Recombinant human acidic sphingomyelinase pharmaceutical compositions and methods

Abstract

Disclosed herein are compositions comprising recombinant acidic sphingomyelinase (rASM) having desired purity, specific activity, and / or rASM subtypes. Also provided are methods for making and purifying such compositions, the methods comprising a chromatography step. Further provided are methods of modulating the specific activity of rASM in compositions, as well as methods of modulating rASM subtypes in compositions. The methods disclosed herein are particularly useful for the manufacture of pharmaceutical compositions comprising rASM for the treatment of acid sphingomyelinase deficiency (ASMD).

Description

Technical Field

[0001] The present invention relates to a composition of recombinant acid sphingomyelinase, such as recombinant human acid sphingomyelinase, and a method for producing the recombinant acid sphingomyelinase, such as recombinant human acid sphingomyelinase. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 321,636 filed on March 18, 2022, the disclosure of which is incorporated herein by reference. Sequence Listing This application contains a sequence listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The electronic copy of the sequence listing was created on March 15, 2023, is named 022548_WO042_SL.xml, and is 32,066 bytes in size. Background Art

[0002] Acid sphingomyelinase deficiency (ASMD) is a rare, life-threatening lysosomal storage disorder. It is an autosomal recessive genetic disease caused by mutations in the SMPD1 gene encoding the lysosomal enzyme acid sphingomyelinase (ASM) (Schuchman et al., Mol Genet Metab. (2017) 120 (1-2): 27-33). ASMD patients are unable to metabolize sphingomyelin, resulting in accumulation in the lysosomes of multiple organs, leading to visceral disease and neurodegeneration in severe cases. Cholesterol and other lipids in the spleen, liver, lungs and bone marrow of ASMD patients increase.

[0003] Olipudase alfa is a recombinant human acid sphingomyelinase that can significantly improve the key clinical manifestations of ASMD in adult and pediatric patients. However, there is still a need to produce pharmaceutical compositions containing orlipudase alfa with desired purity and consistent specific activity on a commercial scale. Summary of the invention

[0004] The present disclosure provides a method for purifying recombinant acid sphingomyelinase (rASM). In some embodiments, the method includes (i) subjecting a protein mixture comprising rASM and host cell protein (HCP) to cation exchange (CEX) chromatography; or subjecting a protein mixture comprising rASM and HCP to immobilized metal affinity chromatography (IMAC); or subjecting a protein mixture comprising rASM and HCP to both CEX chromatography and IMAC. In some embodiments, the method further includes (ii) collecting the eluent from the CEX chromatography or the IMAC to obtain a purified rASM preparation.

[0005] In some embodiments, the protein mixture is subjected to CEX chromatography and IMAC in series, and the eluate obtained from the CEX chromatography is subjected to the IMAC.

[0006] In some embodiments, the rASM is recombinant human acid sphingomyelinase (rhASM).

[0007] In some embodiments, the protein mixture is obtained from Chinese Hamster Ovary (CHO) cells expressing the rASM.

[0008] In some embodiments, the rASM comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0009] In some embodiments, the cation exchange chromatography comprises a resin selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S).

[0010] In some embodiments, the IMAC is a chelating resin.

[0011] In some embodiments, the IMAC is performed with zinc, copper, or nickel.

[0012] In some embodiments, the CEX chromatography method comprises washing the CEX chromatography column with a CEX wash buffer having a first optimal pH and a first optimal salt concentration, wherein the first optimal pH and the first optimal salt concentration are predetermined based on a starting specific activity of the resin and the protein mixture.

[0013] In some embodiments, the CEX chromatography method further comprises eluting the CEX chromatography column with a CEX elution buffer having a second optimal pH and a second optimal salt concentration, wherein rASM bound to the CEX chromatography column after the washing step is removed from the column at the second optimal pH and the second optimal salt concentration.

[0014] In some embodiments, the CEX wash buffer is selected from Table 2a, and the CEX elution buffer is selected from Table 2b.

[0015] In some embodiments, the IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, wherein the third optimal pH and the third optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

[0016] In some embodiments, the IMAC further comprises eluting the IMAC column with an IMAC elution buffer having a fourth optimal pH and a fourth optimal salt concentration, wherein rASM bound to the IMAC column after the washing step is removed from the column at the fourth optimal pH and the fourth optimal salt concentration.

[0017] In some embodiments, the IMAC wash buffer is selected from Table 3, and the IMAC elution buffer is selected from Table 4.

[0018] In some embodiments, the specific activity of the purified rASM preparation is about 5 to 50 U / mg. In some embodiments, the specific activity of the purified rASM preparation is about 10 to 45 U / mg. In some embodiments, the specific activity of the purified rASM preparation is about 10-20 U / mg. The specific activity of the purified rASM preparation is measured according to Example 4.

[0019] In some embodiments, the purity of the rASM preparation obtained is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0020] In some embodiments, the host cell protein (HCP) level of the rASM preparation obtained is no more than 1.0 μg / mg, no more than 2.0 μg / mg, no more than 3.0 μg / mg, no more than 4.0 μg / mg, or no more than 5.0 μg / mg.

[0021] In some embodiments, the purified rASM preparation comprises rASM isoforms with modifications that collectively do not exceed 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the entire rASM population.

[0022] In some embodiments, the modified rASM isoform comprises one or more modifications selected from C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

[0023] In some embodiments, the protein mixture is produced in a bioreactor at a production scale of at least 100 L.

[0024] In some embodiments, the protein mixture is produced in a bioreactor at a production scale of at least 500L.

[0025] In some embodiments, the method is performed partially or completely under refrigeration conditions of 8° C.±3° C. In some embodiments, the method is performed partially or completely at ambient temperature.

[0026] Also provided herein is a method for regulating the relative amount of a subtype of a recombinant acid sphingomyelinase (rASM) in an initial rASM composition, wherein the initial rASM composition comprises an unmodified rASM subtype and at least one rASM subtype having one or more modifications selected from C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation. In some embodiments, the method includes subjecting the initial rASM composition to cation exchange (CEX) chromatography; or subjecting the initial rASM composition to immobilized metal affinity chromatography (IMAC); or subjecting the initial rASM composition to both CEX chromatography and IMAC. In some embodiments, the method further includes collecting the eluent from the CEX chromatography or the IMAC to obtain a purified rASM preparation.

[0027] In some embodiments, the initial rASM composition is subjected to CEX chromatography and IMAC in series, and the eluate obtained from the CEX chromatography is subjected to the IMAC.

[0028] In some embodiments, the initial rASM composition is subjected to IMAC and CEX chromatography in series, and the eluate obtained from the IMAC is subjected to the CEX.

[0029] In some embodiments, the initial rASM composition is subjected to both the CEX chromatography and the IMAC separately with one or more additional steps in between.

[0030] In some embodiments, the rASM is recombinant human acid sphingomyelinase (rhASM).In some embodiments, the rASM comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0031] In some embodiments, the cation exchange chromatography comprises a resin selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S).

[0032] In some embodiments, the IMAC is a chelating resin column.

[0033] In some embodiments, the IMAC is performed with zinc, copper, or nickel.

[0034] In some embodiments, the CEX chromatography method comprises washing the CEX chromatography column with a CEX wash buffer having a first optimal pH and a first optimal salt concentration, wherein the first optimal pH and the first optimal salt concentration are predetermined based on a starting specific activity of the resin and the protein mixture.

[0035] In some embodiments, the CEX chromatography further comprises eluting the CEX chromatography column with a CEX elution buffer having a second optimal pH and a second optimal salt concentration, wherein at the second optimal pH and the second optimal salt concentration, all species bound on the CEX chromatography column after the washing step are removed from the column.

[0036] In some embodiments, the CEX wash buffer is selected from Table 2a, and the CEX elution buffer is selected from Table 2b.

[0037] In some embodiments, the IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, wherein the third optimal pH and the third optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

[0038] In some embodiments, the IMAC further comprises eluting the IMAC column with an IMAC elution buffer having a fourth optimal pH and a fourth optimal salt concentration, wherein at the fourth optimal pH and the fourth optimal salt concentration, all species bound on the IMAC column after the washing step are removed from the column.

[0039] In some embodiments, the IMAC wash buffer is selected from Table 3, and the IMAC elution buffer is selected from Table 4.

[0040] In some embodiments, the specific activity of the rASM preparation obtained is about 5 to 50 U / mg. In some embodiments, the specific activity of the rASM preparation obtained is about 10 to 45 U / mg. In some embodiments, the specific activity of the rASM preparation obtained is about 10 to 20 U / mg. The specific activity of the purified rASM preparation is measured according to Example 4.

[0041] In some embodiments, the host cell protein (HCP) level of the rASM preparation obtained is no more than 1.0 μg / mg, no more than 2.0 μg / mg, no more than 3.0 μg / mg, no more than 4.0 μg / mg, or no more than 5.0 μg / mg.

[0042] In some embodiments, the purified rASM preparation comprises rASM isoforms with modifications that collectively do not exceed 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the entire rASM population.

[0043] In some embodiments, the modification is selected from C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

[0044] In some embodiments, the starting composition comprising rASM is produced in a bioreactor at a production scale of at least 100L or at least 500L.

[0045] In some embodiments, the method is performed partially or completely under refrigeration conditions of 8° C.±3° C. In some embodiments, the method is performed partially or completely at ambient temperature.

[0046] A method for regulating the specific activity of a recombinant acid sphingomyelinase (rASM) in a liquid composition is further provided, the liquid composition comprising an unmodified rASM isoform and at least one rASM isoform having one or more modifications selected from C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation. In some embodiments, the method comprises subjecting the liquid composition to cation exchange (CEX) chromatography; or subjecting the liquid composition to immobilized metal affinity chromatography (IMAC); or subjecting the liquid composition to both CEX chromatography and IMAC. In some embodiments, the method further comprises collecting an eluate from the CEX chromatography or the IMAC to obtain a purified rASM preparation.

[0047] In some embodiments, the liquid composition is subjected to CEX chromatography and IMAC in series and the eluate obtained from the CEX chromatography is subjected to the IMAC, or wherein the liquid composition is subjected to IMAC and CEX chromatography in series and the eluate obtained from the IMAC is subjected to the CEX chromatography.

[0048] In some embodiments, the initial rASM composition is subjected to both the CEX chromatography and the IMAC separately with one or more additional steps in between.

[0049] In some embodiments, the rASM is recombinant human acid sphingomyelinase (rhASM).In some embodiments, the rASM comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0050] In some embodiments, the cation exchange chromatography comprises a resin selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S).

[0051] In some embodiments, the IMAC is a chelating resin column.

[0052] In some embodiments, the IMAC is performed with zinc, copper, or nickel.

[0053] In some embodiments, the CEX chromatography method comprises washing the CEX chromatography column with a CEX wash buffer having a first optimal pH and a first optimal salt concentration, wherein the first optimal pH and the first optimal salt concentration are predetermined based on a starting specific activity of the resin and the protein mixture.

[0054] In some embodiments, the CEX chromatography further comprises eluting the CEX chromatography column with a CEX elution buffer having a second optimal pH and a second optimal salt concentration, wherein at the second optimal pH and the second optimal salt concentration, all species bound on the CEX chromatography column after the washing step are removed from the column.

[0055] In some embodiments, the CEX wash buffer is selected from Table 2a, and the CEX elution buffer is selected from Table 2b.

[0056] In some embodiments, the IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, wherein the third optimal pH and the third optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

[0057] In some embodiments, the IMAC further comprises eluting the IMAC column with an IMAC elution buffer having a fourth optimal pH and a fourth optimal salt concentration, wherein at the fourth optimal pH and the fourth optimal salt concentration, all species bound on the IMAC column after the washing step are removed from the column.

