 3-20g / L (that is, about 5-15g / L, about 10-15g / L, about 10-20g / L) loading.In some embodiments, loading capacity is significantly greater than 4.3g / L (for example, for or greater than about 10g / L, 12.5g / L, 15g / L, 17.5g / L or 20g / L).In certain embodiments, the binding capacity of resin is between about 75-100AU / L (for example, about 75AU / L, about 80AU / L, about 85AU / L, about 90AU / L, about 95AU / L).In certain embodiments, loading capacity is greater than about 80AU / L.In some embodiments, high loading capacity post is TMAE post.In specific embodiment, post 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.

[0165] In a specific embodiment of the invention, the TMAE column is pre-equilibrated with a buffer comprising about 20 mM MES-Tris and 1000 mM NaCl at a pH of 7.0. In certain embodiments, the column is equilibrated with a buffer comprising 50 mM MES-Tris at a pH of 7.0. In some embodiments, the loading flow rate of the TMAE column is about 75-125 cm / h (i.e., about 75-115 cm / h, about 75-110 cm / h, about 75-105 cm / h, about 75-100 cm / h, about 85-115 cm / h, about 85-110 cm / h, about 85-105 cm / h, about 85-100 cm / h, about 95-115 cm / h, about 95-110 cm / h, about 95-105 cm / h, about 95-100 cm / h, about 100-120 cm / h, about 100-115 cm / h, about 100-110 cm / h, about 100 cm / h). Loading conditions can be optimized and assessed by A280 absorbance as described herein.

[0166] In certain embodiments utilizing a TMAE column (e.g., a Fractogel TMAE column), very little product is lost in the flow-through during loading, even at loading capacities greater than 15 g / L. Increasing loading capacity while reducing flow-through losses is a significant improvement in a purification process. In certain embodiments of the invention, the amount of product lost in the flow-through is less than 30% of loading (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%).

[0167] In some embodiments, after loading, TMAE posts are washed at least once. In specific embodiments, posts are washed twice. The first and second wash buffers can include the sodium chloride of optimization level. In some embodiments, the amount of sodium chloride as the first or second wash buffer is between about 50-150mM (e.g., about 50-140mM, about 50-130mM, about 50-120mM, about 50-110mM, about 50-100mM, about 50-90mM, about 50-80mM, about 80-150mM, about 80-140mM, about 80-130mM, about 80-120mM, about 80-110mM, about 80-100mM, about 80-90mM, about 80mM or about 120mM). In some embodiments, the first wash buffer includes 50mM MES-Tris at pH 7.0. In some embodiments, the second wash buffer comprises 20 mM MES-Tris, 100 mM NaCl at pH 7.0. Further optimization of wash conditions, particularly the second wash conditions, is encompassed within the embodiments of the present invention. For example, increasing the salt concentration of the second wash can improve host cell protein (HCP) log reduction value (LRV) and overall purity, but reduce activity and A280 yield. As described herein, the elution conditions described below must be used to balance specific wash conditions to provide an optimized combination of purity, activity, and yield.

[0168] In an embodiment of the present invention, an elution buffer is used to elute the recombinant ASA bound to the TMAE column. In some embodiments, the amount of sodium chloride in the elution buffer is optimized. In a specific embodiment, the amount of sodium chloride in the elution buffer is between about 150-300 mM (e.g., about 150-290 mM, about 150-280 mM, about 150-270 mM, about 150-260 mM, about 150-250 mM, about 150-240 mM, about 150-230 mM, about 150-220 mM, about 150-210 mM, about 170-290 mM, about 170-280 mM, about 170-270 mM, about 170 In some embodiments, the elution buffer comprises 50 mM MES-Tris and 1 M NaCl at a pH of 7.0. In some embodiments, the A280 yield after elution is greater than 60% loading (e.g., about 60%, about 70%, about 80% or more). Further optimization of elution conditions is encompassed within the embodiments of the present invention. For example, increasing the elution salt concentration (i.e., conductivity) provides better yields, but results in poorer purity and HCP removal. And as shown above, elution conditions must be used to balance specific wash conditions to provide an optimized combination of purity, activity, and yield.

[0169] Cation exchange chromatography

[0170] In some embodiments, the method further comprises subjecting the supply of arylsulfatase A to cation exchange chromatography, e.g., sulfopropyl (SP) cation exchange chromatography as described herein. In some embodiments, prior to cation exchange chromatography, the sample of arylsulfatase A is subjected to anion exchange chromatography. In typical embodiments, the cation exchange chromatography comprises sulfopropyl (SP) cation exchange chromatography, but other cation chromatography membranes or resins may be used, MUSTANG TM S-film, S-SEPHAROSE TM Resin or blue SEPHAROSE TMResin. In some embodiments, the method further comprises concentrating and / or filtering a sample of arylsulfatase A, for example, by ultrafiltration and / or diafiltration (e.g., by tangential flow ultrafiltration). Cation exchange chromatography can be performed, for example, at an optimized temperature as described herein, thereby increasing target binding and / or reducing impurity binding. For example, cation exchange chromatography can be performed at a temperature of about 23° C., 18° C., 16° C., or lower.

[0171] In one embodiment, the cation exchange chromatography comprises sulfopropyl (SP) cation exchange chromatography. In another embodiment, the cation exchange chromatography is a grinding step. Cation exchange chromatography (e.g., sulfopropyl (SP) cation exchange chromatography) can be performed using, for example, one or more of the following: SP-650, SP-550, SP-3PW, SP-5PW、SP SEPHAROSE TM Fast Flow Column, SP SEPHAROSE TM High-performance column, SP SEPHAROSE TM XL, SARTO S membrane, HS50、UNOSPHERE TM S and MACROCAP TM S.

[0172] The aqueous solution that comprises arylsulfatase A and pollutant can be loaded on the cationic resin using loading buffer, and this loading buffer has salt concentration and / or pH that makes polypeptide and pollutant combine cation exchange resin.Then use the equilibrium buffer or loading buffer of one or more column volumes but wash resin, then optionally be the washing buffer of one or more column volumes that salt concentration increases wherein.Finally, elutable arylsulfatase A in elution buffer.The part that contains arylsulfatase A activity can be collected, and merged for further purification.

[0173] In typical embodiments, the NaCl concentration and / or pH of the loading buffer, wash buffer, and / or elution buffer can be optimized, for example, as described herein, to increase 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 loading buffer has a pH of 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.

[0174] In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography comprises: loading the sample of arylsulfatase A onto a cation chromatography column (e.g., a sulfopropyl (SP) cation exchange column); washing the cation exchange chromatography column; and eluting arylsulfatase A from the column. In some embodiments, the column can be equilibrated using more than 3 (e.g., 5 to 10) column volumes of 0.01 M NaAc, 0.01 M NaCl, 0.03 M acetic acid, pH 4.2.

[0175] In some embodiments, sample can be loaded in the buffer of step before purification process, or use loading buffer to load sample.In one embodiment, loading buffer contains sodium chloride.For example, the sodium chloride concentration of loading buffer is about 1mM to about 25mM, for example, and about 5mM to about 20mM, for example, about 5mM, about 10mM, about 15mM or about 20mM.In another embodiment, loading buffer contains sodium acetate.For example, the sodium acetate concentration of loading buffer is about 10mM to about 100mM, for example, about 20mM, about 40mM or about 60mM.In some embodiments, at about 3.0 to about 6.0 pH (for example, about 4.0 to about 5.0, for example, about 4.0, about 4.3 or about 4.5), carry out the sample of arylsulfatase A being loaded on cation exchange chromatography column. In some embodiments, a sample of arylsulfatase A is loaded onto a cation exchange chromatography column at a binding capacity of about 15 AU / L resin or less (e.g., about 14 AU / L resin or less or about 12 AU / L resin or less; e.g., between about 10 AU / L resin to about 14 AU / L resin or between about 10 AU / L resin to about 12 AU / L resin).

[0176] In some embodiments, one or more wash buffers are used to wash the cation exchange chromatography column. For example, washing the cation exchange column may include two or more (for example, first and second) washing steps, and each step uses different wash buffers. The buffer for balance can be used to wash the column using 1-10 column volumes. Alternatively, any other balance, load and wash buffer described herein for cation exchange chromatography can be balanced, loaded and washed. In one embodiment, wash buffer contains sodium chloride. For example, the sodium chloride concentration of wash buffer is about 1mM to about 25mM, for example, about 5mM to about 20mM or about 10mM to about 15mM; for example, about 5mM, about 10mM, about 15mM or about 20mM. In another embodiment, wash buffer contains sodium acetate. For example, the sodium acetate concentration of loading buffer is about 10mM to about 100mM, for example, about 20mM, about 40mM or about 60mM. In some embodiments, washing of the cation exchange chromatography column is performed at a pH of about 3.0 to about 6.0 (eg, about 4.0 to about 5.0, eg, about 4.0, about 4.3, or about 4.5).

[0177] In some embodiments, elution buffer is used to elute arylsulfatase A from a cation exchange chromatography column. In one embodiment, the elution buffer comprises sodium chloride. For example, the sodium chloride concentration of the elution buffer is from about 25mM to about 75mM, for example, from about 45mM to about 60mM; for example, from about 45mM, about 50mM, about 55mM or about 55mM. In some embodiments, elution of arylsulfatase A from a cation exchange chromatography column is performed at a pH of from about 3.0 to about 6.0 (for example, from about 4.0 to about 5.0; for example, from about 4.0, about 4.3 or about 4.5). Therefore, as a specific example, the sample is eluted in a buffer comprising 0.02M NaAc, 0.05MNaCl, pH 4.5. Alternatively, the sample can be eluted in any other elution buffer described herein for cation exchange chromatography.

[0178] In some embodiments, elution of arylsulfatase A from a cation exchange chromatography column includes one or more steps of elution peak collection. For example, as determined by spectrophotometry (e.g., at 280 nM), for example, elution peak collection starts from about 50 mAU at the rising side to about 50 mAU at the falling side, for example, from about 100 mAU at the rising side to about 50 mAU at the falling side; from about 200 mAU at the rising side to about 50 mAU at the falling side; from about 50 mAU at the rising side to about 100 mAU at the falling side; from about 50 mAU at the rising side to about 200 mAU at the falling side or from about 100 mAU at the rising side to about 100 mAU at the falling side. The collected eluate peaks can be combined.

[0179] The loading buffer, wash buffer, and elution buffer described herein may include one or more buffers. For example, the buffer may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffer is between about 1 mM and about 500 mM, for example, 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; for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0180] In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography is performed at a temperature of about 23° C. or less, about 18° C. or less, or about 16° C. or less (e.g., about 23° C., about 20° C., about 18° C., or about 16° C.). In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography is performed at between about 23° C. and about 16° C. (e.g., about 23° C., about 20° C., about 18° C., or about 16° C.), and loading the sample of arylsulfatase A onto a cation exchange chromatography column is performed at a pH of between about 4.5 and about 4.3 (e.g., about 4.5, about 4.4, or about 4.3). In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography is performed at about 23° C., and loading the sample of arylsulfatase A onto a cation exchange chromatography column is performed at a pH of about 4.5. In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography is performed at about 23° C., and loading the sample of arylsulfatase A onto a cation exchange chromatography column is performed at a pH of about 4.3. In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography is performed at about 18° C., and loading the sample of arylsulfatase A onto a cation exchange chromatography column is performed at a pH of about 4.5. In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography is performed at about 18° C., and loading the sample of arylsulfatase A onto a cation exchange chromatography column is performed at a pH of about 4.3.

[0181] The productive rate after cation exchange chromatography can be changed. In some embodiments, the arylsulfatase A activity productive rate is at least about 75%, for example, at least about 80%, for example, between about 80% and about 105%. In some embodiments, for example, as determined by spectrophotometry (for example, at 280nm), the protein productive rate (AU or absorbance unit) is about 65% to 100%, for example, about 70% to about 95%.