[0058] In some embodiments, the specific activity of the rASM preparation obtained is about 5 to 50 U / mg. In some embodiments, the specific activity of the rASM preparation obtained is about 10 to 45 U / mg. In some embodiments, the specific activity of the rASM preparation obtained is about 10 to 20 U / mg. The specific activity of the purified rASM preparation is measured according to Example 4.

[0059] In some embodiments, the purity of the rASM in the formulation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0060] In some embodiments, the host cell protein (HCP) level of the rASM preparation obtained is no more than 1.0 μg / mg, no more than 2.0 μg / mg, no more than 3.0 μg / mg, no more than 4.0 μg / mg, or no more than 5.0 μg / mg.

[0061] In some embodiments, the purified rASM preparation comprises rASM isoforms with modifications that collectively do not exceed 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the entire rASM population.

[0062] In some embodiments, the modification is selected from C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

[0063] In some embodiments, the liquid composition comprising rASM is produced in a bioreactor at a production scale of at least 100 L or at least 500 L.

[0064] In some embodiments, the method is performed partially or completely under refrigeration conditions of 8° C.±3° C. In some embodiments, the method is performed partially or completely at ambient temperature.

[0065] The present disclosure also provides a recombinant acid sphingomyelinase (rASM) preparation, the rASM preparation comprising an unmodified rASM isoform and at least one rASM isoform with one or more modifications selected from C-terminal cysteinylation, S-glutathionylation, dimerization and truncation. In some embodiments, the unmodified rASM isoform accounts for at least 50%, 55%, 60%, 65%, 70%, 75% or 80% of the total rASM population in the rASM preparation. In some embodiments, the unmodified rASM isoform accounts for at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more of the total rASM population in the rASM preparation.

[0066] In some embodiments, all modified rASM isoforms together account for no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, or no more than 40% of the total rASM population in the rASM preparation.

[0067] In some embodiments, all modified rASM isoforms collectively account for no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5% or less of the total rASM population in the rASM preparation.

[0068] In some embodiments, the rASM isoform with C-terminal cysteinylation accounts for no more than 10% of the total rASM population in the rASM preparation. In some embodiments, the rASM isoform with C-terminal cysteinylation accounts for no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5% or less of the total rASM population in the rASM preparation.

[0069] In some embodiments, the rASM isoform with C-terminal S-glutathionylation accounts for no more than 5% of the total rASM population in the rASM preparation. In some embodiments, the rASM isoform with C-terminal S-glutathionylation accounts for no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% or less of the total rASM population in the rASM preparation.

[0070] In some embodiments, the rASM isoform with C-terminal dimerization accounts for no more than 0.2% of the total rASM population in the rASM preparation.

[0071] In some embodiments, the rASM isoform with C-terminal S-dimerization accounts for no more than 0.1% of the total rASM population in the rASM preparation.

[0072] In some embodiments, the rASM isoforms having a C-terminal truncation comprise no more than 8% of the total rASM population in the rASM preparation.

[0073] In some embodiments, the rASM isoform with a C-terminal truncation accounts for no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3% or less of the total rASM population in the rASM preparation.

[0074] In some embodiments, the rASM preparation is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more pure.

[0075] In some embodiments, the specific activity of the rASM preparation is about 5 to 50 U / mg. In some embodiments, the specific activity of the rASM preparation is about 10 to 20 U / mg. The specific activity of the purified rASM preparation was measured according to Example 4.

[0076] In some embodiments, the purity of the rASM in the formulation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0077] In some embodiments, the host cell protein (HCP) level of the rASM preparation obtained is no more than 1.0 μg / mg, no more than 2.0 μg / mg, no more than 3.0 μg / mg, no more than 4.0 μg / mg, or no more than 5.0 μg / mg.

[0078] In some embodiments, the rASM preparation is manufactured using the methods described herein.

[0079] Also provided is a pharmaceutical composition prepared by using the recombinant acid sphingomyelinase (rASM) preparation as described herein.

[0080] Further provided is a method of treating acid sphingomyelinase deficiency in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition as described herein. In some embodiments, the method further comprises the step of subjecting the purified rASM to a buffer exchange. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Different subtypes of rhASM with or without modifications are depicted.

[0082] Figure 2 (SEQ ID NO: 13-20) depicts the structure near the C-terminus of rhASM with or without modification. The C-terminal status of rhASM was monitored by LC-MS of native Asp-N digests of rhASM after MMTS labeling. Only the C-terminal amino acid of rhASM is shown.

[0083] Figure 3 The specific activities of enriched rhASM monomer and enriched rhASM dimer are depicted. The percent purity of each enriched population is shown above the corresponding bar.

[0084] Figure 4 The specific activities of rASM compositions containing various relative abundances of total C-terminal modifications are depicted.

[0085] Figure 5 Depicted are HCP clearance (upper graph) or recovery (lower graph) under different salt (NaCl) and pH conditions during the CEX chromatography step.

[0086] Figure 6 Contour plots depicting HCP clearance (left) and recovery (right) under different salt (NaCl) and pH conditions in the IMAC step.

[0087] Figure 7 HCP clearance (upper panel) or specific activity (lower panel) under different salt (NaCl) and pH conditions during the CEX chromatography step are depicted.

[0088] Figure 8 Contour plots of HCP clearance (left) and specific activity (right) under different salt (NaCl) and pH conditions in the IMAC step are depicted.

[0089] Fig. 9 Representative specific activities of rhASM in the load material, wash fractions and elution fractions of a CEX run are depicted. Multiple wash conditions (sodium chloride 45 mM at pH 6.3, pH 6.5 and pH 6.7) were tested as specified.

[0090] Fig. 10A The specific activity of rhASM in the load material, wash fraction and elution fraction of the IMAC method is depicted.

[0091] Fig. 10B The purity of rhASM in the loading material, wash fractions and elution fractions of the IMAC method is depicted. Multiple wash conditions were tested (mild: 10 mM sodium phosphate at pH 6.6; moderate: 10 mM sodium phosphate, 20 mM sodium chloride at pH 6.0; strong: 10 mM sodium phosphate, 80 mM sodium chloride at pH 5.8).

[0092] Fig.11 Depicted are representative normalized abundances of rhASM isoforms in the loading material, wash fractions, and elution fractions of a CEX run. Variant 1: unmodified rhASM isoform; Variant 2: modified rhASM isoform with C-terminal cysteine ​​cysteinylation; Variant 3: modified rhASM isoform with C-terminal S-glutathionylation; Variant 4: dimerized form 1; Variant 5: C-terminally truncated form 1. See Table 1 for detailed information on these isoforms.

[0093] Fig.12 Depicted are representative normalized abundances of rhASM isoforms in the loading material, wash fractions, and elution fractions of an IMAC run. Variant 1: unmodified rhASM isoform; Variant 2: modified rhASM isoform with C-terminal cysteine ​​cysteinylation; Variant 3: modified rhASM isoform with C-terminal S-glutathionylation; Variant 4: dimerized form 1; Variant 5: C-terminally truncated form 1. See Table 1 for detailed information on these isoforms.

[0094] Fig.13 A clinical study using purified rhASM (orlipase alpha) to treat ASMD patients is described.

[0095] Fig.14 Describe the predicted DL in patients treated with oriplasmase alfa or placebo CO% change. DLCO = diffusion capacity for carbon monoxide; FVC = forced vital capacity.

[0096] Fig.15 Depicted are high-resolution computed tomography (HRCT) scans of the lung before (left) and after (right) oripase alfa treatment. Sphingomyelin-filled macrophages are observed as ground-glass opacities.

[0097] Fig.16 Depicted are HRCT ground-glass appearance scores (left) and interstitial lung disease scores (right) in both lungs, which showed mean improvement in oripase alfa-treated patients but not in placebo-treated patients.

[0098] Fig.17 Depicted are the % change in spleen volume (left panel) and the change in splenomegaly-related scores (right panel) in oripase alfa-treated patients but not in placebo-treated patients.

[0099] Fig.18 Changes in % liver volume are depicted. ALT: alanine aminotransferase; AST: aspartate aminotransferase; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; MN: multiple of normal.

[0100] Fig.19 Depicted are the percentage of tissue area occupied by sphingomyelin in tissues obtained from patients treated with placebo or orlipase alfa.

[0101] Fig. 20 Depicted are representative toluidine blue stained images of liver biopsies from patients treated with placebo or orlipase alfa (sphingomyelin appears as a dark stain).

[0102] Fig.21 Depicted are normalized plasma chitotriosidase in patients treated with placebo and patients treated with oripase alfa (left panel), as well as pre-infusion plasma lyso-sphingomyelin levels in both populations. DETAILED DESCRIPTION

[0103] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described; because such methods and conditions are variable. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims.

[0104] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood.

[0105] As used herein, "about" with respect to a measurable value (e.g., an amount, a duration, etc.) is intended to encompass variations of +20% or ±10% (including ±5%, ±1%, and ±0.1%) relative to the specified value, as such variations are suitable for performing the disclosed methods.

[0106] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. Polypeptides can be of natural (tissue-derived) origin, recombinant or naturally expressed by prokaryotic or eukaryotic cell preparations, or chemically produced via synthetic methods. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids. Non-natural residues are described in detail in the scientific and patent literature.

[0107] As used herein, "peptide" includes peptides that are conservative variants of those peptides specifically exemplified herein. As used herein, "conservative variant" means that an amino acid residue is replaced with another biologically similar residue. Examples of conservative variants include, but are not limited to, replacing one hydrophobic residue (such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine) with another, or replacing one polar residue with another, such as replacing lysine with arginine, replacing aspartic acid with glutamic acid, or replacing asparagine with glutamine. Neutral hydrophilic amino acids that can replace each other include asparagine, glutamine, serine and threonine. "Conservative variant" also includes using substituted amino acids to replace unsubstituted parent amino acids, provided that antibodies that immunoreact with the unsubstituted polypeptide also produce immunoreactions with the substituted polypeptide. Such conservative substitutions are within the definition of the category of the peptides of the present invention.

[0108] "Recombinant" when used with respect to a protein means that the protein is produced by introducing a heterologous nucleic acid into a host cell.

[0109] As used herein, "loading" is a composition loaded onto a chromatographic material. Loading buffer is a buffer for loading a composition comprising a target product onto a chromatographic material. Before loading the composition to be purified, the chromatographic material can be balanced with an equilibrium buffer. In some examples, a wash buffer is used after the composition is loaded onto the chromatographic material and before the target polypeptide is eluted from the stationary phase. However, some target products (e.g., polypeptides) can be removed from the chromatographic material (i.e., in the flow-through) by a wash buffer.

[0110] As used herein, "elution" is to remove a product, such as a polypeptide, from a chromatographic material. An elution buffer is a buffer used to elute a polypeptide or other product of interest from a chromatographic material. In many cases, the elution buffer has a physical characteristic different from a loading buffer. For example, the elution buffer may have a conductivity different from the loading buffer or a pH different from the loading buffer. In some embodiments, the elution buffer has a conductivity lower than the loading buffer. In some embodiments, the elution buffer has a conductivity higher than the loading buffer. In some embodiments, the elution buffer has a pH lower than the loading buffer. In some embodiments, the elution buffer has a pH higher than the loading buffer. In some embodiments, the elution buffer has a conductivity different from the loading buffer and a different pH. The elution buffer may have any combination of higher or lower conductivity and higher or lower pH.

[0111] "Conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, electric current flows by ion transport. Therefore, as the amount of ions present in an aqueous solution increases, the solution will have a higher conductivity. The basic units of measurement for conductivity are Siemens (or mho), mho (mS / cm), and can be measured using a conductivity meter (such as various models of Orion conductivity meters). Since electrolytic conductivity is the ability of ions in a solution to carry an electric current, the conductivity of a solution can be changed by changing the concentration of ions in the solution. For example, the concentration of the buffer in the solution and / or the concentration of a salt (e.g., sodium chloride, sodium acetate, or potassium chloride) can be changed to achieve the desired conductivity. Preferably, the salt concentration of the various buffers is changed to achieve the desired conductivity.

[0112] "Host cell protein" (HCP) is a protein from a cell in which a polypeptide is produced. For example, CHOP is a protein from a host cell, i.e., Chinese hamster ovary protein. The amount of CHOP can be measured by enzyme-linked immunosorbent assay ("ELISA") or mass spectrometry. In some embodiments of any method described herein, the amount of HCP (e.g., CHOP) is reduced by greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. The amount of HCP can be reduced by about 10% to 99%, 30% to 95%, 30% to 99%, 50% to 95%, 50% to 99%, 75% to 99% or 85% to 99% between any one. In some embodiments, the amount of HCP is reduced by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98%. In some embodiments, the reduction is determined by comparing the amount of HCP in the composition recovered from one or more purification steps with the amount of HCP in the composition before one or more purification steps.

[0113] The present disclosure provides compositions comprising recombinant ASM, such as recombinant human ASM (rhASM). In some embodiments, the rhASM is oripase alpha. The compositions of the present disclosure have excellent consistency and purity. In some embodiments, the compositions of the present invention are pharmaceutical compositions, i.e., compositions that are in such a form or can be prepared to be in such a form as to allow the biological activity of the active ingredient to be effective while not containing additional ingredients that are significantly toxic in the patient or otherwise cause harmful side effects unrelated to the active ingredient. The terms "pharmaceutical composition" and "pharmaceutical preparation" are used interchangeably herein. The pharmaceutical compositions of the present invention can be used to treat patients suffering from ASM deficiency.

[0114] Drug substance consistency is an important consideration for the final step in manufacturing a drug substance / active pharmaceutical ingredient. It ensures consistent potency between batches, thus ensuring quality. Studies are necessary to ensure that the entire contents of a batch are homogeneous and consistent between batches.

[0115] During the purification process, conditions that lead to excellent host cell protein clearance may affect the specific activity of the product. The inventors have conducted research to identify key steps to ensure that the specific activity in the final product is well controlled during the purification process. As a result, the inventors have found that the ratio of rhASM subtypes (e.g., rhASM subtypes with C-terminal cysteine ​​modifications (including cysteinylation, S-glutathionylation, dimerization, and C-terminal truncation)) in the rhASM product is controlled by adjusting the relative amount of unmodified rhASM and modified rhASM subtypes, thereby controlling the specific activity of the final product. The discovery leads to a robust and effective control of product quality and process performance. Recombinant human acid sphingomyelinase

[0116] ASM is an enzyme that catalyzes the decomposition of sphingomyelin into ceramide and phosphorylcholine. "Recombinant human ASM" refers to human ASM prepared by recombinant means with or without certain amino acid modifications relative to the wild-type sequence. For example, recombinant human ASM can be expressed in cultured mammalian host cells (e.g., COS, CHO, HeLa, 3T3, 293T, NSO, SP2 / 0 or HuT 78 cells, etc.) or in animals in which the human ASM coding sequence is transgenic.

[0117] In some embodiments, the recombinant human ASM is oripase alpha. Oripase alpha is the glycoform alpha of human ASM (EC-3.1.4.12) produced in CHO cells. Mature oripase alpha is a 570 amino acid polypeptide that retains the enzymatic and lysosomal targeting activities of the native human protein. The amino acid sequence of oripase alpha, including its leader sequence (residues 1-57), is shown below as SEQ ID NO: 1, wherein the leader sequence is italicized and bold. The mature oripase alpha sequence (SEQ ID NO: 2, which spans residues 58-627 of SEQ ID NO: 1) does not have a leader sequence.

[0118] In other embodiments, human ASM useful in the present invention is 99%, 98%, 97%, 96% or 95% identical to oripase alpha in terms of amino acid sequence. For example, the human ASM in the composition may have the sequence shown in U.S. Pat. No. 6,541,218, the disclosure of which is incorporated herein by reference in its entirety. The sequence (SEQ ID NO: 3) is shown below, with the leader sequence (residues 1-59) in italics and bold, where the mature protein (SEQ ID NO: 4, which spans residues 60-629 of SEQ ID NO: 3) has no leader sequence.

[0119] The human ASM in the composition may also be identical in amino acid sequence to the human ASM or polymorphic variant thereof disclosed as sequence P17405-1 in the UNIPROT database. The P17405-1 sequence is shown below (SEQ ID NO: 5), the leader sequence (residues 1-59) is italicized and bold, and the mature protein (SEQ ID NO: 6, which spans residues 60-629 of SEQ ID NO: 5) has no leader sequence.

[0120] The rhASM DNA, diagnostic methods, and rhASM protein are covered by US Patent 5,773,278, US Patent 5,686,240, and US Patent 6,541,218, each of which is incorporated herein by reference in its entirety.

[0121] Recombinant human ASM (rhASM) produced in a host cell may exist as a mixture of one or more isoforms. In some embodiments, the isoforms are Figure 1 and Figure 2 The C-terminal status and the relative proportions of the species of C-terminal modifications can be determined by LC-MS analysis. Table 1. rhASM subtypes Cell culture methods

[0122] Compositions and methods for expressing recombinant ASM in host cells (such as Chinese hamster ovary cells) are described in U.S. Patent No. 5,773,278, which is incorporated herein by reference in its entirety. In order to express biologically active ASM, the coding sequence, functional equivalent or modified sequence of the enzyme can be inserted into an appropriate expression vector, that is, a vector containing the elements necessary for transcribing and translating the inserted coding sequence in an appropriate host cell. Host cell expression systems with cellular mechanisms and elements for appropriate processing (i.e., signal cleavage, glycosylation, phosphorylation and protein sorting) can be used. For example, mammalian host cell expression systems can be used for the expression of biologically active enzymes that are appropriately folded and processed. When administered in humans, such expression products should exhibit appropriate tissue targeting and should not exhibit adverse immune responses.

[0123] Methods well known to those skilled in the art can be used to construct expression vectors containing the ASM coding sequence and appropriate transcription / translation control signals. These methods include in vitro recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning A Laboratory Manual, Cold spring Harbor Laboratory, NY, Chapter 12 (1982).

[0124] In bacterial systems, a variety of expression vectors can be advantageously selected according to the purpose of the ASM protein expressed. For example, when a large amount of ASM is to be produced, a vector that directs the expression of a high level of a fusion protein product that is easily purified may be required. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. (1983) 2: 1791), in which the ASM coding sequence can be linked in frame to a vector with a lac Z coding region to produce a hybrid AS-lac Z protein; pIN vectors (Inouye and Inouye, Nucleic acids Res. (1985) 13: 3101-9; Van Heeke and Schuster, J Biol Chem. (1989) 264: 5503-9); etc.

[0125] Various eukaryotic host-vector systems can be used to express the ASM coding sequence. Although prokaryotic systems have the unique advantages of being easy to operate and having low amplification costs, their major drawback in ASM expression is the lack of appropriate post-translational modifications of expressed mammalian proteins. Eukaryotic systems and preferably mammalian expression systems allow for appropriate modifications. Eukaryotic cells with cell mechanisms that appropriately process primary transcripts (such as glycosylation, phosphorylation and favorable secretion of gene products) should be used as host cells for expressing ASM. Mammalian cell lines are preferred. Such host cell lines can include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, -293, WI38, etc.

[0126] For the long-term high yield production of recombinant protein, stable expression can be used. For example, after introducing foreign DNA, engineered cells can be grown in enriched medium for 1 to 2 days and then transferred to selective medium. Different from using expression vectors containing viral replication origins, host cells can be transformed with ATN or DNA controlled by suitable expression control elements (such as promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selective markers. The selective marker in the recombinant plasmid confers resistance to selection, and allows cells to stably integrate plasmids into their chromosomes and grow to form colonies, and then can be cloned and expanded into cell lines. A variety of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell (1977) 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc Natl Acad Sci. USA (1962) 48:2026), and adenine phosphoribosyltransferase (Lowy et al., Cell (1980) 22:817) genes, which can be used for tk-, hgprt-, or aprt- cells, respectively. Similarly, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci. USA (1980) 77:3567; O'Hare et al., Proc Natl Acad Sci. USA (1981) 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc Natl Acad Sci. USA (1981) 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J Mol Biol. (1981) 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., Gene (1984) 30:147). Recently, additional selection genes have been described, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine (Hartman and Mulligan, Proc Natl Acad Sci. USA (1988) 85:8047); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor (2-(difluoromethyl)-DL-ornithine, DFMO) (McConlogue L., in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed. (1987)).

[0127] Alternative eukaryotic expression systems that can be used to express the ASM enzyme are yeast transformed with a recombinant yeast expression vector containing the ASM coding sequence; insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing the ASM coding sequence; or plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the ASM coding sequence or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the ASM coding sequence.

[0128] In yeast, a variety of vectors containing constitutive or inducible promoters are available. For reviews, see Current Protocols in Molecular Biology, Vol. 2, 1988, ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Chapter 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, Methods in Enzymology, ed. Wu and Grossman, 31987, Acad. Press, NY, Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., DC, Chapter 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, ed. Berger and Kimmel, Acad. Press, NY, Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, ed. Strathern et al., Cold Spring Harbor Laboratory Press, 1986. Harbor Press, Volume I and Volume II. In order to carry out complementary determination in yeast, the cDNA of ASM can be cloned into a yeast episome plasmid (YEp), which replicates autonomously in yeast due to the presence of yeast 2μ rings. The cDNA can be cloned behind a constitutive yeast promoter (such as ADH or LEU2) or an inducible promoter (such as GAL) (Cloning in Yeast, Chapter 3, R.Rothstein in: DNA Cloning Volume 11, A Practical Approach, Editor DM Glover, 1986, IRL Press, Wash., DC). The construct may contain 5' and 3' untranslated regions of homologous ASM mRNA or those regions corresponding to yeast genes. The YEp plasmid is transformed with high efficiency, and the plasmid is very stable. Alternatively, it can be used to promote the integration of foreign DNA sequences into the vector in the yeast chromosome.

[0129] In the case of using plant expression vectors, the expression of the ASM coding sequence can be driven by any of a variety of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., Nature (1984) 310: 511-514), or the coat protein promoter of TMV (Takamatsu et al., EMBO J. (1987) 6: 307-311) can be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., EMBO J. (1984) 3: 1671-1680; Broglie et al., Science (1984) 224: 838-843) can be used; or heat shock promoters such as soybean hsp17.5-E or hsp17.3-B (Gurley et al., Mol Cell Biol. (1986) 6: 559-565). These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors; direct DNA transformation; microinjection, electroporation, etc. For a review of such techniques, see, e.g., Weissbach and Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson and Corey, 1988, Plant Molecular Biology, 2nd ed., Blackie, London, Chapters 7-9.