[0182] In some embodiments, the purity and activity after cation exchange chromatography are greatly improved. In some embodiments, the host cell protein (HCP) logarithmic reduction value (LRV) is between about 1.0 to about 2.5, for example, between about 1.5 to about 2.0 or between about 1.7 to about 1.9. For example, as measured by methods described herein, the specific activity of purified arylsulfatase A can be at least about 50U / mg to about 140U / mg, for example, at least about 70U / mg, at least about 90U / mg, at least about 100U / mg or at least about 120U / 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 determined by, for example, one or more of the following methods: host cell protein (HCP) Western blotting, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse phase HPLC, and size exclusion HPLC. In certain embodiments, reducing the salt concentration of the loading buffer and lowering its pH increase the binding of ASA to the cation exchange column without affecting the binding of impurities. In other words, optimizing the balance of salt concentration and pH as shown above after cation exchange chromatography can increase yield without adversely affecting purity.

[0183] In some embodiments, the pH of the cation exchange combined eluate 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 comprising 0.25M sodium phosphate, 1.33M sodium chloride, 0.34M sodium citrate, pH 7.0. In certain embodiments, the pH-adjusted SP combined eluate is virus filtered on a Planova 20N filter. In some embodiments, after virus filtration of the pH-adjusted cation exchange eluate, the yield relative to the input, as measured by A280 absorbance, is between about 90–100%, i.e., about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater. The virus filtration yield is important because it demonstrates that pH adjustment to about 6.0 allows the octamer of ASA (which has a diameter of about 20 nm) to be decomposed into a dimeric form. Therefore, the pore size of the virus filter can be selected to ensure that only the dimeric form is filtered (i.e., the octameric form may be retained by the filter or cause the virus filter to clog). For example, a virus filter with a pore size of 20 nm will retain the octameric form of ASA, but not the dimeric form.

[0184] Mixed-mode chromatography

[0185] 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 multiple separation modes, typically liquid chromatography, are applied to resolve a mixture of different molecules. For example, a mixed-mode separation may include a combination of phases having both ion exchange and reversed-phase properties. These stationary phases, which possess more than one type of interaction, are available from a variety of column manufacturers.

[0186] In one aspect, the disclosure features a method of purifying arylsulfatase A from a sample, where the method includes, for example, providing a sample of arylsulfatase A (e.g., recombinant arylsulfatase A) and subjecting the sample of arylsulfatase A to mixed-mode chromatography, e.g., mixed-mode chromatography described herein, e.g., a method including ceramic hydroxyapatite (HA) chromatography (e.g., hydroxyapatite type I or type II chromatography). In some embodiments, the mixed-mode chromatography is performed using one or more of: CHT TM Ceramic hydroxyapatite type I medium, CHT TM Ceramic hydroxyapatite type II medium, BIO- HT Hydroxyapatite and BIO- HTP hydroxyapatite.

[0187] In some embodiments, subjecting the sample of arylsulfatase A to mixed-mode chromatography comprises: loading the sample of arylsulfatase A onto a mixed-mode chromatography column (e.g., HA chromatography); washing the mixed-mode chromatography column; and eluting arylsulfatase A from the column. In some embodiments, subjecting the sample of arylsulfatase A to mixed-mode exchange chromatography is performed at a temperature of about 23° C. or less, about 18° C. or less, or about 16° C. or less (e.g., about 23° C., about 20° C., about 18° C., or about 16° C.).

[0188] In some embodiments, a sample of arylsulfatase A is loaded onto a mixed mode chromatography column 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, for example, from about 1 mM to about 5 mM, from about 5 mM to about 10 mM; for example, from 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, for example, from about 200 to about 300 mM, for example, from about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0189] In some embodiments, loading the sample of arylsulfatase A onto the mixed mode chromatography column is performed at a pH of about 5 to about 9 (eg, about 6 to about 8, eg, about 7).

[0190] In some embodiments, mixed-mode chromatography includes ceramic hydroxyapatite (HA) chromatography. Hydroxyapatite (HAP) generally refers to a crystalline form of calcium phosphate. The mechanism of action of HAP involves nonspecific interactions between negatively charged protein carboxyl groups on the resin and positively charged calcium ions, and between positively charged protein amino groups on the resin and negatively charged phosphate ions. Basic or acidic proteins can be selectively absorbed onto the column by adjusting the pH of the buffer; elution can be achieved by varying the salt concentration of the buffer. Furthermore, it is apparent that a variety of buffer compositions and combinations of buffers can be employed. However, typically, the column is equilibrated using 1–10 column washes of a buffer comprising 0.001 M NaPO₄, 0.02 M MES-Tris, 0.26 M NaCl, pH 7.0. For convenience, the sample can be loaded in the buffer from a previous step in the purification process or using a loading buffer. The column can be washed using 1-10 column volumes of a buffer for equilibration, followed by a wash buffer comprising 0.005 M NaPO4, 0.02 M MES-Tris, 0.26 M NaCl, pH 7.0. Alternatively, the column can be equilibrated, loaded, and washed using any other equilibration, loading, and wash buffers described herein for mixed-mode chromatography. The sample can be eluted in a buffer comprising 0.04 M NaPO4, pH 7.0. Optionally, the column can be eluted using 1-10 column volumes of 0.4 M NaPO4, pH 12. Alternatively, the sample can be eluted in any other elution buffer described herein for mixed-mode chromatography.

[0191] In some embodiments, washing the mixed mode chromatography column is performed using one or more wash buffers.For example, washing the mixed mode chromatography column can include two or more (eg, first and second) wash steps, each step using a different wash buffer.

[0192] In one embodiment, the wash buffer contains sodium phosphate. For example, the wash buffer has a sodium phosphate concentration of about 1 mM to about 10 mM, for example, about 1 mM to about 5 mM, about 5 mM to about 10 mM; for example, about 1 mM, about 5 mM, or about 10 mM. In another embodiment, the wash buffer contains sodium chloride. For example, the wash buffer has a sodium chloride concentration of about 50 mM to about 600 mM, for example, about 100 mM to about 500 mM, or about 200 to about 400 mM; for example, about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0193] In some embodiments, washing of the mixed-mode chromatography column is performed at a pH of about 5 to about 9 (eg, about 6 to about 8, eg, about 7).

[0194] In some embodiments, elution of arylsulfatase A from a mixed-mode chromatography column is performed at a pH of about 5 to about 9 (e.g., about 6 to about 8, e.g., about 7). In some embodiments, elution of arylsulfatase A from a mixed-mode chromatography column comprises one or more steps of elution peak collection. For example, as determined by spectrophotometry (e.g., at 280 nM), for example, elution peak collection starts from about 50 mAU on the rising side to about 50 mAU on the falling side, e.g., about 100 mAU on the rising side to about 50 mAU on the falling side; about 200 mAU on the rising side to about 50 mAU on the falling side; about 50 mAU on the rising side to about 100 mAU on the falling side; about 50 mAU on the rising side to about 200 mAU on the falling side or about 100 mAU on the rising side to about 100 mAU on the falling side.

[0195] The loading buffer, wash buffer, and elution buffer described herein may include one or more buffers. For example, the buffer may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffer is between about 1 mM and about 500 mM, for example, 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; for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0196] In some embodiments, purification of ASA by mixed mode chromatography is performed after purification by ion exchange chromatography (e.g., anion exchange chromatography). However, it is contemplated that these steps can be performed in the reverse order.

[0197] The productive rate after mixed mode chromatography can change.In some embodiments, arylsulfatase A activity productive rate is at least about 80%, for example, at least about 90%, for example, between about 80% to about 115%.In some embodiments, for example, as determined by spectrophotometry (for example, under 280nm), protein productive rate (AU or absorbance unit) is about 30% to 80%, for example, about 35% to about 75% or about 50% to about 70%.

[0198] Purity is greatly improved after mixed mode chromatography. In some embodiments, for example, as measured by methods described herein, the specific activity of the purified arylsulfatase A is at least about 50 U / mg to about 140 U / mg, for example, 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 determined by, for example, one or more of the following methods: host cell protein (HCP) Western blotting, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reversed-phase HPLC, and size exclusion HPLC. In some embodiments, the host cell protein (HCP) log reduction value (LRV) is between about 0.3 to about 0.6, for example, between about 0.4 to 0.5.

[0199] Hydrophobic Interaction Chromatography (HIC)

[0200] The purification methods described herein may comprise subjecting a sample of arylsulfatase A to hydrophobic interaction chromatography (HIC). In one embodiment, hydrophobic interaction chromatography comprises phenyl chromatography. In other embodiments, hydrophobic interaction chromatography comprises butyl chromatography or octyl chromatography. In some embodiments, subjecting a sample of arylsulfatase A to HIC is performed at a temperature of about 23°C or less, about 18°C ​​or less, or about 16°C or less (e.g., about 23°C, about 20°C, about 18°C, or about 16°C). In some embodiments, a sample of arylsulfatase A is subjected to mixed mode chromatography prior to HIC.

[0201] Hydrophobic interaction chromatography exploits the adsorption of a given molecule for polar or nonpolar environments, and depending on the protein, this tendency is dictated by the hydrophobicity or hydrophilicity of the residues on the exposed external surface of the protein. Proteins are therefore fractionated according to their varying degrees of adsorption to hydrophobic matrices (typically inert supports with alkyl linker arms of 2–18 carbon chains). The stationary phase consists of a hydrophilic polymer backbone (e.g., cross-linked Sepharose) attached to the hydrophilic polymer backbone. TM , dextran or agarose) are composed of small non-polar groups (butyl, octyl or phenyl). Therefore, HIC columns are usually butyl SEPHAROSE TM Column or Phenyl SEPHAROSE TM Columns, most commonly phenyl SEPHAROSE TM In some embodiments, hydrophobic interaction chromatography comprises phenyl chromatography using one or more of the following: Phenyl SEPHAROSE TMHigh performance, phenyl SEPHAROSE TM 6 Fast Flow (low resolution) or Phenyl SEPHAROSE TM 6 Fast streams (high resolution).

[0202] In some embodiments, subjecting a sample of arylsulfatase A to hydrophobic interaction chromatography comprises: loading the sample of arylsulfatase A onto a HIC column; washing the HIC column; and eluting the arylsulfatase A from the column. Loading, washing, and eluting in HIC are essentially in accordance with the same principles of ion exchange chromatography as described above, but conditions that are almost opposite to those used in ion exchange chromatography are generally applied. Therefore, the HIC process comprises using a high salt loading buffer that disassembles the protein to expose hydrophobic sites. The protein is retained by a hydrophobic ligand on the column, and the protein is exposed to a buffer comprising a decreasing salt concentration gradient. When the salt concentration decreases, the protein returns to its native conformation and is ultimately eluted from the column. Alternatively, PEG can be used to elute the protein.

[0203] In some embodiments, loading buffer is used to carry out the sample of arylsulfatase A being loaded onto the HIC post. In one embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration of the loading buffer is about 0.5M to about 2.5M, for example, about 1M or about 1.5M. In another embodiment, the loading buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is about 10mM to about 100mM, for example, about 25mM, about 50mM or about 75mM. In some embodiments, loading buffer is used to carry out the sample of arylsulfatase A being loaded onto the HIC post at a pH of about 5 to about 7 (for example, about 5.5 to about 6.5, for example, about 5.5, about 6.0 or about 6.5). In some embodiments, a sample of arylsulfatase A is loaded onto a HIC column at a binding capacity of about 12 AU / L resin or less (e.g., about 10 AU / L resin or less, about 9 AU / L resin or less, about 7 AU / L resin or less, or about 5 AU / L resin or less; e.g., between about 5 AU / L resin to about 9 AU / L resin or between about 5 AU / L resin to about 7 AU / L resin).

[0204] Using Phenyl SEPHAROSE as the Solid Phase in HIC TM Commonly used in this disclosure. Furthermore, it will be apparent that a wide variety of possibilities exist with respect to the precise conditions and buffers and buffer combinations 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 of a previous step in the purification process, or the sample can be loaded using a loading buffer.

[0205] In some embodiments, one or more wash buffers are used to wash the HIC column. For example, washing the HIC column may include two or more (e.g., first and second) washing steps, each step using different wash buffers. In some embodiments, the wash buffer contains sodium chloride. For example, the sodium chloride concentration of the wash buffer is from about 100mM to about 1.5M, for example, from about 250mM to about 1M; for example, from about 250mM, about 500mM, about 750mM, or about 1M. In another embodiment, the wash buffer contains sodium phosphate. For example, the sodium phosphate concentration of the loading buffer is from about 10mM to about 100mM, for example, from about 25mM, about 50mM, or about 75mM. In some embodiments, washing the HIC column is performed at a pH of about 5 to about 7 (e.g., from about 5.5 to about 6.5, for example, from about 5.5, about 6.0, or about 6.5). For example, washing can be performed using 1-2 column washes of equilibration buffer, followed by 1-5 column volumes of 0.02 M MES, 0.05 M NaPO4, 0.5 M NaCl, pH 5.5. Alternatively, the column can be equilibrated, loaded, and washed using any other equilibration, loading, and wash buffers described herein for HIC.

[0206] In some embodiments, elution of arylsulfatase A from a HIC column is performed using an elution buffer. In some embodiments, the elution buffer comprises sodium chloride. For example, the sodium chloride concentration of the elution buffer is from about 30 mM to about 100 mM, for example, from about 45 mM to about 85 mM; for example, from about 50 mM, from about 60 mM, from about 70 mM, or from about 80 mM. In some embodiments, elution of arylsulfatase A from a HIC column is performed at a pH of from about 5 to about 9 (e.g., from about 6 to about 8, for example, from about 7). For example, elution of arylsulfatase A can be performed 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.

[0207] In some embodiments, elution of arylsulfatase A from the HIC column includes one or more steps of elution peak collection. For example, as determined by spectrophotometry (e.g., at 280 nM), elution peak collection starts from about 50 mAU on the rising side to about 50 mAU on the falling side, for example, about 100 mAU on the rising side to about 50 mAU on the falling side; about 200 mAU on the rising side to about 50 mAU on the falling side; about 50 mAU on the rising side to about 100 mAU on the falling side; about 50 mAU on the rising side to about 200 mAU on the falling side, or about 100 mAU on the rising side to about 100 mAU on the falling side.

[0208] In some embodiments, purification of arylsulfatase A by HIC is performed after purification by ion exchange chromatography (eg, anion exchange chromatography) and / or mixed mode chromatography. However, it is contemplated that these steps can be performed in the reverse order.

[0209] The loading buffer, wash buffer, and elution buffer described herein may include one or more buffers. For example, the buffer may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffer is between about 1 mM and about 500 mM, for example, 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; for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0210] The yield after HIC can vary. In some embodiments, the arylsulfatase A activity yield is at least about 60%, for example, at least about 70%, for example, between about 70% and about 100%. In some embodiments, for example, as determined by spectrophotometry (e.g., at 280 nm), the protein yield (AU or absorbance units) is about 45% to 100%, for example, about 50% to about 95% or about 55% to about 90%.

[0211] Purity is greatly improved after HIC. In some embodiments, for example, as determined by the methods described herein, the specific activity of the purified arylsulfatase A is at least about 50 U / mg to about 140 U / mg, for example, 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.

[0212] 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 determined, for example, by one or more of the following methods: host cell protein (HCP) Western blotting, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reversed-phase HPLC, and size exclusion HPLC. In some embodiments, the host cell protein (HCP) log reduction value (LRV) is between about 0.6 and about 1.2, for example, between about 0.7 and 0.95.

[0213] Ultrafiltration / diafiltration

[0214] Purification methods as described herein may include one or more steps of downstream ultrafiltration and / or diafiltration.In some embodiments, the method further comprises, for example, concentrating and / or filtering the sample of arylsulfatase A by ultrafiltration and / or diafiltration (e.g., by tangential flow ultrafiltration).

[0215] Ultrafiltration refers to a membrane separation process driven by a pressure gradient, in which the membrane separates liquid components based on their solvated size and structure. Diafiltration is a specific type of ultrafiltration process in which the retentate is diluted with water before ultrafiltration, thereby reducing the concentration of soluble permeate components and further increasing the concentration of retained components. Ultrafiltration and diafiltration are often combined into an ultrafiltration / diafiltration (UFDF) purification step.

[0216] Embodiments of the present invention utilize at least one, at least two, at least three or more downstream UFDF purification steps. One or more diafiltrations can occur in a UFDF step (e.g., UFDFDF). In some embodiments, as determined by spectrophotometry (e.g., at 280nM), compared with the amount before the purification step, after downstream UFDF, protein yield (AU or absorbance unit) is about 90% to 105%, e.g., about 95% to about 100%, e.g., about 97% to about 99%. In some embodiments, there is substantially no loss protein in UFDF.

[0217] In some embodiments of the invention, the downstream UFDF yields rASA that is at least about 95%, at least about 97%, at least about 98%, at least about 99%, or more pure as determined by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and / or reverse phase high performance liquid chromatography (RP-HPLC). In some embodiments, the arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. The purity of the arylsulfatase A can be determined, for example, by one or more of the following methods: host cell protein (HCP) Western blotting, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse phase HPLC, and size exclusion HPLC. For example, the specific activity of rASA as determined by a sulfatase release assay as described below is at least about 60 U / mg to about 100 U / mg, e.g., at least about 65 U / mg, at least about 90 U / mg, at least about 70 U / mg, or at least about 90 U / mg.

[0218] In some embodiments, arylsulfatase A is purified by separation from contaminants according to their size by tangential flow filtration in an acidic environment. At low pH, arylsulfatase A forms an octamer with a theoretical molecular weight of 480 kDa and is thus 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).

[0219] In typical embodiments, the diafiltration buffer comprises 0.01 M sodium phosphate-citrate, 0.137 M NaCl, pH 6.0.

[0220] In some embodiments, when the starting material for the process is a suspension of arylsulfatase A eluted from a chromatography column in a previous step of the process, the pH of the suspension is adjusted by adding 0.2–1 M sodium acetate, pH 4.5. Diafiltration is then performed through 1–10 buffer volumes of sodium acetate, pH 4–5, in a manner well known to those skilled in the art. Filtration can be performed using a variety of different filter types with nominal cutoff values ​​ranging from 20–450 kDa, but filters with a cutoff value range of 100–300 kDa are typically used. For further processing of the solution containing arylsulfatase A, the pH is adjusted to a value in the range of 7 to 8 by adding Tris-base to a final concentration of approximately 20–50 mM.

[0221] As an alternative to acidic tangential flow filtration as described above, separation of ASA from contaminants can be achieved by acidic gel filtration using essentially the same conditions and buffer composition. Filtration is performed at low pH through a gel filtration column that has been equilibrated with a solution (e.g., a 0.2-0.9 M solution of sodium acetate at pH 4-5) at low pH. Alternatively, tangential flow filtration through a 20-50 kDa filter prior to gel filtration can concentrate the solution of arylsulfatase A. The concentration range can vary considerably, such that arylsulfatase A can be concentrated from about 0.1 mg / ml to about 50 mg / ml, preferably to about 5 mg / ml.

[0222] In some embodiments, the sample pool is concentrated by Biomax A-screen, 30 kDa. Diafiltration is performed through 3-5 column washes of 20 mM sodium acetate, pH 5.4-5.7.

[0223] Characterization of purified ASA protein

[0224] Purified recombinant ASA protein can be characterized using a variety of methods.

[0225] purity

[0226] The purity of the purified recombinant ASA protein is typically determined by the levels of various impurities (e.g., host cell proteins or host cell DNA) present in the final product. For example, host cell protein (HCP) levels can be determined 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 some embodiments, the purified recombinant ASA protein contains less than about 150 pg / mg, 140 pg / mg, 130 pg / mg, 120 pg / mg, 110 pg / mg, 100 pg / mg, 90 pg / mg, 80 pg / mg, 70 pg / mg, 60 pg / mg, 50 pg / mg, 40 pg / mg, 30 pg / mg, 20 pg / mg, or 10 pg / mg of host cell DNA.

[0227] 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 SDS-PAGE using Coomassie Brilliant Blue staining. In some embodiments, the purified recombinant ASA protein has no HCP bands with an intensity greater than 15 kDa of the assay control and 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%, 0.5%, or 1.0% of the assay control when subjected to SDS-PAGE using Western blotting for HCP. 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 an assay control when subjected to SDS-PAGE using silver stain. In some embodiments, the host cell protein (HCP) log reduction value (LRV) is between about 0.3 and about 0.6, for example, between about 0.4 and 0.5. Various assay controls can be used, particularly those acceptable to regulatory agencies such as the FDA.

[0228] 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 liquid 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 increase resolution include gradient conditions, organic modifiers, counterions, temperature, column pore size and particle size, composition, and flow rate. As known to those skilled in the art, the level of purity can be discerned by the percentage of the main peak. For example, purity can be determined by integrating the observed main peak and side peaks and calculating the percentage of the main peak in the total area. In some embodiments of the present invention, the purity of ASA purified by the methods disclosed herein and as determined by the percentage of the main peak by 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 as determined by the percentage of the main peak by RP-HPLC is greater than or equal to 98% (ie, about 98%, about 99% or higher).

[0229] Specific activity

[0230] Purified recombinant ASA protein can also be characterized by evaluating its function and / or biological activity. The enzymatic activity of a recombinant ASA composition can be determined using methods known in the art. Typically, the method involves detecting the removal of sulfate from a synthetic substrate, a method known as a sulfate release assay. One example of an enzyme activity assay involves the use of ion chromatography. This method quantifies the amount of sulfate ions released by the enzyme in the 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 a functional equivalent thereof. Typically, the released sulfate ions are analyzed by ion chromatography using a conductivity detector. In this example, the result can be expressed as U / mg of protein, where 1 unit represents the amount of enzyme required to release 1 micromole of sulfate ions from the substrate per hour. In some embodiments, the purified recombinant ASA has a specific activity of at least about 50 U / mg, 60 U / mg, 70 U / mg, 80 U / mg, 90 U / mg, 100 U / mg, 110 U / mg, 120 U / mg, 130 U / mg, 140 U / mg. In some embodiments, the purified recombinant ASA has a specific activity in the range of about 50-200 U / mg (e.g., about 50-190 U / mg, 50-180 U / mg, 50-170 U / mg, 50-160 U / mg, 50-150 U / mg, 50-140 U / mg, 50-130 U / mg, 50-120 U / mg, 50-110 U / mg, 50-100 U / mg, 60-200 U / mg). U / 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 / or 130 U / mg).

[0231] In another example, the enzymatic activity of a recombinant ASA composition can be determined by measuring the removal of sulfate from a 4-methylumbelliferyl-sulfate (4-MUF-sulfate) substrate to form fluorescent methylumbelliferyl. In this example, using a 4-MUF standard, the fluorescent signal generated by the test sample can be used to calculate the enzyme activity (in mU / mL). One milliunit of activity is defined as the amount of enzyme required to convert 1 nanomole of 4-MUF-sulfate to 4-MUF in 1 minute at 37°C. Specific activity can then be calculated by dividing the enzyme activity by the protein concentration.