[0130] An alternative expression system that can be used to express ASM is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus is grown in Spodoptera frugiperda cells. The ASM sequence can be cloned into a non-essential region of the virus (e.g., a polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter). Successful insertion of the coding sequence will result in the inactivation of the polyhedrin gene and the generation of non-closed recombinant viruses (i.e., viruses lacking a protein coat encoded by a polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells expressing the inserted gene (see, e.g., Smith et al., J Viol. (1983) 46:584; Smith, U.S. Patent No. 4,215,051). Methods for purifying rASM and regulating its specific activity and isoforms

[0131] The present disclosure provides the unexpected discovery that by changing the purification method, one can adjust the specific activity of rASM (e.g., rhASM) and the ratio of rASM isoforms in a composition comprising rASM. This innovation can be used to adjust the specific activity of a batch of rASM to within a target range. This method of adjusting specific activity can be implemented more directly without significant impact on process performance relative to other potential methods (e.g., changing the cell culture method).

[0132] Therefore, the present disclosure provides the following methods: 1. A method for purifying rASM from a protein mixture. In some embodiments, the protein mixture comprises rASM and at least another protein. In some embodiments, the protein mixture comprises rASM and host cell proteins, such as CHO cell proteins. In some embodiments, the method comprises subjecting a protein mixture comprising rASM and host cell proteins (HCP) to cation exchange (CEX) chromatography, as described herein. In some embodiments, the method further comprises collecting the eluent from the CEX chromatography, thereby obtaining a purified rASM preparation. In some embodiments, the method comprises subjecting a protein mixture comprising rASM and HCP to immobilized metal affinity chromatography (IMAC), as described herein. In some embodiments, the method further comprises collecting the eluent from the IMAC chromatography, thereby obtaining a purified rASM preparation. In other embodiments, the method comprises subjecting a protein mixture comprising rASM and HCP to both CEX chromatography and IMAC, either individually or in series. The order of CEX and IMAC can be changed. For example, in some embodiments, the method includes (i) subjecting a protein mixture comprising rASM and host cell proteins (HCP) to CEX chromatography, as described herein. In some embodiments, the method further includes (ii) subjecting the eluent obtained from the CEX chromatography to IMAC directly or indirectly, as described herein. As used herein, the term "directly" means that the eluent obtained from the CEX chromatography is directly subjected to IMAC without being processed by another step, and the term "indirectly" means that before the eluent obtained from the CEX chromatography is subjected to IMAC, the eluent is processed by one or more additional steps. For example, before the eluent obtained from the CEX chromatography is subjected to IMAC, the eluent is passed through another purification step (e.g., another purification column). In some embodiments, the method further includes collecting the eluent from the IMAC, thereby obtaining a purified rASM preparation. Optionally, the order of CEX chromatography and IMAC can be exchanged. For example, in some embodiments, the method comprises (i) subjecting a protein mixture comprising rASM and HCP to IMAC, as described herein. In some embodiments, the method further comprises (ii) subjecting the eluate obtained from the IMAC directly or indirectly to CEX chromatography, as described herein. As used herein, the term "directly" means subjecting the eluate obtained from the IMAC directly to CEX chromatography without being processed by another step, while the term "indirectly" means that the eluate obtained from the IMAC is processed by one or more additional steps before being subjected to CEX chromatography.For example, before the eluent obtained from the IMAC is subjected to CEX chromatography, the eluent is passed through another purification step (e.g., another purification column). In some embodiments, the method further includes collecting the eluent from the CEX chromatography to obtain a purified rASM preparation. In some embodiments, in order to remove impurities (e.g., HCP) from the sample, one or more additional purification steps may be included before and / or after subjecting the sample to CEX chromatography and / or IMAC. Such purification steps are discussed in U.S. Patent Nos. 8,796,419, 9,481,706, 10,259,842 and PCT Publication Nos. WO2008 / 085988 A1 and WO 2019 / 121846A1, each of which is incorporated by reference in its entirety. II. Method for adjusting the relative amount of the subtype of recombinant acid sphingomyelinase (rASM) in the initial rASM composition. The relative amount of rASM subtypes in the composition is equal to the percentage of the normalized abundance of the subtype compared to the total rASM abundance when all rASM subtypes are combined. For example, if the normalized abundance of unmodified rASM in the composition is 9 million, and the total rASM abundance of all rASM subtypes in the composition is 10 million, the relative amount of unmodified rASM in the composition is 90%. The initial rASM composition may include unmodified rASM subtypes and at least one rASM subtype with one or more modifications selected from C-terminal cysteinylation, S-glutathionylation, dimerization and truncation. In some embodiments, the method includes subjecting the initial rASM composition to CEX chromatography, as described herein. In some embodiments, the method further includes collecting the eluent from the CEX chromatography, thereby obtaining the rASM preparation of the rASM subtype with the relative amount adjusted. In some embodiments, the method includes subjecting the initial rASM composition to immobilized metal affinity chromatography (IMAC), as described herein. In some embodiments, the method further includes collecting the eluent from the IMAC chromatography to obtain an rASM preparation with an adjusted relative amount of rASM subtypes. In other embodiments, the method includes subjecting the initial rASM composition to both CEX chromatography and IMAC individually or in series. The order of CEX and IMAC can be changed. For example, in some embodiments, the method includes (i) subjecting the initial composition comprising rASM to cation exchange (CEX) chromatography, as described herein. In some embodiments, the method further includes (ii) collecting the eluent from the cation exchange (CEX) chromatography to obtain an rASM preparation with an adjusted relative amount of rASM subtypes. In some embodiments, the method further includes subjecting the eluent obtained from the CEX chromatography to IMAC directly or indirectly. In some embodiments, the method further comprises collecting the eluate from the IMAC to obtain an rASM preparation having an adjusted relative amount of rASM isoforms. Optionally, the order of CEX chromatography and IMAC can be exchanged. For example, in some embodiments, the method comprises (i) subjecting an initial composition comprising rASM to IMAC, as described herein. In some embodiments, the method further comprises (ii) collecting the eluate from the IMAC to obtain an rASM preparation having an adjusted relative amount of rASM isoforms. In some embodiments, the method further comprises subjecting the eluate obtained from the IMAC to CEX chromatography directly or indirectly.In some embodiments, the method further comprises collecting the eluate obtained from the CEX chromatography, thereby obtaining a rASM preparation having an adjusted relative amount of rASM isoforms. In some embodiments, such methods increase the relative amount of unmodified rASM isoforms in the composition, while reducing the relative amount of at least one modified rASM isoform selected from C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation, as described herein. In some embodiments, the relative amount of the unmodified rASM isoform in the resulting composition is increased by at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2.0%, at least 2.0%, at least 2.5%, at least 2.0%, at least 2.5%, at least 2.0%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2.5%, at least 2 .1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3.0%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4.0%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5.0% or more. In some embodiments, the relative amount of the modified rASM isoform is reduced by at least 5%, at least 10%, 15%, at least 20%, 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more. In some embodiments, the relative amount of unmodified rASM isoform in the resulting composition is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more. III. A method for regulating the specific activity of a recombinant acid sphingomyelinase (rASM) in a liquid composition comprising an unmodified rASM isoform and at least one rASM isoform having one or more modifications selected from C-terminal cysteinylation, S-glutathionylation, dimerization and truncation. In some embodiments, the method includes subjecting the liquid composition to cation exchange (CEX) chromatography, as described herein. In some embodiments, the method further includes (ii) collecting the eluent from the cation exchange (CEX) chromatography, thereby obtaining an rASM preparation with regulated specific activity. In some embodiments, the method includes subjecting the liquid composition to immobilized metal affinity chromatography (IMAC), as described herein. In some embodiments, the method further includes collecting the eluent from the IMAC chromatography, thereby obtaining an rASM preparation with regulated specific activity. In other embodiments, the method includes subjecting the initial rASM composition to both CEX chromatography and IMAC individually or in series. The order of CEX and IMAC can be changed. For example, in some embodiments, the method includes (i) subjecting the liquid composition to cation exchange (CEX) chromatography, as described herein. In some embodiments, the method further includes (ii) collecting the eluent from the cation exchange (CEX) chromatography, so as to obtain a rASM preparation with a regulated specific activity. In some embodiments, the method further includes subjecting the eluent obtained from the CEX chromatography to IMAC directly or indirectly. In some embodiments, the method further includes collecting the eluent from the IMAC, so as to obtain a rASM preparation with a further regulated specific activity. Optionally, the order of CEX chromatography and IMAC can be exchanged. For example, in some embodiments, the method includes (i) subjecting the liquid composition to IMAC, as described herein. In some embodiments, the method further includes (ii) collecting the eluent from the IMAC, so as to obtain a rASM preparation with a regulated specific activity. In some embodiments, the method further includes subjecting the eluent obtained from the IMAC to CEX chromatography directly or indirectly. In some embodiments, the method further includes collecting the eluent from the CEX chromatography, thereby obtaining an rASM preparation with a further adjusted specific activity. In certain embodiments, the rASM preparation obtained has a reduced specific activity. In some embodiments, the specific activity in the rASM preparation obtained is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more.In some embodiments, the specific activity in the obtained rASM preparation is about 5-50 U / mg, such as about 10-40 U / mg, about 15-45 U / mg, about 10-30 U / mg, about 15-35 U / mg or about 10-20 U / mg. In some embodiments, the specific activity in the obtained rASM preparation is about 5 U / mg, about 10 U / mg, about 15 U / mg, about 20 U / mg, about 25 U / mg, about 30 U / mg, about 35 U / mg, about 40 U / mg, about 45 U / mg, about 50 U / mg.

[0133] In some embodiments, the rASM of the present disclosure can be produced in a host cell. For example, a host cell expressing rASM and / or a cell culture comprising rASM is collected to produce a harvest. In the methods described herein, the harvested cells and / or cell cultures can be used as desired or concentrated as is. In some embodiments, the harvest is concentrated. In some embodiments, before purification, the harvest is clarified by a suitable method (e.g., filtering) to produce a clarified harvest. In some embodiments, the harvest is cracked to produce a lysate.

[0134] In some embodiments, cation exchange (CEX) chromatography is used in the process to reduce host cell proteins and / or provide viral clearance. As used herein, the terms "CEX", "cation exchange medium", "cation exchange resin" and "cation exchange material" refer to a negatively charged solid phase, and it therefore has free cations for exchange with cations in an aqueous solution passing through or through the solid phase. The negatively charged ligand attached to the solid phase to form a cation exchange resin can be, for example, a carboxylate or a sulfonate. Commercially available cation exchange resins include, but are not limited to, carboxymethyl cellulose, sulfopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW from Pharmacia) and sulfonates. TM Or SP-SEPHAROSE HIGH PERFORMANCE TM ) and sulfonyl groups immobilized on agarose (e.g. S-SEPHAROSE FAST FLOW from Pharmacia TM). In some embodiments, the cation exchange chromatography comprises a resin selected from carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). In some embodiments, the CEX chromatography comprises (1) loading a liquid composition comprising rASM onto a CEX chromatography membrane or column; (2) washing the CEX chromatography membrane or column with a wash buffer; (3) eluting rASM from the CEX chromatography membrane or column with an elution buffer; and (4) collecting the eluate comprising rASM. The term "equilibrium buffer" refers to a buffer used to equilibrate a chromatography resin before loading a sample onto a chromatography. The term "wash buffer" refers to a buffer used to wash a chromatography resin after loading a sample onto a chromatography. In some embodiments, the wash buffer and the equilibration buffer are the same or different. In some cases, the wash buffer and the loading buffer may be the same. "Washing" the chromatography medium means passing an appropriate buffer through the medium or through the medium after loading the sample onto the chromatography medium. "Elution buffer" is used to elute the target protein from the solid phase. The conductivity and / or pH of the elution buffer usually causes the target protein to elute from the chromatographic resin. "Eluting" molecules (e.g., target polypeptides or impurities) from the chromatographic resin is intended to make the buffer compete with the target molecule for binding to the chromatographic resin by changing the solution conditions, or to weaken the binding interaction between the target molecule and the resin, resulting in the dissociation of the target molecule and removing the molecule from the resin. A non-limiting example is to make the buffer compete with the molecules for the charged sites on the ion exchange material by changing the ionic strength of the buffer around the ion exchange material, thereby eluting the molecules from the ion exchange resin. As used herein, the term "eluent" refers to a solution containing the target molecule obtained via elution and a flow-through fraction containing the target protein obtained due to the flow-through purification. In some embodiments, the term "eluent" refers to an elution pool collected from the binding and elution chromatography steps.

[0135] In some embodiments, the CEX chromatography method comprises (1) loading a composition comprising rASM onto a CEX chromatography membrane or a CEX chromatography column. In some embodiments, the composition comprises an unmodified rASM isoform and at least one modified rASM isoform, as described herein.

[0136] In some embodiments, the CEX chromatography further comprises (2) washing the membrane or the column with a wash buffer having a first optimal pH and a first optimal salt concentration. The first optimal pH and the first optimal salt concentration are predetermined based on the initial specific activity of the resin and the composition. In some embodiments, the wash buffer comprises a pH buffer system based on phosphate (such as sodium phosphate). In some embodiments, the sodium phosphate concentration is about 5 to 100 mM, such as about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM or about 100 mM.

[0137] In some embodiments, the wash buffer comprises a salt at an optimal salt concentration. In some embodiments, the salt is sodium chloride. In some embodiments, the sodium chloride concentration is about 5 to 100 mM, such as about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM or about 100 mM.

[0138] In some embodiments, the first optimal salt concentration and the first optimal pH are selected from the conditions in Table 2a below. Table 2a. Salt (NaCl) / pH conditions for CEX wash buffer

[0139] In general, a higher pH and / or higher salt concentration in the wash buffer results in higher purity (e.g., higher HCP clearance), but results in lower specific activity and lower recovery. Thus, depending on the target purity and specific activity, CEX wash conditions can be selected from CEX W1 to CEX W250. In some embodiments, the conditions are selected from CEX W1 to CEX W25; in some embodiments, the conditions are selected from CEX W26 to CEX W50; in some embodiments, the conditions are selected from CEX W51 to CEX W75; in some embodiments, the conditions are selected from CEX W76 to CEX W100; in some embodiments, the conditions are selected from CEX W101 to CEX W125; in some embodiments, the conditions are selected from CEX W126 to CEX W150; in some embodiments, the conditions are selected from CEX W151 to CEX W175; in some embodiments, the conditions are selected from CEX W176 to CEX W200; in some embodiments, the conditions are selected from CEX W201 to CEX W225; in some embodiments, the conditions are selected from CEX W226 to CEX W250. In some embodiments, the conditions are selected from CEX W36, CEX W61, CEX W86, CEX W111, CEX W37, CEX W62, CEX W87, CEX W112, CEX W38, CEX W63, CEX W88, CEX W113, CEX W39, CEX W64, CEX W89, CEX W114, CEX W40, CEX W65, CEX W90, and CEX W115. In some embodiments, the conditions are selected from CEX W66, CEX W91, CEX 116, CEX W67, CEX W92, CEX W117, CEX W68, CEX W93, CEX W118, CEX W69, CEX W94, CEX W119, CEX W70, CEX W95, and CEX W120. In some embodiments, the conditions are selected from CEX W141, CEX W166, CEX W191, CEX W142, CEX W167, CEX W192, CEX W143, CEX W168, CEX W193, CEX W144, CEX W169, CEX W194, CEX W145, CEX W170, and CEX W195.In some embodiments, the conditions are selected from CEX W136, CEX W161, CEX W186, CEX W137, CEX W162, CEX W187, CEX W138, CEX W163, CEX W188, CEX W139, CEX W164, CEX W189, CEX W140, CEX W165, and CEX W190.

[0140] In some embodiments, the CEX chromatography further comprises (3) eluting the membrane or the column with an elution buffer having a second optimal pH and a second optimal salt concentration. In some embodiments, the second optimal pH is the same or similar to the first optimal pH used in the wash buffer. In some embodiments, the second optimal salt concentration is predetermined according to the first optimal salt concentration in the wash buffer. Typically, a salt concentration higher than the first optimal salt concentration is used to elute the rASM bound to the membrane or column. In some embodiments, the salt concentration in the elution buffer is about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM, about 210mM, about 220mM, about 230mM, about 240mM or about 250mM. In some embodiments, the elution buffer comprises a pH buffer system based on phosphate (such as sodium phosphate). In some embodiments, the sodium phosphate concentration is about 5 to 100 mM, such as about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM. In some embodiments, the sodium phosphate concentration is the same or similar to the sodium phosphate concentration in the CEX wash buffer.

[0141] In some embodiments, the elution buffer comprises a salt at an optimal salt concentration. In some embodiments, the salt is sodium chloride.

[0142] In some embodiments, the second optimal salt concentration and the second optimal pH of the elution buffer are selected from the conditions in Table 2b below. Table 2b. Salt (NaCl) / pH conditions for CEX elution buffer

[0143] In some embodiments, the conditions are selected from CEX E1 to CEX E25; in some embodiments, the conditions are selected from CEX E26 to CEX E50; in some embodiments, the conditions are selected from CEX E51 to CEX E75; in some embodiments, the conditions are selected from CEX E76 to CEX E100; in some embodiments, the conditions are selected from CEX E101 to CEX E125; in some embodiments, the conditions are selected from CEX E126 to CEX E150; in some embodiments, the conditions are selected from CEX E151 to CEX E175; in some embodiments, the conditions are selected from CEX E176 to CEX E200; in some embodiments, the conditions are selected from CEX E201 to CEX E225; in some embodiments, the conditions are selected from CEX E226 to CEX E250. In some embodiments, the conditions are selected from CEX E36, CEX E61, CEX E86, CEX E111, CEX E37, CEX E62, CEX E87, CEX E112, CEX E38, CEX E63, CEX E88, CEXE113, CEX E39, CEX E64, CEX E89, CEX E114, CEX E40, CEX E65, CEX E90, and CEX E115. In some embodiments, the conditions are selected from CEX E66, CEX E91, CEX 116, CEX E67, CEX E92, CEX E117, CEX E68, CEX E93, CEX E118, CEX E69, CEX E94, CEX E119, CEX E70, CEX E95, and CEX E120. In some embodiments, the conditions are selected from CEX E141, CEX E166, CEX E191, CEX E142, CEX E167, CEX E192, CEX E143, CEX E168, CEX E193, CEX E144, CEX E169, CEX E194, CEX E145, CEX E170, and CEX E195. In some embodiments, the conditions are selected from CEX E136, CEX E161, CEX E186, CEX E137, CEX E162, CEX E187, CEX E138, CEX E163, CEX E188, CEX E139, CEX E164, CEX E189, CEX E140, CEX E165, and CEX E190.

[0144] In some embodiments, the CEX chromatography comprises any of the following wash / elution condition combinations: (1) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; (2) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; (3) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; (4) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; (5) CEX wash conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; (6) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; (7) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; (8) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; (9) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250.

[0145] The CEX chromatography step as described herein can be performed under refrigerated conditions (eg, 8°C ± 3°C) or at ambient temperature.

[0146] In some embodiments, the methods of the present disclosure further include immobilized metal affinity chromatography (IMAC) for reducing host cell proteins. As used herein, the term "IMAC" refers to an immobilized metal affinity chromatography (IMAC) based on transition metal ions (such as Zn 2 , Cu 2 、Ni 2 + and Co 2+) in aqueous solution for histidine or cysteine ​​affinity. Types of IMAC are described in Block et al., Methods in Enzymology (2009) 463: 439-73. In other embodiments, the IMAC resin is loaded with divalent ions. In still other embodiments, the divalent metal ion is nickel, copper, cobalt or zinc. In more specific embodiments, the divalent metal ion is zinc.

[0147] In some embodiments, the IMAC chromatography method comprises (1) loading a liquid composition comprising rASM onto an IMAC chromatography membrane or column; (2) washing the IMAC chromatography membrane or column with a wash buffer; (3) eluting rASM from the IMAC chromatography membrane or column with an elution buffer; and (4) collecting the eluate comprising rASM.

[0148] In some embodiments, the eluate obtained in the CEX chromatography is subjected to IMAC chromatography in bind and elute mode.

[0149] In some embodiments, the IMAC comprises (1) loading a composition comprising rASM onto an IMAC chromatography membrane or an IMAC chromatography column. In some embodiments, the composition comprises an unmodified rASM isoform and at least one modified rASM isoform, as described herein.

[0150] In some embodiments, the IMAC chromatography further comprises (2) washing the membrane or the column with a wash buffer having the third optimum pH and the third optimum salt concentration. The third optimum pH and the third optimum salt concentration are predetermined according to the initial specific activity of the resin and the composition. In some embodiments, the wash buffer comprises a pH buffer system based on phosphate (such as sodium phosphate). In some embodiments, the sodium phosphate concentration is about 1 to 100mM, such as about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM or about 100mM. In some embodiments, the third optimal salt concentration and the third optimal pH are selected from the conditions in Table 3 below. Table 3. Salt (NaCl) / pH conditions for IMAC wash buffer

[0151] In general, lower pH and / or higher salt concentration in the IMAC wash buffer results in higher purity (e.g., higher HCP clearance), but results in lower specific activity and lower recovery. Thus, depending on the target purity and specific activity, the IMAC wash conditions may be selected from IMAC W1 to IMAC W250. In some embodiments, the conditions are selected from IMAC W1 to IMAC W25; in some embodiments, the conditions are selected from IMAC W26 to IMAC W50; in some embodiments, the conditions are selected from IMAC W51 to IMAC W75; in some embodiments, the conditions are selected from IMAC W76 to IMAC W100; in some embodiments, the conditions are selected from IMAC W101 to IMAC W125; in some embodiments, the conditions are selected from IMAC W126 to IMAC W150; in some embodiments, the conditions are selected from IMAC W151 to IMAC W175; in some embodiments, the conditions are selected from IMAC W176 to IMAC W200; in some embodiments, the conditions are selected from IMAC W201 to IMAC W225; in some embodiments, the conditions are selected from IMAC W226 to IMAC W250. In some embodiments, the conditions are selected from IMAC W1, IMAC W26, IMAC W51, IMAC W76, IMAC W101, IMAC W2, IMAC W27, IMAC W52, IMAC W77, IMAC W102, IMAC W3, IMAC W28, IMAC W53, IMAC W78, IMAC W103, IMAC W4, IMAC W29, IMAC W54, IMAC W79, IMAC W104, IMAC W5, IMAC W30, IMAC W55, IMAC W80, and IMAC W105. In some embodiments, the conditions are selected from IMAC W126, IMAC, W151, IMAC W176, IMAC W201, IMAC W226, IMAC W127, IMAC W152, IMAC W177, IMAC W202, IMAC W227, IMAC W128, IMAC W153, IMAC W178, IMAC W203, IMAC W228, IMAC W129, IMAC W154, IMAC W179, IMAC W204, IMAC W229, IMAC W130, IMAC W155, IMAC W180, IMAC W205, and IMAC W230.In some embodiments, the conditions are selected from IMAC W6, IMAC W31, IMAC W56, IMAC W81, IMAC W106, IMAC W7, IMAC W32, IMAC W57, IMAC W82, IMAC W107, IMAC W8, IMAC W33, IMAC W58, IMAC W83, IMAC W108, IMAC W9, IMAC W34, IMAC W59, IMAC W84, IMAC W109, IMAC W10, IMAC W35, IMAC W60, IMAC W85, and IMAC W110. In some embodiments, the conditions are selected from IMAC W131, IMAC W156, IMAC W181, IMAC W206, IMAC W231, IMAC W132, IMAC W157, IMAC W182, IMAC W207, IMAC W232, IMAC W133, IMAC W158, IMAC W183, IMAC W208, IMAC W233, IMAC W134, IMAC W159, IMAC W184, IMAC W209, IMAC W234, IMAC W135, IMAC W160, IMAC W185, IMAC W210, and IMAC W235.