[0232] In some embodiments, activity is determined by hydrolysis of a synthetic chromogenic substrate, p-nitrocatechol sulfate (pNCS), with the end product p-nitrocatechol (pNC) absorbing light at 515 nm. The following equation can be used to calculate enzyme activity: pNCS hydrolyzed in moles / min x ml (=units / ml):

[0233] V total (ml) ×ΔA=unit / ml(1)

[0234] εM / 1000×Vsample (ml)×incubation time (min), where:

[0235] ΔA = absorbance of sample – absorbance of blank

[0236] Vtotal (ml) = total reaction volume in ml (0.15 ml in this case)

[0237] Vsample (ml) = added sample in ml (0.05 ml in this case)

[0238] εM = molar absorptivity of the product pNC, which in this case is 12400 M-1 cm-1.

[0239] Equation 1 can be simplified to:

[0240] ΔA×(0.15 / (12400 / 1000×0.05x 30))=

[0241] X μmol / (minute × ml) (=unit / ml) (1)

[0242] To calculate the specific activity in μmol pNC consumed / (min×mg) (=mononucleotides / mg), divide equation 1 by the protein concentration of the sample:

[0243] Equation 1 / protein concentration (mg / ml) = Y μmol / (minute × mg) = unit / mg (2)

[0244] In any example, 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, protein concentration is determined by ultraviolet absorbance assays. These absorbance assays are typically performed at a wavelength of about 280 nm (A280).

[0245] In some embodiments, the purified recombinant ASA has a kinase activity on 4-methylumbelliferone substrate in the range of about 1.0×10 3 mU / mg to 100.0×10 3 mU / mg, about 1.0×10 3 mU / mg to 50.0×10 3 mU / mg, about 1.0×10 3 mU / mg to 40.0×10 3 mU / mg, about 1.0×10 3 mU / mg to 30.0×10 3 mU / mg, about 1.0×10 3 mU / mg to 20.0×10 3 mU / mg, about 1.0×10 3 mU / mg to 10.0×10 3 mU / mg, about 4.0×10 3 mU / mg to 8.0×10 3 mU / mg, about 4.0×10 3 mU / mg to 10.0×10 3 mU / mg, about 4.5×10 3mU / mg to 10.0×10 3 mU / mg, about 5.0×10 3 mU / mg to 10.0×10 3 mU / mg, about 5.5×10 3 mU / mg to 15.0×10 3 mU / mg or approximately 4.0×10 3 mU / mg to 20.0×10 3 In some embodiments, the purified recombinant ASA has a specific activity of about 1.0×10 3 mU / mg, about 2.0×10 3 mU / mg, about 3.0×10 3 mU / mg, about 4.0×10 3 mU / mg, about 5.0×10 3 mU / mg, about 10.0×10 3 mU / mg, about 15.0×10 3 mU / mg, about 20.0×10 3 mU / mg, about 25.0×10 3 mU / mg, about 30.0×10 3 mU / mg, about 35.0×10 3 mU / mg, about 40.0×10 3 mU / mg, about 45.0×10 3 mU / mg, about 50.0×10 3 mU / mg or greater specific activity.

[0246] Charge curve

[0247] The charge curve associated with the protein can characterize the purified recombinant ASA. Typically, the protein charge curve reflects the profile of the residue side chain charges (typically present on the protein surface). The charge curve can be determined by performing ion exchange (IEX) chromatography (e.g., HPLC) on the protein. In some embodiments, a "charge curve" refers to a set of values ​​representing the amount of protein eluted from an ion exchange column at a time point after addition of a mobile phase column containing exchange ions.

[0248] In general, suitable ion exchange columns are anion exchange columns. For example, charge curves can be determined by using the strong anion exchange (SAX) chromatogram of a high performance liquid chromatography (HPLC) system. Generally, recombinant ASA is adsorbed on the fixed positive charge of the strong anion exchange column, and the gradient elution using the ionic strength increase of the mobile phase under a predetermined flow rate is used to elute the recombinant ASA species from a column proportional to the ionic interaction strength between the positively charged columns. The ASA species with more negative charge (more acidic) elutes later than the ASA species with less negative charge (less acidic). The concentration of protein in the eluent is detected by ultraviolet absorbance (at 280 nm).

[0249] In some embodiments, recombinant ASA is adsorbed to the fixed positive charge of a Mini Q PE column in 20 mM TRIS-HCl at about pH 8.0, and the recombinant ASA species are eluted from the column in proportion to the strength of the ionic interactions between the positively charged species and the column using a gradient of increasing ionic strength of a mobile phase consisting of 20 mM TRIS-HCl, 1 M sodium chloride, pH 8.0 at a flow rate of 0.8 ml / min.

[0250] In some embodiments, the charge curve can be shown by a chromatogram of absorbance units versus time after elution from an HPLC column. The chromatogram can include a group of one or more peaks, each of which identifies a subpopulation of recombinant ASA having a composition with a similar surface charge.

[0251] Glycan map

[0252] In some embodiments, the purified recombinant ASA protein can be characterized by its proteoglycan composition (often referred to as its glycan profile). Without being bound by any theory, it is believed that the shape and complexity of glycan linkages and branching structures can affect in vivo clearance, lysosomal targeting, bioavailability, and / or potency.

[0253] Typically, the glycan profile can be determined by enzymatic digestion and subsequent chromatographic analysis. Various enzymes can be used for enzymatic digestion, and these enzymes include but are not limited to suitable glycosylases, peptidases (e.g., endopeptidases, exopeptidases), proteases, and phosphatases. In some embodiments, the suitable enzyme is alkaline phosphatase. In some embodiments, the suitable enzyme is neuraminidase. Glycans (e.g., phosphate glycans) can be detected by chromatographic analysis. For example, phosphate glycans can be detected by high-performance anion exchange chromatography (HPAE-PAD) or size exclusion high-performance liquid chromatography (HPLC) using pulsed amperometric detection. According to methods known in the art and disclosed herein, the amount of glycans (e.g., phosphate glycans) represented by each peak on the glycan profile can be calculated using a standard curve of glycans (e.g., phosphate glycans).

[0254] In some embodiments, the purified recombinant ASA protein according to the present invention is characterized by a glycan profile comprising at least seven peak groups indicative of neutral (peak group 1), monosialylated (peak group 2), blocked mannose-6-phosphorylated (peak group 3), disiallylated (peak group 4), monomannose-6-phosphorylated (peak group 5), hybrid (peak group 6), and dimannose-6-phosphorylated (peak group 7) ASA protein, respectively.

[0255] Peptide Mapping

[0256] In some embodiments, peptide maps can be used to characterize amino acid composition, post-translational modifications, and / or cellular processing; for example, cleavage and / or glycosylation of signal peptides. Typically, recombinant proteins can be broken into discrete peptide fragments by controlled or random destruction to produce patterns or peptide maps. In some cases, the purified ASA protein can be enzymatically digested prior to analytical analysis. Prior to analytical analysis, digestion can be performed using peptidases, glycoside hydrolases, phosphatases, lipases, or proteases, and / or combinations thereof. The structural composition of the peptides can be determined using methods well known in the art. Exemplary methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR), or HPLC.

[0257] Metal Analysis

[0258] In some embodiments, the purified recombinant ASA protein can be characterized by metal analysis. Various methods for analyzing trace metals in purified pharmaceutical substances are known in the art and can be used in the practice of the present invention.

[0259] In some embodiments, residual phosphorus is measured and compared with a reference sample. Without being limited by any specific theory, it is envisioned that residual phosphorus helps maintain the pH of the pharmaceutical substance. In some embodiments of the present invention, residual phosphorus is between about 10-50ppm (that is, between about 10-45ppm, about 10-40ppm, about 10-30ppm, about 20-50ppm, about 20-45ppm, about 20-40ppm, about 20-30ppm, about 30-50ppm, about 30-40ppm). In some embodiments, the pH range of the recombinant ASA purified according to the methods disclosed herein is between about 5-7 (that is, 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).

[0260] In some embodiments, recombinant ASA purified according to the methods disclosed herein contains calcium. Without being bound by any particular theory, it is hypothesized that calcium ions present in the active site of ASA may be essential for enzyme activity. In some embodiments of the invention, calcium is present at a level of 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).

[0261] Pharmaceutical compositions and administration

[0262] Purified recombinant ASA protein can be administered to MLD patients according to known methods. For example, purified recombinant ASA protein can be delivered intravenously, subcutaneously, intramuscularly, parenterally, transdermally, or transmucosally (e.g., orally or nasally).

[0263] In some embodiments, recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by intravenous administration.

[0264] In some embodiments, recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by intrathecal administration. As used herein, the term "intrathecal administration" or "intrathecal injection" refers to injection into the spinal canal (the intrathecal space surrounding the spinal cord). Various techniques including, but not limited to, intracerebroventricular injection via a burr hole or transcerebral puncture or lumbar puncture can be used. In some embodiments, "intrathecal administration" or "intrathecal delivery" according to the present invention refers to IT administration or delivery via the lumbar region or area, i.e., lumbar IT administration or delivery. As used herein, the term "lumbar region" refers to the area between the third and fourth lumbar (lower back) vertebrae, more generally, the L2-S1 region of the spine. In some embodiments, recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by intrathecal administration as described in PCT International Applications WO2011 / 163648 and WO2011 / 163650, which are incorporated herein by reference in their entirety.

[0265] In some embodiments, recombinant ASA or a pharmaceutical composition containing the same is administered to a subject by subcutaneous administration (i.e., under the skin). For this purpose, the formulation is injectable using a syringe. However, other devices for administering the formulation are available, such as injection devices (e.g., Inject-ease and Genject devices); injection pens (e.g., GenPen); needle-free devices (e.g., MediJector and BioJector); and subcutaneous patch delivery systems.

[0266] In some embodiments, intrathecal administration can be combined with other routes of administration, eg, intravenous, subcutaneous, intramuscular, parenteral, transdermal, or transmucosal (eg, oral or nasal).

[0267] The present invention encompasses single administration as well as multiple administrations of a therapeutically effective amount of recombinant ASA as described herein or a pharmaceutical composition containing the same. Depending on the nature, severity, and extent of the subject's condition (e.g., lysosomal storage disease), recombinant ASA or a pharmaceutical composition containing the same may be administered at regular intervals. In some embodiments, a therapeutically effective amount of recombinant ASA or a pharmaceutical composition containing the same may be administered regularly 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 every two months), once a month (once every month), once every two weeks (once every two weeks), once a week, once a day, or continuously).

[0268] Recombinant ASA or a pharmaceutical composition containing the same can be formulated using a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and therapeutic agent can be sterile. The formulation should be suitable for the mode of administration.

[0269] Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose, or starch), sugars (e.g., mannitol, sucrose, or other sugars), dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, aromatic oils, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like, and combinations thereof. If desired, the pharmaceutical preparations may be mixed with auxiliary agents that do not adversely affect the active compounds or interfere with their activity (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances, etc.). In some embodiments, a water-soluble carrier suitable for intravenous administration is used.

[0270] If desired, compositions or medicaments also can contain a small amount of wetting agent or emulsifier or pH buffer.Composition can be liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release preparation or powder.Composition can also be formulated as suppository using common adhesive and carrier (for example triglyceride).Oral formulations can include standard carrier, for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate polyvinyl pyrrolidone, saccharin sodium, cellulose, magnesium carbonate etc.

[0271] Compositions or medicaments can be formulated according to conventional procedures for pharmaceutical compositions suitable for human administration. For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. Where necessary, compositions may also include solubilizers and local anesthetics to alleviate injection site pain. In general, ingredients can be provided in unit dosage form alone or mixed together, for example, as lyophilized powders or anhydrous concentrates in sealed containers such as ampoules or sachets indicating the active dose. In the case of administering the composition by infusion, an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water can be used to disperse the composition. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0272] In some embodiments, arylsulfatase A is formulated in an isotonic solution (e.g., 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 another embodiment, ASA is formulated in a physiological buffer, e.g.,

[0273] a) Formulation buffer I containing (in mM): Na2HPO4 (3.50-3.90), NaH2PO4 (0-0.5), glycine (25-30), mannitol (230-270) and water for injection; or

[0274] b) Formulation buffer II containing (in mM): Tris-HCl (10), glycine (25-30), mannitol (230-270) and water for injection.