[0152] Optionally, the IMAC chromatography includes other washing steps (IMAC washing 2). In some embodiments, the second washing step includes using a pH lower than the pH used in the first IMAC washing step. In some embodiments, the second washing step includes using a pH lower than the pH used in the first IMAC washing step by about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0 or about 1.2. In some embodiments, the second washing step includes using the same or similar salt conditions as the salt conditions used in the first IMAC washing step. In some embodiments, the second washing step includes a salt concentration (e.g., NaCl) higher than the first washing. In some embodiments, the second washing step comprises at least 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM or more sodium chloride concentration.

[0153] In some embodiments, the second wash step comprises using a wash buffer having a salt concentration that is the same or similar to the salt concentration used in the first IMAC wash step.

[0154] In some embodiments, the IMAC chromatography further includes (3) washing the membrane or the column with an elution buffer having a fourth optimal pH and a fourth optimal salt concentration. In some embodiments, the elution buffer comprises a pH buffer system based on phosphate (such as sodium phosphate). In some embodiments, the sodium phosphate concentration is about 5 to 100mM, such as about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM or about 100mM. In some embodiments, the sodium phosphate concentration is the same or similar to the sodium phosphate concentration in the IMAC washing buffer. In some embodiments, the fourth optimal salt concentration and the fourth optimal pH are predetermined according to the third optimal salt concentration and the third optimal pH in the IMAC washing buffer.

[0155] In some embodiments, the fourth optimal salt concentration and the fourth optimal pH are selected from the conditions in Table 4 below. Table 4. Salt (NaCl) / pH conditions for IMAC elution buffer

[0156] Depending on the target purity and specific activity, the IMAC elution conditions can be selected from IMAC E1 to IMAC E250. In some embodiments, the conditions are selected from IMAC E1 to IMAC E25; in some embodiments, the conditions are selected from IMAC E26 to IMAC E50; in some embodiments, the conditions are selected from IMAC E51 to IMAC E75; in some embodiments, the conditions are selected from IMAC E76 to IMAC E100; in some embodiments, the conditions are selected from IMAC E101 to IMACE125; in some embodiments, the conditions are selected from IMAC E126 to IMAC E150; in some embodiments, the conditions are selected from IMAC E151 to IMAC E175; in some embodiments, the conditions are selected from IMAC E176 to IMACE200; in some embodiments, the conditions are selected from IMAC E201 to IMAC E225; in some embodiments, the conditions are selected from IMAC E226 to IMAC E250. In some embodiments, the conditions are selected from IMAC E13, IMAC E38, IMAC E63, IMAC E88, IMAC E113, IMAC E14, IMAC E39, IMAC E64, IMAC E89, IMAC E114, IMAC E15, IMAC E40, IMAC E65, IMAC E90, IMAC E115, IMAC E16, IMAC E41, IMAC E66, IMACE91, IMAC E116, IMAC E17, IMAC E42, IMAC E67, IMAC E92, IMAC E117, IMAC E18, IMACE43, IMAC E68, IMAC E93, and IMAC E118. In some embodiments, the conditions are selected from IMAC E138, IMACE163, IMAC E188, IMAC E213, IMAC E238, IMAC E139, IMAC E164, IMAC E189, IMAC E214, IMAC E239, IMAC E140, IMAC E165, IMAC E190, IMAC E215, IMAC E240, IMAC E141, IMACE166, IMAC E191, IMAC E216, IMAC E241, IMAC E142, IMAC E167, IMAC E192, IMAC E217, and IMAC E242.In some embodiments, the conditions are selected from IMAC E19, IMAC E44, IMAC E69, IMAC E94, IMAC E119, IMAC E20, IMAC E45, IMAC E70, IMAC E95, IMAC E120, IMAC E21, IMAC E46, IMAC E71, IMAC E96, IMAC E121, IMAC E22, IMAC E47, IMAC E72, IMAC E97, IMAC E122, IMAC E23, IMAC E48, IMAC E73, IMAC E98, and IMAC E123. In some embodiments, the conditions are selected from IMAC E143, IMAC E168, IMAC E193, IMAC E218, IMAC E243, IMAC E144, IMAC E169, IMACE194, IMAC E219, IMAC E244, IMAC E145, IMAC E170, IMAC E195, IMAC E220, IMAC E245, IMAC E146, IMAC E171, IMAC E196, IMAC E221, IMAC E246, IMAC E147, IMAC E172, IMACE197, IMAC E222, and IMAC E247.

[0157] In some embodiments, the IMAC comprises any of the following combinations of wash / elution conditions: (1) IMAC wash conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (2) IMAC wash conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (3) IMAC wash conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (4) IMAC wash conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (5) IMAC wash conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (6) IMAC wash conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (7) IMAC wash conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (8) IMAC wash conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (9) IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250.

[0158] The IMAC step as described herein can be performed under refrigerated conditions (eg, 8°C ± 3°C) or at ambient temperature.

[0159] In some embodiments, the method as described herein comprises both CEX chromatography and IMAC in series or individually (regardless of the order of CEX chromatography and IMAC), wherein the method comprises any of the following wash / elution conditions: (1) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (2) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (3) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (4) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (5) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (6) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (7) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (8) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (9) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (10) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; (11) IMAC wash conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (12) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (13) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (14) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (15) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (16) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMACE250; (17) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (18) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (19) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMACE250; (20) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (21) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (22) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (23) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (24) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (25) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (26) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (27) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (28) CEX washing conditions of any one of CEX W1 to CEX W25, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (29) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (30) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (31) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (32) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (33) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (34) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMACE250; (35) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (36) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (37) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMACE250; (38) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMACE75; (39) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (40) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMACE250; (41) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (42) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (43) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMACE250; (44) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (45) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (46) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (47) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMACE75; (48) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (49) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (50) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (51) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (52) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (53) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (54) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (55) CEX washing conditions of any one of CEX W101 to CEX W125, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (56) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (57) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (58) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (59) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (60) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (61) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (62) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (63) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (64) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E1 to CEX E25; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (65) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMACE75; (66) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (67) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (68) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (69) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (70) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (71) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMAC E75; (72) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (73) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E101 to CEX E125; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (74) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E51 to IMACE75; (75) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E126 to IMACE150; (76) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W1 to IMAC W25, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (77) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E51 to IMACE75; (78) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (79) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W76 to IMAC W100, and IMAC elution conditions of any one of IMAC E226 to IMAC E250; (80) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E51 to IMACE75; (81) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E126 to IMAC E150; (82) CEX washing conditions of any one of CEX W226 to CEX W250, and CEX elution conditions of any one of CEX E226 to CEX E250; IMAC washing conditions of any one of IMAC W176 to IMAC W200, and IMAC elution conditions of any one of IMAC E226 to IMAC E250. Recombinant acid sphingomyelinase composition

[0160] The present disclosure provides compositions comprising recombinant acid sphingomyelinase (rASM). In some embodiments, the rASM is recombinant human acid sphingomyelinase (rhASM). In some embodiments, the rhASM is orlipase alpha. In some embodiments, the rhASM comprises a polypeptide having SEQ ID NO: 1, 2, 3, 4, 5 or 6. In some embodiments, the rhASM comprises a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with any one of SEQ ID NO: 1-6, or a mixture thereof.

[0161] In some embodiments, the composition as described herein is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated according to the methods described herein. In some embodiments, the composition is in the form of a liquid formulation. In some embodiments, the composition is a lyophilized formulation.

[0162] In some embodiments, the composition is a rASM preparation, such as a rhASM preparation. In some embodiments, the preparation is a final product ready for therapeutic use or commercial sale. In some embodiments, the preparation is an intermediate product for downstream manufacturing.

[0163] In some embodiments, the disclosure provides containers (eg, vials) containing a composition described herein.

[0164] In some embodiments, the compositions of the present disclosure contain rhASM and exhibit excellent rhASM isoform identity and purity with well-controlled specific activity.

[0165] In some embodiments, the purity of the rASM composition of the present disclosure is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99%, at least 99.5% or more. The purity of the composition of the present disclosure can be determined by suitable methods known in the art. In some embodiments, purity is determined by HPLC (such as RP-HPLC).

[0166] In some embodiments, the rASM compositions of the present disclosure have unmodified rASM isoforms that account for at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the total rASM population in the rASM preparation. In some embodiments, the unmodified rASM isoforms in the rASM composition account for at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 85%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more. In some embodiments, all modified rASM isoforms collectively account for no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5% or less of the total rASM population in the rASM preparation.

[0167] In some embodiments, the rASM isoform with C-terminal cysteinylation accounts for no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5% or less of the total rASM population in the rASM preparation.

[0168] In some embodiments, the rASM isoform with C-terminal S-glutathionylation accounts for no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of the total rASM population in the rASM preparation. In some embodiments, the rASM isoform with C-terminal S-glutathionylation accounts for no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% or less of the total rASM population in the rASM preparation.

[0169] In some embodiments, the rASM subtype with C-terminal dimerization accounts for no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3% or no more than 0.2% of the total rASM population in the rASM preparation. In some embodiments, the rASM subtype with C-terminal dimerization accounts for no more than 0.1% of the total rASM population in the rASM preparation. The subtypes with C-terminal dimerization include, but are not limited to, those described in Table 1.

[0170] In some embodiments, the rASM subtype with C-terminal truncation accounts for no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8% of the total rASM population in the rASM preparation. In some embodiments, the rASM subtype with C-terminal truncation accounts for no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% or less of the total rASM population in the rASM preparation. The subtypes with C-terminal truncation include, but are not limited to, those described in Table 1.

[0171] The specific activity of the rASM composition is about 5 U / mg, about 6 U / mg, about 7 U / mg, about 8 U / mg, about 9 U / mg, about 10 U / mg, about 11 U / mg, about 12 U / mg, about 13 U / mg, about 14 U / mg, about 15 U / mg, about 16 U / mg, about 17 U / mg, about 18 U / mg, about 19 U / mg, about 20 U / mg, about 21 U / mg, about 22 U / mg, about 23 U / mg, about 24 U / mg, about 25 U / mg, about 26 U / mg, about 27 U / mg, about 28 U / mg, about 29 U / mg, about 30 U / mg, about 31 U / mg, about 32 U / mg, about 33 U / mg, about 34 U / mg, about 35 U / mg, about 36 U / mg, about 37 U / mg, about 38 U / mg, about 39 U / mg, about 40 U / mg, about 41 U / mg, about 42 U / mg, about 43 U / mg, about 44 U / mg, about 45 U / mg, about 46 U / mg, about 47 U / mg, about 48 U / mg, about 49 U / mg, about 50 U / mg, about 51 U / mg, about 52 U / mg, about 53 U / mg, about 54 U / mg, about 55 U / mg, about 56 U / mg, about 57 U / mg, about 58 U / mg, about 59 U / mg, about 60 U / mg, about 61 U / mg, about 62 U / mg, about 63 U / mg, about 64 U / mg U / mg, about 28U / mg, about 29U / mg, about 30U / mg, about 31U / mg, about 32U / mg, about 33U / mg, about 34U / mg, about 35U / mg, about 36U / mg, about 37U / mg, about 38U / mg, about 39U / mg, about 40U / mg, about 41U / mg, about 42U / mg, about 43U / mg, about 44U / mg, about 45U / mg, about 46U / mg, about 47U / mg, about 48U / mg, about 49U / mg or about 50U / mg. In some embodiments, the specific activity is about 5 to 50U / mg. In some embodiments, the specific activity is about 10 to 40U / mg. In some embodiments, the specific activity is about 10 to 30U / mg. In some embodiments, the specific activity is about 10 to 20U / mg. In some embodiments, the specific activity in the obtained rASM preparation is about 5-50U / mg, such as about 10-40U / mg, about 15-45U / mg, about 10-30U / mg, about 15-35U / mg or about 10-20U / mg. In some embodiments, the specific activity in the obtained rASM preparation is about 5U / mg, about 10U / mg, about 15U / mg, about 20U / mg, about 25U / mg, about 30U / mg, about 35U / mg, about 40U / mg, about 45U / mg, or about 50U / mg. The specific activity of the rASM composition can be determined by a suitable method known in the art. In some embodiments, the specific activity of the rASM composition is determined by a determination as described in Example 4.