[0275] Arylsulfatase A purified by the methods herein can be used as an agent for reducing the level of the sphingolipid 3-O-sulfated galactosylceramide (galactosylsulfolipid) in peripheral nervous system cells and / or in the central nervous system in subjects suffering from and / or diagnosed with metachromatic leukodystrophy. The administration of ASA will result in reduced impairment of motor learning skills and increased neuromotor conduction velocity and / or neurotransmission amplitude. As used herein, the term "therapeutically effective amount" is primarily determined based on the total amount of therapeutic agent contained in the pharmaceutical composition of the present invention. In general, a therapeutically effective amount is sufficient to provide a subject with a meaningful benefit (e.g., to treat, regulate, cure, prevent and / or alleviate the primary disease or condition). For example, a therapeutically effective amount can be an amount sufficient to achieve the desired therapeutic and / or preventive effect, such as an amount sufficient to regulate lysosomal enzyme receptors or their activity to treat such lysosomal storage diseases or their symptoms (e.g., to reduce or eliminate the appearance or incidence of "zebra bodies" or cell vacuolization after administration of the composition of the present invention to a subject). In general, the amount of the therapeutic agent (e.g., recombinant lysosomal enzyme) administered to a subject in need thereof depends on the subject's characteristics. These characteristics include the subject's condition, disease severity, general health, age, sex, and body weight. One of ordinary skill in the art can easily determine appropriate dosage based on these and other relevant factors. Additionally, objective and subjective assays can be optionally used to determine the optimal dosage range.

[0276] A therapeutically effective amount is typically administered in a dosage regimen that can include multiple unit doses. For any particular protein, the therapeutically effective amount (and / or appropriate unit dose within an effective dosage regimen) may vary, for example, depending on the route of administration, combination with other pharmaceutical products. Moreover, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on various factors, including the condition to be treated and the severity of the condition; the activity of the specific pharmaceutical product employed; the specific composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and / or the excretion rate or metabolism of the specific fusion protein employed; the duration of treatment, and other factors well known in the medical field.

[0277] It is further understood that for any particular subject, specific dosage regimens should be adjusted according to the individual need and the professional judgment of the administrator or monitor of the administration of enzyme replacement therapy, and that the dosage ranges set forth herein are examples only and are not intended to limit the scope or practice of the claimed invention.

[0278] The present invention also includes the following embodiments:

[0279] Embodiment 1. A method for purifying recombinant arylsulfatase A (ASA) protein, the method comprising

[0280] purifying recombinant arylsulfatase A (ASA) protein from impure preparations by performing one or more chromatography steps;

[0281] combining the eluates from the one or more chromatography steps;

[0282] adjusting the pH of the combined eluates to a pH of about 6.0 or greater than about 6.0; and

[0283] The pH adjusted eluate is subjected to ultrafiltration and / or diafiltration.

[0284] Embodiment 2. The method of Embodiment 1, wherein the pH is adjusted to about 6.0.

[0285] Embodiment 3. The method of embodiment 1 or 2, wherein the pH is adjusted using a buffer comprising sodium phosphate, sodium chloride and sodium citrate at pH 7.0.

[0286] Embodiment 4. The method of embodiment 3, wherein the buffer comprises approximately 0.1-0.5 M sodium phosphate, 0.5-2.5 M sodium chloride, and 0.1-0.6 M sodium citrate, and the pH of the buffer is 7.0.

[0287] Embodiment 5. The method of any of the preceding embodiments, wherein virus filtration is performed before the ultrafiltration and diafiltration steps.

[0288] Embodiment 6. The method of any of the preceding embodiments, wherein a single step of ultrafiltration and / or diafiltration is performed.

[0289] Embodiment 7. The method of Embodiment 5, wherein the single step of ultrafiltration and / or diafiltration comprises only one diafiltration.

[0290] Embodiment 8. The method of any of the preceding embodiments, wherein the ultrafiltration is tangential flow ultrafiltration.

[0291] Embodiment 9. The method of any of the preceding embodiments, wherein the one or more chromatography steps comprise cation exchange chromatography.

[0292] Embodiment 10. The method of Embodiment 9, wherein the cation exchange chromatography is the last chromatography step and the eluates from the cation exchange chromatography are combined before pH adjustment.

[0293] Embodiment 11. The method of embodiment 10, wherein one or more of anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography are performed before performing the cation exchange chromatography.

[0294] Embodiment 12. The method of Embodiment 11, wherein the anion exchange chromatography is Q chromatography.

[0295] Embodiment 13. The method of Embodiment 11, wherein the anion exchange chromatography comprises TMAE resin.

[0296] Embodiment 14. The method of embodiment 13, wherein once the impure product is loaded, the TMAE resin is washed with a first wash buffer comprising MES-Tris at pH 7.0.

[0297] Embodiment 15. The method of Embodiment 14, wherein the first wash buffer comprises approximately 20-75 mM MES-Tris.

[0298] Embodiment 16. The method of any one of Embodiments 13-15, wherein the TMAE resin is washed with a second wash buffer comprising MES-Tris and NaCl at pH 7.0.

[0299] Embodiment 17. The method of embodiment 16, wherein the second wash buffer comprises about 5-75 mM MES-Tris and about 50-150 mM NaCl, and the pH of the second buffer is 7.0.

[0300] Embodiment 18. The method of any one of Embodiments 13-17, wherein once the impure product is loaded, the TMAE resin is eluted using an elution buffer comprising MES-Tris and NaCl at pH 7.0.

[0301] Embodiment 19. The method of Embodiment 18, wherein the elution buffer comprises about 5-75 mM MES-Tris and about 150-300 mM NaCl, and the pH of the elution buffer is 7.0.

[0302] Embodiment 20. The method of embodiment 11, wherein the anion exchange chromatography utilizes a column selected from the group consisting of: Q Sepharose TM Fast Flow Column, Q Sepharose TM High-performance columns, Q Sepharose TM XL, Capto TM Q, DEAE, TOYOPEARL Q. TMAE, Eshmuno TM Q. Nuvia TM Q or UNOsphere TM Q.

[0303] Embodiment 21. The method of any one of Embodiments 11-20, wherein the mixed-mode chromatography is hydroxyapatite (HA) chromatography.

[0304] Embodiment 22. The method of any of Embodiments 11-21, wherein the hydrophobic interaction chromatography is phenyl chromatography.

[0305] Embodiment 23. The method of any one of Embodiments 9-22, wherein performing one or more chromatography steps comprises performing anion exchange chromatography using TMAE resin, HA chromatography, phenyl chromatography, and cation exchange SP chromatography in the following order.

[0306] Embodiment 24. The method of any of Embodiments 9-23, wherein the anion exchange chromatography comprises using a column having a loading capacity greater than about 4.5 g / L.

[0307] Embodiment 25. The method of Embodiment 24, wherein the column has a loading capacity of about 5-20 g / L.

[0308] Embodiment 26. The method of any of the preceding embodiments, wherein ultrafiltration of the impure preparation is performed prior to the one or more chromatography steps.

[0309] Embodiment 27. The method of Embodiment 26, wherein the ultrafiltration is tangential flow ultrafiltration.

[0310] Embodiment 28. The method of Embodiment 26 or 27, wherein the ultrafiltration utilizes a membrane filter comprising a pore size having a molecular weight cut-off of at least 10 kDA.

[0311] Embodiment 29. The method of Embodiment 26 or 27, wherein the ultrafiltration utilizes a membrane filter comprising a pore size having a molecular weight cutoff of at least 30 kDA.

[0312] Embodiment 30. The method of Embodiment 26 or 27, wherein the ultrafiltration utilizes a membrane filter comprising a pore size having a molecular weight cutoff of at least 50 kDA.

[0313] Embodiment 31. The method of embodiment 26 or 27, wherein at least 75% of the recombinant ASA remains in the impure preparation.

[0314] Embodiment 32. The method of embodiment 26 or 27, wherein at least 75% of the recombinant ASA permeates through the filter.

[0315] Embodiment 33. The method of Embodiment 26 or 27, wherein the ultrafiltration comprises a polyethersulfone or cellulose membrane.

[0316] Embodiment 34. The method of any one of Embodiments 26-33, wherein the ultrafiltration is followed by one or more steps of depth filtration and / or viral inactivation.

[0317] Embodiment 35. The method of embodiment 34, wherein the one or more steps of depth filtration and / or viral inactivation are followed by one or more chromatography steps.

[0318] Embodiment 36. The method of embodiment 34 or 35, wherein the step of inactivating viruses comprises adding a detergent to the impure preparation.

[0319] Embodiment 37. The method of any of the preceding embodiments, wherein the recombinant ASA protein is produced by mammalian cells cultured in suspension.

[0320] Embodiment 38. The method of Embodiment 37, wherein the mammalian cells are cultured in a bioreactor.

[0321] Embodiment 39. The method of embodiment 37 or 38, wherein the culture medium is serum-free.

[0322] Embodiment 40. The method of embodiment 39, wherein the serum-free medium is a chemically defined medium.

[0323] Embodiment 41. The method of any one of Embodiments 37-40, wherein the mammalian cell is a human cell.

[0324] Embodiment 42. The method of any of the preceding embodiments, wherein the impure product is a feed stream from a perfusion bioreactor.

[0325] Embodiment 43. The method of any one of Embodiments 1-41, wherein the impure preparation is prepared from a serum-free medium containing recombinant ASA protein secreted by mammalian cells.

[0326] Embodiment 44. The method of embodiment 43, wherein the impure preparation is thawed from a frozen culture medium preparation.

[0327] Embodiment 45. The method of any of the preceding embodiments, wherein the ultrafiltration and / or diafiltration steps comprise exchanging the purified recombinant ASA protein into a pharmaceutical formulation buffer.

[0328] Embodiment 46. The method of any one of the preceding embodiments, wherein the recombinant ASA protein has an amino acid sequence that is at least 70% identical to SEQ ID NO: 1 (corresponding to wild-type human ASA protein).

[0329] Embodiment 47. The method of any one of the preceding embodiments, wherein the recombinant ASA protein has an amino acid sequence identical to SEQ ID NO: 1.

[0330] Embodiment 48. The method of any one of the preceding embodiments, wherein the purified recombinant ASA protein contains less than 100 ng / mg HCP.

[0331] Embodiment 49. The method of any one of the preceding embodiments, wherein the purified recombinant ASA protein contains less than 60 ng / mg HCP.

[0332] Embodiment 50. The method of any of the preceding embodiments, wherein the purified recombinant ASA protein has no new bands with an intensity greater than 1.0% of the assay control when subjected to SDS-PAGE using Coomassie Brilliant Blue staining.

[0333] Embodiment 51. The method of any one of the preceding embodiments, wherein the purified recombinant ASA protein contains less than 100 pg / mg host cell DNA.

[0334] Embodiment 52. The method of any one of the preceding embodiments, wherein the purified recombinant ASA protein contains less than 50 pg / mg of host cell DNA.

[0335] Embodiment 53. A method for purifying recombinant arylsulfatase A (ASA) protein, the method comprising

[0336] purifying a recombinant arylsulfatase A (ASA) protein from an impure preparation by performing one or more chromatography steps, wherein a first chromatography step uses a column having a loading capacity of about 4.5 g protein / L resin or greater;

[0337] combining the eluates from the one or more chromatography steps;

[0338] adjusting the pH of the combined eluate to a pH of about 6.0 or greater than about 6.0; and

[0339] The pH adjusted eluate is subjected to ultrafiltration and / or diafiltration.

[0340] Embodiment 54. The method of Embodiment 53, wherein performing one or more chromatography steps comprises performing, in the following order: anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and cation exchange chromatography.

[0341] Embodiment 55. The method of Embodiment 54, wherein the anion exchange chromatography uses a column with TMAE resin.

[0342] Embodiment 56. The method of Embodiment 55, wherein the TMAE column has a loading capacity of about 5-20 g protein / L resin.

[0343] Embodiment 57. The method of any one of Embodiments 53-56, wherein a single step of ultrafiltration and / or diafiltration is performed after the one or more chromatography steps.

[0344] Embodiment 58. A recombinant arylsulfatase A (ASA) protein purified according to any one of the preceding embodiments.

[0345] Embodiment 59. A pharmaceutical composition comprising the recombinant ASA protein of embodiment 58.

[0346] Embodiment 60. A method of treating metachromatic leukodystrophy, comprising administering the pharmaceutical composition of embodiment 59 to a subject in need of treatment.

[0347] Embodiment 61. A composition comprising purified recombinant arylsulfatase A (ASA) having an amino acid sequence at least 70% identical to SEQ ID NO: 1,

[0348] wherein the purified recombinant ASA has a specific activity of at least about 50 U / mg; and

[0349] Furthermore, the purified recombinant ASA contains less than 150 ng / mg host cell protein (HCP) and / or 150 pg / mg host cell DNA (HCD).

[0350] Embodiment 62. The composition of embodiment 61, wherein the purified recombinant ASA contains less than 100 ng / mg HCP.

[0351] Embodiment 63. The composition of embodiment 61 or 62, wherein the purified recombinant ASA contains less than 60 ng / mg HCP.

[0352] Embodiment 64. The composition of any one of Embodiments 61-63, wherein the purified recombinant ASA contains less than 100 pg / mg HCD.

[0353] Embodiment 65. The composition of any of Embodiments 61-64, wherein the purified recombinant ASA contains less than 50 pg / mg HCD.

[0354] Embodiment 66. The composition of any of Embodiments 61-65, wherein the purified recombinant ASA has a specific activity of at least about 70 U / mg.

[0355] Embodiment 67. The composition of any of Embodiments 61-66, wherein the purified recombinant ASA has a specific activity of at least about 100 U / mg.

[0356] Embodiment 68. The composition of any one of Embodiments 61-67, wherein the purified recombinant ASA has a specific activity of at least about 120 U / mg.

[0357] Embodiment 69. The composition of any one of Embodiments 61-68, wherein the purified recombinant ASA has a specific activity in the range of about 50-200 U / mg.

[0358] Embodiment 70. The composition of any of Embodiments 61-69, wherein the purified recombinant ASA has a specific activity ranging from about 50-140 U / mg.

[0359] Embodiment 71. A composition comprising purified recombinant arylsulfatase A (ASA) having an amino acid sequence at least 70% identical to SEQ ID NO: 1

[0360] wherein the purified ASA is characterized by a glycan profile comprising seven or fewer peak groups, the peak groups being selected from peak groups indicating neutrality (peak group 1), monosialylation (peak group 2), blocked mannose-6-phosphorylation (peak group 3), disialylation (peak group 4), monomannose-6-phosphorylation (peak group 5), hybridization (peak group 6), and dimannose-6-phosphorylation (peak group 7).

[0361] Embodiment 72. The composition of embodiment 71, wherein the purified recombinant ASA has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

[0362] Embodiment 73. The composition of embodiment 71 or 72, wherein the purified recombinant ASA has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.

[0363] Embodiment 74. The composition of any one of Embodiments 71-73, wherein the purified recombinant ASA has an amino acid sequence at least 95% identical to SEQ ID NO: 1.

[0364] Embodiment 75. The composition of any one of Embodiments 71-74, wherein the purified recombinant ASA has an amino acid sequence identical to SEQ ID NO: 1.

[0365] Embodiment 76. A formulation comprising the composition of any one of embodiments 71-75 and a physiologically acceptable carrier.

[0366] Embodiment 77. The formulation of Embodiment 76, wherein the formulation is suitable for intravenous administration.

[0367] Embodiment 78. The formulation of Embodiment 76, wherein the formulation is suitable for intrathecal administration.

[0368] Embodiment 79. The formulation of Embodiment 76, wherein the formulation is suitable for subcutaneous administration. Example

[0369] Example 1. Tangential Flow Ultrafiltration-Capture

[0370] This example demonstrates that tangential flow ultrafiltration (also known as cross-flow filtration) can be used to capture recombinant ASA protein directly from a production bioreactor.

[0371] Specifically, unpurified bulk material was captured from the production perfusion bioreactor by tangential flow ultrafiltration. Tangential flow filtration was performed in a 30 kD standard membrane.

[0372] Additional experiments were performed using the Sartorius The ECO membrane performs tangential flow filtration while maintaining the same composition and molecular weight cut-off. The ECO membrane enables the process to utilize lower cross-flow velocities.

[0373] The contour profiler function was used to analyze the permeate flux values ​​(LMH) generated from 12 deviation runs using ECO and standard membranes. This function displays the response contours for two factors at a time. In this study, the feed flow rate and transmembrane pressure ("TMP") for each membrane were compared to the permeate flux. Exemplary results are shown in Figure 3 middle.

[0374] As can be seen from the contour plots of the ECO vs. standard membrane deviation runs, the ECO membrane exhibited a much stronger flux output over the given parameter range tested. The ECO membrane was able to achieve higher flux rates at lower feed flow rates within the operating range.

[0375] More specifically, based on analysis of data from the designed runs, the titer ELISA recoveries ranged from about 92% to 132%. The host cell protein (HCP) log reduction values ​​ranged from about 0.11 to 0.38. Titer ELISA recoveries, host cell protein (HCP) log reduction values ​​(LRVs), and percent activity recovery were determined as described elsewhere in the application.

[0376] The experimental results confirm the feasibility of implementing the UFDF capture step in the arylsulfatase A III phase purification process. According to the side-by-side UFDF deviation operation, the ECO membrane is particularly effective because it can obtain higher flux rates at lower feed flow rates within the operating range. Design experiments were also performed using the ECO membrane to evaluate the potential impact of the operating parameters, TMP, feed flow rate, initial concentration factors and treatment temperature on the performance of the rhASA capture process in the loading test. Host cell protein (HCP) ELISA and activity step productivity were not significantly affected by the operating parameters of the ECO membrane.

[0377] Example 2. Depth Filtration and Virus Inactivation

[0378] This experiment illustrates conditions for depth filtration and viral inactivation that can be implemented in a purification process.

[0379] For example, the captured unpurified bulk material is thawed at 2-8°C for ≤120 hours. The temperature of the bulk material is adjusted to about 18 ± 2°C and filtered through a Cuno Zeta Plus depth filter, then filtered by grinding. A Sartorious Maxicap Sartopore 2, 0.45 ± 0.2 μm polishing filtration capsule is connected to the downstream of the depth filter to remove particulates and reduce bioburden in the product for downstream processing. The filter is rinsed with water for injection ("WFI"), followed by a MES-Tris, pH 7.1 equilibrium solution.

[0380] After filtration, a final rinse was performed to recover the retained material from the filter set. Once filtration was complete, the filter set was rinsed with a 0.05 M ES-Tris, pH 7.0 rinse solution. The collected rinse solution was added to the filtered, combined bulk material.

[0381] The filtered, but unpurified, bulk was then treated with a viral inactivation step by adding polysorbate 80 and tributyl phosphate (TnBP) followed by a subsequent incubation for 3-24 hours as follows. Polysorbate 80 was added to the filtered, combined, unpurified bulk from a 10% (v / v) stock solution in 0.05M MES-Tris, pH 7.1, to a final concentration of 1%. Tributyl phosphate (TnBP) was then added to a final concentration of approximately 0.3% based on the new volume of the unpurified bulk containing polysorbate 80. The resulting pool was mixed to make compatible and then incubated for approximately 3-24 hours under continuous stirring.

[0382] When the virus inactivation is completed, the amalgam is processed through a polyvinylidene fluoride (PVDF) 0.2 μm filter before being loaded onto the first chromatographic column. In this experiment, the filtered virus inactivation amalgam is applied to a Q column (described below) within approximately 28 hours of the virus inactivation incubation start time. The virus inactivation intermediate is filtered, and at the end of filtration, 0.05MMES-Tris, pH 7.0 buffer is used to rinse the filter to maximize product recovery.

[0383] Example 3. Anion Exchange Chromatography

[0384] This example shows exemplary conditions for anion exchange chromatography, in particular, when anion exchange chromatography is used as the first chromatographic step. In this particular example, QSEPHAROSE TM Fast Flow (QFF) resins are used in quaternary ammonium anion exchange operating in a bind and elute mode.

[0385] A Q FF column was prepared using standard methods. ASA was bound to the Q column at a pH of 7.0 and then eluted using 0.02M MES-Tris, 0.26M NaCl at pH 7.1. Loading, washing, and elution were performed at a flow rate of approximately 100. The operating temperature range was 16–23°C. The Q eluate was collected by measuring the absorbance at 280nm (A280) set points from 0.5AU / cm at the rising side of the peak to 0.25AU / cm at the falling side of the peak. Exemplary conditions for anion exchange columns are shown in Table 2.

[0386] Table 2: Exemplary conditions for anion exchange columns

[0387]

[0388]

[0389] Additional experiments were performed to compare the purity, activity, and throughput (eg, target loading capacity) of exemplary anion exchange resins. In particular, to facilitate scale-up, a variety of high capacity anion exchange resins were screened and evaluated in this study.

[0390] Once "filled," the resin was equilibrated, loaded, washed, and eluted using the conditions shown in Table 3. In this study, the binding conditions remained the same as for the QFF process described above. Flow-through (FT), wash (W), and elution / elution (EL / elution) fractions were collected and analyzed using A280 absorbance, titer determination, and SDS-PAGE (Coomassie and Silver staining).

[0391] Table 3. Example conditions

[0392]

[0393] Seven resins, excluding QFF, were evaluated to determine binding capacity, selectivity, and recovery. Overall yields for all resins were comparable, ranging from 80-83%, indicating similar recoveries and consistent experimental operation. Some resins had lower binding capacities than QFF, while others had higher binding capacities. In particular, Fractogel demonstrated the highest binding capacity, the highest selectivity, and comparable recovery of all the resins tested. Furthermore, very little product loss was observed during the Fractogel flow-through at a loading of 15 g / L.

[0394] Exemplary results showing the yield and binding capacity of Q FF and Fractogel resins are shown in Table 4.

[0395] Table 4: Exemplary results of binding capacity and productivity

[0396]

[0397]

[0398] Confirm the operation side by side

[0399] To validate the Fractogel process and evaluate its impact on subsequent purification steps and the quality of the final drug substance ("DS"), a side-by-side purification process of Fractogel™ AE versus Q FF was performed from unpurified bulk material through to the drug substance (including the chromatography steps described in this example). Following anion exchange chromatography, the additional purification steps were as follows (presented in the order in which they were performed): mixed-mode chromatography on an HA column, hydrophobic interaction chromatography on a phenyl column, cation exchange chromatography on an SP column, and two ultrafiltration / diafiltration steps with an intervening virus filtration step.

[0400] A280 and titer productivity were comparable. HCP reduction was also similar between columns.

[0401] Furthermore, SDS-PAGE (silver) gel ( Figure 4) shows similar band profiles for Fractogel and Q FF throughout the entire process. CE-SDS data show that Fractogel is purer than Q FF in the first step, but the results are comparable after the phenyl column step. This is confirmed by the HCP / rhASA ratio.

[0402] The quality attributes of the SP eluates from both columns, as well as from a control run, were evaluated. The Fractogel and QFF processes provided similarly pure and acceptable SP eluates, but some attributes differed slightly from the control due to differences in the unpurified loading material.