[0172] The host cell protein (HCP) level of the recombinant ASM composition as described herein is no more than 5.0 μg / mg. In some embodiments, the HCP level of the rASM composition is no more than 5.0 μg / mg, no more than 4.5 μg / mg, no more than 4.0 μg / mg, no more than 3.5 μg / mg, no more than 3.0 μg / mg, no more than 2.5 μg / mg, no more than 2.0 μg / mg, no more than 1.5 μg / mg, no more than 1.0 μg / mg, no more than 0.9 μg / mg, no more than 0.8 μg / mg, no more than 0.7 μg / mg, no more than 0.6 μg / mg, no more than 0.5 μg / mg or less.

[0173] In some embodiments, the rASM compositions of the present disclosure may have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight features selected from the following: (1) a purity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99%, at least 99.5% or more; (2) the unmodified rASM isoforms in the rASM composition comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more of the total rASM population; (3) no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, or less of the total rASM population; (4) the rASM isoform containing C-terminal S-glutathionylation accounts for no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% or less of the total rASM population; (5) the rASM isoform containing C-terminal dimerization accounts for no more than 2%, no more than 1.9%, no more than 1.8%, no more than 1.7%, no more than 1.6%, no more than 1.5%, no more than 1.4%, no more than 1.3%, no more than 1.2%, no more than 1.1%, no more than 1.0%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2% or no more than 0.1% of the total rASM population; (6) rASM isoforms containing C-terminal truncations account for no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, or less of the total rASM population; (7) a specific activity of about 10 to 50 U / mg, about 15 to 35 U / mg, about 10 to 30 U / mg, or about 10 to 20 U / mg; and (8) Host cell protein (HCP) level not exceeding 5.0 μg / mg, not exceeding 4.5 μg / mg, not exceeding 4.0 μg / mg, not exceeding 3.5 μg / mg, not exceeding 2.0 μg / mg, not exceeding 1.5 μg / mg, not exceeding 1.0 μg / mg, not exceeding 0.5 μg / mg or less.

[0174] In some embodiments, the composition comprising recombinant acid sphingomyelinase (rASM) as described herein is produced by purifying the rASM expressed in a host cell using the method as described herein. In particular, the composition comprising rASM as described herein is produced by a method including a purification method as described herein. In other embodiments, the composition comprising rASM as described herein is purified using CEX chromatography as described herein. In other embodiments, the composition comprising rASM as described herein is purified using CEX chromatography and / or IMAC as described herein. Formulated pharmaceutical compositions comprising purified rhASM

[0175] The present disclosure also provides a pharmaceutical composition comprising a purified rASM (e.g., rhASM) as described herein. In some embodiments, the pharmaceutical composition is prepared by preparing a rASM preparation as described herein. In some embodiments, the preparation process does not change or significantly change the specific activity of rASM in the rASM preparation and / or the relevant ratio of rASM subtypes in the preparation. For example, after the preparation process, the specific activity of rASM in the composition is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least 10x, at least 15x, at least 20x, at least 30x, at least 40x, at least 50x or higher of the specific activity before the preparation process.

[0176] In some embodiments, the composition of the present invention contains one or more pharmaceutically acceptable excipients. "Excipient" refers to an inert substance used as a diluent, vehicle, carrier, preservative, binder or stabilizer for one or more active ingredients of a drug. For example, the composition may contain a buffer, an isotonic agent and / or a stabilizer (such as an antioxidant). In some cases, one reagent can be used for more than one of these purposes. In some embodiments, the composition of the present invention contains recombinant human ASM (such as orlipase α), a buffer (such as sodium phosphate or sodium citrate), a stabilizer (such as L-methionine) and a non-reducing sugar (such as sucrose or trehalose). Due to the specific composition in the composition, human ASM has improved stability. The composition of the present invention can be an aqueous liquid solution or a lyophilized preparation.

[0177] In some embodiments, the composition is an aqueous liquid composition comprising 1-10 mg / mL (e.g., 3-5 mg / mL) rhASM (e.g., oripase alpha); 10-50 mM (e.g., 10-30 mM) sodium phosphate; 70-150 mM (e.g., 80-120 mM) methionine (e.g., L-methionine); and 1%-10% (e.g., 4%-6%) w / v sucrose or trehalose. The pH of the aqueous liquid composition can be 5-8 (e.g., 6-7).

[0178] In some embodiments, the aqueous liquid composition does not contain a detectable amount of mannitol, the most readily used crystallization excipient, as it can significantly increase aggregation of human ASM during or after lyophilization of the aqueous liquid compositions described herein.

[0179] In some embodiments, the aqueous liquid composition comprises 0.004%-0.008%, 0.005%-0.007% or 0.005% w / v of one or more surfactants. Exemplary surfactants include nonionic detergents such as polysorbates (e.g., polysorbates 20 and 80) and poloxamers (e.g., poloxamer 188). In a specific embodiment, the aqueous liquid composition comprises 0.005% polysorbate 80. In some cases, the presence of one or more surfactants can help reduce turbidity in the liquid composition.

[0180] In some embodiments, the aqueous liquid composition comprises no more than 0.05, 0.01 or 0.005 mM of one or more chelating agents, such as EDTA and EGTA; in exemplary embodiments, the aqueous liquid composition does not contain a detectable amount of one or more chelating agents. In some cases, the presence of chelating agents at concentrations higher than, for example, 0.05 mM or 0.1 mM may increase the aggregation of human ASM and reduce its stability, particularly after extended storage times, such as 12-16 weeks, or under non-refrigerated conditions, such as at 25°C. In some embodiments, the aqueous liquid composition may contain 0-50 ppm (e.g., 15-30 ppm) of zinc, which may be carried over from the manufacturing process or added externally, for example.

[0181] In a specific embodiment, the aqueous liquid composition comprises or consists essentially of 4 mg / mL oripase alpha, 20 mM sodium phosphate, 100 mM methionine, and 5% (w / v) sucrose and has a pH of 6.5. The term "consisting essentially of" means that the composition does not contain detectable amounts of other ingredients, or may contain only trace amounts of certain materials derived from the protein manufacturing process, wherein such materials do not affect the biological activity of the enzyme or cause harm in human patients.

[0182] In some embodiments, the composition is an aqueous liquid composition comprising 1-20 mg / mL (e.g., 10 mg / mL) rhASM (e.g., orlipase alpha) and 10-50 mM (e.g., 20 mM) sodium phosphate. In certain embodiments, the aqueous liquid composition further comprises methionine (e.g., L-methionine) and sucrose or trehalose. In certain embodiments, the aqueous liquid composition further comprises 80-120 mM (e.g., 100 mM) methionine and 4%-6% (e.g., 5%) (w / v) sucrose. In specific embodiments, the aqueous liquid composition has a pH of 6.5.

[0183] In some embodiments, the composition is an aqueous liquid composition comprising 1-50 mg / mL (e.g., 3.8, 18 or 49 mg / mL) rhASM (e.g., orlipase alpha) and 10-50 mM (e.g., 20 mM) sodium phosphate. In certain embodiments, the aqueous liquid composition further comprises 1%-15% (e.g., 5%, 6%, 7% or 8%) sucrose or trehalose. In certain embodiments, the aqueous liquid composition further comprises 80-120 mM (e.g., 100 mM) methionine. In specific embodiments, the aqueous liquid composition has a pH of 6.5. The composition may comprise, for example, 3.8 mg / mL rhASM, 20 mM sodium phosphate and 5% sucrose; 18 mg / mL rhASM, 20 mM sodium phosphate and 5% sucrose; or 49 mg / mL rhASM, 20 mM phosphate and 8% sucrose.

[0184] The aqueous liquid composition can be prepared by mixing human ASM produced by recombinant technology and subsequently purified from host cells with excipients described herein in water, and adjusting the resulting mixture to the desired pH. For example, human ASM and the desired excipients can be added to or buffer exchanged into a sodium phosphate buffer having a desired sodium phosphate concentration and pH.

[0185] In some embodiments, the aqueous liquid composition can be prepared by reconstituting the lyophilized composition of the invention as described in further detail below. Reconstitution can be accomplished with a pharmaceutically acceptable liquid such as sterile water, saline (e.g., 0.9% sodium chloride), or phosphate buffered saline.

[0186] The present invention also provides lyophilized compositions. Such compositions can be prepared by lyophilizing the aqueous liquid compositions described herein. The lyophilized compositions are suitable for long-term storage. Lyophilization can be performed according to methods known in the art. For example, the liquid composition can be cooled to a temperature below zero (degrees Celsius) (e.g., -5°C to -80°C) that allows freezing, and then placed in a low pressure (partial vacuum) chamber to allow sublimation (primary drying) to occur; if necessary, the temperature of the composition can be increased in the second stage of drying (secondary drying) to further remove unwanted water molecules. In some embodiments, after the lyophilization process is completed, an inert gas (such as nitrogen) can be introduced into the container (e.g., glass vial) of the composition before the container is sealed.

[0187] In some embodiments, the invention provides powdered compositions, which can be prepared, for example, by spray drying an aqueous liquid composition as described herein. The spray-dried composition is suitable for long-term storage. Spray drying can be performed according to methods known in the art. For example, the liquid composition can be forced to pass through an atomizer or nozzle to disperse it into a hot air stream in a chamber as small droplets of controlled size, so that the liquid composition is quickly dried to a powder. The dried powder can then be collected at the bottom of a drying chamber. Other drying methods for preparing powdered compositions are also contemplated.

[0188] Sucrose (or trehalose) and methionine present in the amounts described herein during lyophilization provide excellent results; the lyophilized product forms an elegant cake while maintaining the stability of human ASM during storage. Under refrigerated conditions (e.g., at 0°C-10°C, 2°C-8°C, or 4°C), the human ASM in the lyophilized composition of the present invention can remain non-aggregated and biologically active for at least 4 months (e.g., at least 6 months or at least 12 months).

[0189] In some embodiments, the composition of the present invention is a lyophilized pharmaceutical composition comprising 4%-50% oripase alpha, 3%-7% sodium phosphate, and 45%-90% sucrose (all w / w percentages). In certain embodiments, the lyophilized composition comprises 5.5% oripase alpha, 20.6% L-methionine, 2.3% disodium hydrogen phosphate heptahydrate, 2.6% sodium dihydrogen phosphate monohydrate, and 69.0% sucrose (all w / w percentages). In certain embodiments, the lyophilized composition comprises 6.6% oripase alpha, 3.0% disodium hydrogen phosphate heptahydrate, 3.3% sodium dihydrogen phosphate monohydrate, and 87.1% sucrose (all w / w percentages). In certain embodiments, the lyophilized composition comprises 25.2% oripase alpha, 2.4% disodium hydrogen phosphate heptahydrate, 2.6% sodium dihydrogen phosphate monohydrate, and 69.9% sucrose (all w / w percentages). In certain embodiments, the lyophilized composition comprises 47.8% Oripase alpha, 1.7% sodium phosphate dibasic heptahydrate, 1.8% sodium phosphate dibasic monohydrate, and 48.8% sucrose (all w / w percentages).