[0403] In summary, as demonstrated by the above results, using Fractogel as the first column provides comparable process performance (yield, HCP reduction) and SP quality attributes to using Q FF as the control process. However, the Fractogel column provides higher loading capacity and increased throughput.

[0404] Finally, side-by-side validation runs showed that the Fractogel TMAE process had comparable performance (yield, HCP removal) to the QFF process, but offered the added benefit of a higher target loading capacity (e.g., at least 10 g rhASA / L). No significant yield loss was observed for the characterized loading range of 5-20 g / L. The quality attributes of the SP eluate from the Fractogel run were also comparable to those of the control QFF run.

[0405] Example 4. Mixed Hydroxyapatite (HA) Chromatography

[0406] This example shows exemplary conditions for mixed-mode hydroxyapatite chromatography that can be used in the purification of recombinant ASA protein.

[0407] Specifically, the anion exchange chromatography eluate prepared above was then purified by hydroxyapatite (HA) chromatography. A commercially available ceramic hydroxyapatite type 1 resin column was prepared. In this example, 0.250 M sodium phosphate buffer, pH 7.0 was used to pre-equilibrate the column.

[0408] The HA column was operated in a bind and elute mode. The anion exchange eluent was first heated to ambient temperature and then adjusted to about 0.001 M sodium phosphate before being loaded onto the HA column. rhASA was bound to the column at pH 7.0 and then eluted using about 0.04 M sodium phosphate, pH 7.1. Loading, washing, and elution were performed at a flow rate of about 150 cm / h. The HA eluate was collected by the absorbance at 280 nm (A280) set point of 0.5 AU / cm at the rising side of the peak to 0.25 AU / cm at the falling side of the peak. Exemplary conditions for the HA column are shown in Table 5.

[0409] Table 5: Exemplary conditions for HA columns

[0410]

[0411] Example 5. Hydrophobic Interaction (Phenyl) Chromatography

[0412] This example shows exemplary conditions for hydrophobic interaction (phenyl) chromatography that can be used in the purification of recombinant ASA protein.

[0413] Specifically, the HA column chromatography eluate prepared above was then purified by phenyl column chromatography.A commercially available Phenyl Sepharose Fast Flow column was packed and prepared for the purification of rhASA.

[0414] Phenyl column is operated in combination and elution mode. After heating until ambient temperature, then before being loaded into the phenyl column, HA eluent is adjusted to about 1.1M NaCl and uses acid-free 0.5M MES to regulate pH to about 5.6. At pH 5.6, rhASA is bound to the column and then 0.02M MES-Tris, 0.06M NaCl, pH 7.1 are used to elute the column. Load, wash and elute at a flow velocity of about 150cm / h. The phenyl eluent is gathered by the absorbance of 280nm (A280) set point of 0.5AU / cm at the rising side of the peak to 0.25AU / cm at the falling side of the peak. The exemplary conditions for the phenyl column are presented in Table 6.

[0415] Table 6: Exemplary conditions for phenyl columns

[0416]

[0417] Example 6. Cation Exchange Chromatography

[0418] This example shows exemplary conditions for cation exchange chromatography that can be used in the purification of recombinant ASA protein.

[0419] Specifically, the phenyl column chromatography eluate prepared above was then purified by cation exchange chromatography. In this example, an SP-650M column (TOYOPEARL) was packed and prepared.

[0420] The SP column was operated in a bind and elute mode. The phenyl column eluate was warmed to 16-23°C, then diluted with WFI to reduce conductivity and adjusted to pH 4.2 with approximately 1 M acetic acid before loading onto the SP column. rhASA bound to the column at pH 4.2 and then eluted with 0.01 M sodium acetate, 0.01 M acetic acid, 0.05 M NaCl, pH 4.5. Loading, washing, and elution were performed at a flow rate of approximately 150 cm / h. The SP eluate was collected by measuring the absorbance at 280 nm (A280) set points from 0.25 AU / cm on the rising side of the peak to 0.25 AU / cm on the falling side of the peak. Exemplary conditions for the SP column are shown in Table 7.

[0421] Table 7: Exemplary conditions for SP columns

[0422]

[0423] Example 7. Ultrafiltration / Diafiltration and Virus Filtration

[0424] This example shows exemplary conditions for ultrafiltration / diafiltration and viral filtration that can be used in the purification of recombinant ASA protein.

[0425] Ultrafiltration / diafiltration1

[0426] The SP elution pool was concentrated and diafiltered using a 10 kDa Hydrosart Sartocon membrane. The product pool was concentrated to approximately 15 mg / mL and diafiltered using 6-8 volumes of 0.01 M sodium phosphate-citrate, 0.137 M NaCl, pH 6.0. The concentrated and diafiltered product pool was then passed through a 0.2 μm filter.

[0427] The concentrated / diafiltered pool is filtered through 0.2 μm and typically stored at 2-8°C for less than about 24 hours.

[0428] Virus filtration

[0429] A virus filtration operation can be performed after ultrafiltration / diafiltration 1 to remove any virus-like particles in the process stream. For example, a filtration stack with a pump, Planova filter, and pressure gauge can be set up. 2 At the target pressure, the filter was flushed with 1 L of 0.154 M NaCl through the retentate side, followed by more than 3 L of flushing through the permeate side. Once complete, the filter was flushed with 4 L of 0.154 M NaCl to increase product recovery. A gold particle test was performed on the filter after use to ensure that filter integrity was maintained.

[0430] Ultrafiltration / diafiltration2

[0431] The material of virus filtration is concentrated to 15-30mg / ml, and then uses 10kDa Hydrosart SartoconUFDF membrane to diafilter through 6-8 volumes of 0.154M NaCl diafiltration buffer. Once diafiltration is completed, the material is further concentrated to 48-50mg / ml. The diafiltration buffer is used to flush the system. Then an amount of system rinse is added back to the product concentrate to obtain the target final concentration of about 40mg / ml.

[0432] Example 8. Single Ultrafiltration / Diafiltration Unit Operation

[0433] The process described in Example 7 includes ultrafiltration / diafiltration ("UFDF") of the SP column eluate (UFDF1), virus filtration of the UFDF1 concentrate, and final UFDF of the virus filtrate (UFDF2) to produce a pre-final drug substance (DS). An exemplary flow diagram of these processes is shown in Figure 1 middle.

[0434] In this example, additional experiments were conducted to determine the feasibility of eliminating one UFDF unit operation to reduce overall process time, cost, and potentially increase process yield. Therefore, a study was conducted to evaluate the feasibility of viral filtration of the pH-adjusted SP eluate followed by a single UFDF unit operation to produce a pre-final filtered drug substance ("DS").

[0435] This study utilized partially purified source material that had previously been purified from unpurified bulk material ("UPB") using the SP eluate as described above. The SP eluate was used as the load for three UFDF runs performed in this study, including two feasibility experimental runs and one control run of the dual UFDF process as described above. The two experimental (also referred to as feasibility) runs consisted of one UFDF and one ultrafiltration / two diafiltrations ("UFDFDF") after pH adjustment of the SP eluate pool. The SP eluate used for the feasibility run was adjusted to pH 6.0 and then filtered for virus through, for example, a Planova 20N virus filter. The experimental runs were compared to a control run of UFDF1-virus filtration-UFDF2 as described above.

[0436] The SP eluate was adjusted to pH 6.0, the virus was filtered, and the mixture was divided into two equal volumes; one of the two virus filtrate pools was used for each experimental feasibility run. An exemplary SP eluate pool pH adjustment buffer formulation was designed as shown in Table 8. A volume / volume addition method of approximately 0.075 L of adjustment buffer / L of SP eluate was determined to achieve a target pH of approximately 6.0.

[0437] Table 8: Exemplary pH Adjustment Buffers

[0438]

[0439] Upon receipt of the SP pooled eluate from the experimental run, a pH adjustment to 6.0 was performed; this adjustment resulted in an SP eluate having a pH of 5.98. The pH adjusted SP eluate was then virus filtered on a Planova 20N filter.

[0440] Table 9 includes a summary of the step yields for the individual unit operations. As observed, all UFDF runs exhibited similar recovery yields based on A280. The experimental virus filtration unit operation produced a step yield of 106.6%, indicating that no A280 adsorbed protein was lost during filtration. The virus filtration yield is important because it demonstrates that pH adjustment to approximately 6.0 allows the 20 nm diameter rhASA octameric molecule to dissociate into a dimeric form. It is believed that under acidic conditions (pH 5.0), the rhASA molecule exists as an octameric form; whereas, at neutral pH, it dissociates into a dimeric form. Because the Planova 20N has a 20 nm pore size, it is important that the rhASA protein molecule is not in the octameric form, as octameric rhASA is likely to be retained / lost within the virus filter. The step yield data demonstrate the feasibility of performing virus filtration prior to the UFDF operation by adjusting the pH of the SP eluate. These yield data indicate that there is no loss of rhASA during virus filtration and that pH adjustment is important for both purification efficiency and targeted pharmaceutical formulation pH.

[0441] Table 9: Summary of step yields

[0442]

[0443] Once virus filtration was complete, the virus filtrate pool was evenly divided into two portions. One portion was fed to experimental run 1 (UFDFDF) and the other portion was fed to experimental run 2 (UFDF).

[0444] As demonstrated below, both experimental runs achieved similar average permeabilities in the UF and initial DF segments. Furthermore, the second DF segment of Experiment 1 exhibited similar average permeabilities to the UF and initial DF segments. Both control runs, UFDF1 and UFDF2, consistently achieved lower average permeability values ​​compared to the feasibility run, likely due to variability within the scale of the utilized equipment.

[0445] Table 10: Average penetration rate and total treatment time

[0446]

[0447]

[0448] The product quality of the drug substance of the control and experimental runs was compared by a battery of analytical assays including: SEC-HPLC, RP-HPLC, activity, SDS-PAGE / HCP Western, SDS-PAGE (Coomassie), glycan mapping, peptide mapping, and metal analysis.

[0449] SEC-HPCL&RP-HPLC

[0450] Table 11 provides size exclusion (SEC-HPLC) and reverse phase (RP-HPLC) results. SEC and RP data indicate negligible differences between the experimental and control runs of the drug substance, indicating that the drug substances produced by the two experimental runs have comparable SEC and RP main peak percentages to those of the control run. These data confirm the feasibility of utilizing either experimental run to replace the dual UFDF control process, as no discernible differences in purity were identified.

[0451] Table 11: SEC-HPLC & RP-HPLC results

[0452]

[0453] active

[0454] Table 12 details the activity (U / mL) and specific activity (U / mg); the concentration (mg / mL) was determined by dividing the absorbance (AU) by the known absorption coefficient of 0.67. Although a lower specific activity value was observed for test run 1 (UFDFDF), it was well within the expected range of 50-140 U / mg in the instructions. Test run 2 (UFDF) yielded a specific activity comparable to the control run. All three runs (two test runs and one control) were comparable, indicating that the method used to obtain the formulated drug substance did not affect the drug substance activity.

[0455] Table 12: Activity and specific activity results

[0456]

[0457] Host cell protein (HCP) Western & SDS-PAGE (Coomassie)

[0458] Figure 5 and 6 SDS-PAGE / HCP Western blotting and SDS-PAGE (Coomassie) gel imaging are shown, respectively. Figure 5It was observed that the experimental (feasibility) and control run samples (lanes 5, 7, 9) exhibited similar banding patterns as the reference standard (lane 3), indicating that the drug substances from all runs exhibited comparable low levels of HCPs. Figure 6 Comparability between the experimental (feasibility) and control runs and the reference standard was also confirmed; no additional band patterns were detected by SDS-PAGE (Coomassie). These data indicate that the product quality of the drug substance from the experimental run conditions is comparable to that of the control run based on SDS-PAGE / HCP Western blotting and SDS-PAGE (Coomassie) analysis. No HCP bands with an intensity greater than the 15 kDa HCP band of the control were observed in the HPC Western blotting; and no new bands greater than 1% of the assay control intensity were observed.

[0459] Glycan map

[0460] Table 13 shows an exemplary glycan profile. All glycosylation forms were found to occur at similar levels in all samples tested. The results indicate that the product quality of the drug substance from the experimental (feasibility) run was comparable to that of the control run.

[0461] Table 13: Glycan mapping results

[0462]

[0463]

[0464] Metal Analysis and pH

[0465] The drug substance (DS) produced by the experiment and the control was also subjected to metal analysis to determine the residual phosphate level. Table 14 shows the residual phosphorus and final pH of each drug sample. It was found that the residual phosphorus was comparable under all operating conditions. It was also found that the pH was similar regardless of the operating conditions. If it is assumed that residual phosphate helps maintain the pH of the drug substance, the results of residual phosphorus are also consistent with the observation that the pH of all three batches of drug substances is maintained. These data show that both experimental (feasibility) runs obtained residual phosphate levels in the drug substance similar to the control run, and all three batches of drug substances were able to maintain a target pH of about 6.0 (i.e., 5.5-6.5) in the final formulation.

[0466] Metal analysis also detected elevated cap levels, as shown in Table 14. Calcium levels were fairly consistent across all samples. Previous studies have shown that calcium ions are present in the active site of rhASA and are important for enzyme activity.

[0467] Table 14: Residual phosphorus, calcium and pH results

[0468]

[0469] Peptide Mapping

[0470] Peptide mapping analysis was performed. Again, all samples gave similar profiles, indicating that the run conditions tested gave rhASA peptide maps comparable to the control.

[0471] in conclusion

[0472] The above experiments evaluated the feasibility of virus filtration before the UFDF operation by adjusting the pH of the SP eluate, the feasibility of a single UFDF step instead of two UFDF steps, and the feasibility of a single diafiltration (vs. double filtration) after the virus filtration step.

[0473] The high step yield of the virus filtration unit operation in the experimental feasibility run confirmed the feasibility of virus filtration before the UFDF operation. Because the virus filtrate makes rhASA molecules available for delivery to the filtrate, two UFDF feasibility runs were performed. Because all product quality and process performance metrics showed that the two feasibility runs were comparable to the control, it was determined that a single UFDF step is feasible. The results also confirmed the feasibility of direct single diafiltration of the final formulated solution (0.9% saline solution) (experimental feasibility run 2), as this process was able to produce a drug substance of comparable quality to that produced by the control and double diafiltration experiments (experimental feasibility run 1).

[0474] Based on these data, it has been determined that adjusting the pH to approximately 6.0, utilizing a buffer formulation such as that contained in Table 4, will produce a pH adjusted SP eluate suitable for virus filtration between final UFDF runs. Figure 2 The process exemplified in has been shown to be an alternative to processes that include a UFDF step followed by virus filtration and then another UFDF step. The pH-adjustment-virus filtration-UFDF process has been demonstrated to produce a pharmaceutical substance of comparable quality and purity to pharmaceutical substances produced by more complex, expensive, and time-consuming processes.

[0475] Example 9. Recombinant human arylsulfatase A (rhASA) pharmaceutical substance

[0476] The rhASA purified according to the above process was formulated at 35-45 mg / ml in 154 mM NaCl buffer with a pH of approximately 5.5-6.5.

[0477] The peptide and glycan profiles of the final pharmaceutical substance were consistent with those described in Example 8. DNA assay determined that residual host cell DNA was present in an amount of ≤100 pg / mg. Purity determined by reverse phase and size exclusion HPLC indicated greater than 98% and greater than 95% main peak area, respectively. Western blotting showed no host cell protein (HCP) bands with an intensity greater than the approximately 15 kDa HCP band intensity in the assay control. Only three HCP bands were detected. SDS-PAGE (Coomassie; reducing) analysis was consistent with the reference standard, with no new bands having an intensity greater than 1% of the assay control. The specific activity determined was 50-140 U / mg.

[0478] Equivalent form and scope

[0479] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.It is not intended that the scope of the invention be limited by the above description, but rather by the claims that follow.

[0480] The use of ordinal terms (such as "first," "second," "third," etc.) that modify claim elements in the claims does not in itself imply any priority, superiority, or order of one claim element over another or a temporal order in which method acts are performed, but serves merely as a label to distinguish one claim element having a certain name from another element having the same name (but using ordinal terms), thereby distinguishing the claim elements.

[0481] Unless expressly stated to the contrary, the articles "a" and "an" as used herein in the specification and claims should be understood to include plural referents. Unless the text indicates otherwise or otherwise clearly states, claims or descriptions including "or" between one or more members of a group should be considered to satisfy that one, more than one, or all of the group members are present, employed, or associated with a given product or method. The present invention includes embodiments in which only one member of the group is present, employed, or associated with a given product or method. The present invention also includes embodiments in which more than one or all of the group members are present, employed, or associated with a given product or method. Moreover, it is understood that, unless otherwise stated or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency would arise, the present invention covers all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive modifiers, etc., of one or more of the illustrated claims, introduced into another claim (or any other claim as if related) that is subordinate to the same base claim. When elements are presented as a list (e.g., in a Markush group or similar format), it is understood that it also discloses various subgroups of elements, and any element can be removed from the group. In general, it will be understood that where the present invention or aspects of the present invention include particular elements, features, etc., certain embodiments of the present invention or aspects of the present invention consist of or consist essentially of these elements, features, etc. For the sake of simplicity, those embodiments will not be specifically described in this document with so much verbiage in each case. It will be understood that any embodiment or aspect of the present invention may be explicitly excluded from the claims, regardless of whether such specific exclusion is stated in the specification. Publications, websites, or other reference materials cited herein that describe the background of the present invention and provide additional detailed details about its implementation are incorporated herein by reference.

Claims

1. A method for purifying recombinant arylsulfatase A (ASA) protein, the method comprising purifying recombinant arylsulfatase A (ASA) protein from impure preparations by performing one or more chromatography steps; combining the eluates from the one or more chromatography steps; Adjust the pH of the combined eluate to 5.8-6.5; and The pH adjusted eluate is subjected to a single step of ultrafiltration, diafiltration or a combined ultrafiltration and diafiltration step (UFDF) following chromatography to exchange the purified recombinant ASA protein into a saline based pharmaceutical formulation.

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

0.

3. The method of claim 1, wherein the pH is adjusted using a buffer comprising sodium phosphate, sodium chloride, and sodium citrate at pH 7.

0.

4. The method of claim 3, wherein the buffer comprises 0.1-0.5 M sodium phosphate, 0.5-2.5 M sodium chloride and 0.1-0.6 M sodium citrate, and the pH of the buffer is 7.

0.

5. The method of claim 1, wherein the ultrafiltration, diafiltration, or combined ultrafiltration and diafiltration step (UFDF) is diafiltration. The method of claim 1 , wherein the ultrafiltration is tangential flow ultrafiltration.

7. The method of claim 1, wherein the one or more chromatography steps comprise cation exchange chromatography.

8. The method of claim 7, wherein the cation exchange chromatography is the last chromatography step and the eluates from the cation exchange chromatography are combined before pH adjustment.

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

10. The method of claim 9, wherein the anion exchange chromatography is Q chromatography.

11. The method of claim 9, wherein the anion exchange chromatography comprises TMAE resin.

12. The method of claim 11, wherein once the impure product is loaded, the TMAE resin is washed using a first wash buffer comprising MES-Tris at pH 7.

0.

13. The method of claim 12, wherein the first wash buffer comprises 20-75 mM MES-Tris.

14. The method of claim 11, wherein the TMAE resin is washed with a second wash buffer comprising MES-Tris and NaCl at pH 7.

0.

15. The method of claim 14, wherein the second wash buffer comprises 5-75 mM MES-Tris and 50-150 mM NaCl, and the pH of the second buffer is 7.

0.

16. The method of claim 11, wherein once the impure product is loaded, the TMAE resin is further eluted using an elution buffer comprising MES-Tris and NaCl at pH 7.

0.

17. The method of claim 16, wherein the elution buffer comprises 5-75 mM MES-Tris and 150-300 mM NaCl, and the pH of the elution buffer is 7.

0.

18. The method of claim 9, wherein the anion exchange chromatography utilizes a column selected from the group consisting of: Q Sepharose TM Fast Flow Column, Q Sepharose TM High-performance columns, Q Sepharose TM XL, Capto TM Q, DEAE, TOYOPEARL Q. TMAE, Eshmuno TM Q. Nuvia TM Q or UNOsphere TM Q.

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

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

21. The method of claim 7, wherein performing one or more chromatography steps comprises performing anion exchange chromatography using TMAE resin, HA chromatography, phenyl chromatography, and cation exchange SP chromatography in the following order.

22. The method of claim 21, wherein the anion exchange chromatography using TMAE resin comprises using a column having a loading capacity greater than 4.5 g / L.

23. The method of claim 22, wherein the column has a loading capacity of 5-20 g / L.

24. The method of claim 1, further comprising ultrafiltration of the impure preparation, wherein ultrafiltration of the impure preparation is performed prior to the one or more chromatography steps.

25. The method of claim 24, wherein the ultrafiltration is tangential flow ultrafiltration.

26. The method of claim 24, wherein the ultrafiltration utilizes a membrane filter comprising a pore size having a molecular weight cut-off of at least 10 kDA.

27. The method of claim 24, wherein the ultrafiltration comprises ultrafiltration using a polyethersulfone or cellulose membrane.

28. The method of claim 24, wherein the ultrafiltration is followed by one or more steps of depth filtration and / or viral inactivation.

29. The method of claim 28, wherein the one or more steps of depth filtration and / or viral inactivation are followed by one or more chromatography steps.

30. The method of claim 28, wherein the step of inactivating viruses comprises adding a detergent to the impure preparation.

31. The method of claim 30, wherein the recombinant ASA protein is produced by mammalian cells cultured in suspension.

32. The method of claim 31 , wherein the mammalian cells are cultured in a bioreactor.

33. The method of claim 31, wherein the culture medium is serum-free.

34. The method of claim 33, wherein the serum-free medium is a chemically defined medium.

35. The method of claim 31, wherein the mammalian cell is a human cell.

36. The method of claim 1, wherein the impure product is feed from a perfusion bioreactor.

37. The method of claim 1, wherein the impure preparation is prepared from a serum-free medium containing recombinant ASA protein secreted by mammalian cells.

38. The method of claim 37, wherein the impure preparation is thawed from a frozen culture medium preparation.

39. The method of claim 1, wherein the ultrafiltration, diafiltration, or combined ultrafiltration and diafiltration step (UFDF) comprises exchanging the purified recombinant ASA protein into a pharmaceutical formulation buffer.

40. The method of claim 1, wherein the recombinant ASA protein has an amino acid sequence that is at least 70% identical to SEQ ID NO:

1.

41. The method of claim 1, wherein the recombinant ASA protein has an amino acid sequence identical to SEQ ID NO:

1.

42. The method of claim 1, wherein the purified recombinant ASA protein contains less than 100 ng / mg HCP.

43. The method of claim 1, wherein the purified recombinant ASA protein contains less than 60 ng / mg HCP.

44. The method of claim 1, wherein the purified recombinant ASA protein has no new bands greater than 1.0% of the intensity of the assay control when subjected to SDS-PAGE using Coomassie Brilliant Blue staining.

45. The method of claim 1, wherein the purified recombinant ASA protein contains less than 100 pg / mg host cell DNA.

46. ​​The method of claim 1, wherein the purified recombinant ASA protein contains less than 50 pg / mg host cell DNA.

47. A composition comprising recombinant ASA produced by the method of claim 1.

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