[0190] In some embodiments, the composition of the present invention is a lyophilized pharmaceutical composition comprising 4%-7% oripase alpha, 15-25% L-methionine, 3%-7% sodium phosphate, and 65%-75% sucrose (all w / w percentages). In a specific embodiment, the lyophilized composition comprises 5.5% oripase alpha, 20.5% L-methionine, 2.3% disodium hydrogen phosphate heptahydrate, 2.6% sodium dihydrogen phosphate monohydrate, and 68.6% sucrose (all w / w percentages). In certain embodiments, the lyophilized composition may also comprise, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0% moisture.

[0191] In some embodiments, the present invention provides a vial containing a lyophilized pharmaceutical composition comprising 15-25 mg of oripase alpha, 75-85 mg of L-methionine, 15-25 mg of sodium phosphate, and 250-300 mg of sucrose. Prior to use, the composition can be reconstituted in 4-6 mL of sterile water.

[0192] In some embodiments, the vial contains a lyophilized pharmaceutical composition comprising or consisting of 21.2 mg, 20.1 mg, 95.4 mg, or 259.7 mg of orlplase alpha; 9.0 mg of disodium hydrogen phosphate heptahydrate; 10.0 mg of sodium dihydrogen phosphate monohydrate; and 265 mg of sucrose. The lyophilized composition may optionally include 79.1 mg of L-methionine. The lyophilized pharmaceutical composition may optionally include 0-0.3 mg (e.g., 0.08-0.16 mg) of zinc, which may be carried over from the manufacturing process or added externally, for example. In certain embodiments, the vial may have an internally sterile nitrogen-filled atmosphere. In a specific embodiment, the lyophilized composition may be reconstituted in 5.1 mL of sterile water to produce an orlplase alpha concentration of about 4.0 mg / mL, 3.8 mg / mL, 18 mg / mL, or 49 mg / mL, respectively. Based on the dose to be administered, the reconstituted composition may be further diluted to a specific volume in 0.9% sodium chloride solution.

[0193] In a specific embodiment, the vial contains a lyophilized pharmaceutical composition comprising or consisting of 21.2 mg of orlplase alpha, 79 mg of L-methionine, 9.0 mg of disodium hydrogen phosphate heptahydrate, 10.0 mg of sodium dihydrogen phosphate monohydrate, and 265 mg of sucrose. The lyophilized pharmaceutical composition may optionally include 0-0.3 mg (e.g., 0.08-0.16 mg) of zinc, which may be carried from the manufacturing process or added externally, for example. In certain embodiments, the lyophilized pharmaceutical composition is in the form of a cake or a lyophilized powder. In certain embodiments, the vial may have an internally sterile nitrogen-filled atmosphere. In a specific embodiment, the lyophilized composition may be reconstituted in 5.1 mL of sterile water to produce an orlplase alpha concentration of about 4.0 mg / mL. Based on the dose to be administered, the reconstituted composition may be further diluted to a specific volume in 0.9% sodium chloride solution.

[0194] In some embodiments, the present invention provides a vial containing a lyophilized pharmaceutical composition comprising 3-5 mg oripase alpha, 15-17 mg L-methionine, 3-5 mg sodium phosphate, and 50-60 mg sucrose. Prior to use, the composition can be reconstituted in 0.8-1.2 mL of sterile water.

[0195] In a specific embodiment, the vial contains a lyophilized pharmaceutical composition comprising or consisting of 4.8 mg of oripase alpha, 17.9 mg of L-methionine, 2.0 mg of disodium hydrogen phosphate heptahydrate, 2.3 mg of sodium dihydrogen phosphate monohydrate, and 60 mg of sucrose. In certain embodiments, the lyophilized pharmaceutical composition is in the form of a cake or a lyophilized powder. The lyophilized composition may optionally include 0-0.06 mg of zinc, which may be carried over from the manufacturing process or added externally, for example. In certain embodiments, the vial may have an internally sterile nitrogen-filled atmosphere. In a specific embodiment, the lyophilized composition may be reconstituted in 1.1 mL of sterile water to produce an oripase alpha concentration of about 4.0 mg / mL. Based on the dose to be administered, the reconstituted composition may be further diluted to a specific volume in 0.9% sodium chloride solution. Products

[0196] The rASM purified by the methods described herein and / or the formulation comprising the rASM purified by the methods described herein may be included in the product. The product may include a container containing the rASM and / or the rASM formulation. In certain embodiments, the product comprises: (a) a container containing a composition containing the rASM and / or the rASM formulation described herein; and (b) a drug instruction sheet having instructions for administering the formulation to a subject.

[0197] The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds or contains the preparation and can have a sterile access port (for example, the container can be an intravenous fluid bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is the polypeptide. The label or drug instructions indicate the use of the composition in the subject, as well as specific instructions on the dosage and interval of the polypeptide and any other drugs provided. The article can further include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the container is a syringe. In some embodiments, the syringe is further contained in an injection device. In some embodiments, the injection device is an automatic syringe.

[0198] "Product package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product and / or warnings about the use of such therapeutic products. Example

[0199] The following examples are put forward to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions featured in the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Example 1: Effect of purification on the specific activity of rhASM

[0200] During process characterization studies, efforts were made to better understand the factors that influence the specific activity of rhASM. The goal of such process characterization studies is to understand how the manufacturing process affects the specific activity of the rhASM product and to identify the critical steps that adequately control the specific activity of the product.

[0201] There are several different rhASM isoforms in the ASM population produced in the bioreactor. The detailed information of the rhASM isoforms is described in Table 1. Studies have shown that the dimerized form of rhASM has a higher specific activity ( Figure 3). Similarly, isoforms of orlipase α with a chemical modification at the C-terminal cysteine ​​(C570) had higher specific activity than the unmodified form, likely due to the association of C570 with the active site. The study further demonstrated a correlation between the abundance of the C-terminal modified form and increased specific activity ( Figure 4 ). Essentially, if a higher abundance of modified rhASM is present in a composition, the composition will have a higher specific activity.

[0202] The sample containing rhASM was subjected to cation exchange (CEX) chromatography. The rhASM was bound to the resin while impurities passed through. Post-loading washes achieved further impurity reduction and then Orlipase alpha was eluted from the column by increasing the salt concentration (compared to the salt concentration of the wash buffer).

[0203] The cation exchange (CEX) chromatography eluent is further purified by immobilized metal affinity chromatography (IMAC). The resin is loaded with metal ions. The cation exchange (CEX) chromatography eluent is adjusted to the target pH and loaded into the balanced IMAC column. The rhASM is then eluted with a high salt elution buffer.

[0204] The immobilized metal affinity chromatography (IMAC) eluates were collected, mixed and concentrated. The bulk samples obtained were then formulated as described in Example 2.

[0205] It was observed that the purity of the rhASM preparation (as measured by RP-HPLC) was well controlled during the CEX chromatography and / or IMAC steps. Figure 5 As shown in , in the CEX chromatography step, greater HCP clearance is achieved at higher pH and higher salt concentrations, but greater HCP clearance results in lower recovery yields. Figure 6 As shown, in the IMAC step, greater HCP clearance was achieved at lower pH and higher salt concentrations, but greater HCP clearance also resulted in lower yields. Recovery was measured by both A280 absorbance and activity levels. Assay and ELISA to measure HCP levels.

[0206] Surprisingly, conditions that resulted in excellent HCP clearance had a negative impact on specific activity. Figure 7 As shown in Figure 2, in the CEX chromatography step, although greater HCP clearance was obtained under higher pH, higher salt conditions, the resulting rhASM preparation had lower specific activity. Figure 8 As shown, in the IMAC step, although greater HCP clearance was obtained under lower pH, higher salt conditions, the resulting rhASM preparation had a lower specific activity.

[0207] The inventors hypothesized that the highly active rhASM isoforms bind weakly to the chromatography column and are lost during the washing steps of the chromatography step.

[0208] To test this hypothesis, during the CEX chromatography step, the loading material (e.g., product pool - rhASM sample before the CEX chromatography step), the wash fraction obtained during the CEX chromatography step, and the elution fraction obtained during the CEX chromatography step were analyzed to determine their specific activities in vitro, as described in Example 4. Indeed, the wash fraction collected from the CEX run exhibited a greater specific activity ( 1.3 % ) compared to the initial loading material and the elution fraction. Fig. 9 , under the indicated wash conditions). Several other salt concentration / pH wash conditions were also tested and resulted in similar results.

[0209] In the IMAC step, the loading material (e.g., product pool - rhASM sample before the IMAC chromatography step), the wash fraction obtained in the IMAC chromatography step, and the elution fraction obtained in the IMAC chromatography step were also analyzed to determine their specific activity in vitro. Similarly, the wash fraction collected from the IMAC run exhibited a greater specific activity ( Fig. 10A , under specified washing conditions). Fig. 10B The IMAC operation was shown to further improve the purity of rhASM in the eluted fraction. Purities exceeding 98% or 99% were achieved. Several other salt concentration / pH conditions were also tested, which led to similar results.

[0210] For further validation, the loading material, wash fractions, and elution fractions from the CEX run and IMAC run were submitted for mass spectrometry analysis to quantify the unmodified and modified isoforms. Prior to analyzing the ASM isoform progeny, the free cysteine ​​residues of rhASM were labeled. The relative abundance of the unmodified and modified isoforms is depicted in Fig.11 (CEX operations) and Fig.12 (IMAC operation). Indeed, in both operations, the rhASM isoforms with C-terminal modifications were enriched in the wash fractions.

[0211] It was also observed that the relative abundance of unmodified rhASM isoforms and modified rhASM isoforms changed in both the wash fraction and the elution fraction by CEX operation. LC-MS analysis showed that the relative abundance of unmodified rhASM isoforms was enriched in the elution fraction compared with the loading material. The total amount of modified rhASM isoforms was lower in the elution population compared with the loading material. Therefore, CEX operation can adjust the ratio between unmodified rhASM isoforms and modified rhASM isoforms in the rhASM product.

[0212] In summary, the optimization of the two binding and elution chromatography steps in the manufacturing process of Oripase α produced unexpected effects on the specific activity and rhASM subtype population. It was observed that the specific activity of both operations was reduced, and high specific activity was observed in the wash fractions of both operations. Mass spectrometry identified weakly bound "active" rhASM subtypes, which were enriched in the wash fractions and reduced in the elution fractions. The analytical results of both studies showed that the specific activity in the CEX operation or IMAC operation eluate can be reduced by selectively removing highly active C-terminal modified species in the washing step. In addition, the operation can adjust the ratio between unmodified rhASM subtypes and modified rhASM subtypes in the rhASM preparation, thereby improving the uniformity of the rhASM population in the final product. The method described herein allows the specific activity and the ratio of unmodified rhASM subtypes and modified rhASM subtypes to be adjusted during the manufacturing process, thereby obtaining an rhASM preparation with a desired specific activity. Example 2: Recombinant human acid sphingomyelinase formulation

[0213] The formulation steps are performed to achieve the final oripase alfa and excipient concentrations in the drug substance.

[0214] The formulation was performed according to the method described in WO 2019 / 227029 A1, which is incorporated herein by reference in its entirety. The rhASM preparation obtained from the purification process in Example 1 was filtered to remove viral contaminants and concentrated to increase protein concentration before formulation. The formulated bodies were mixed and the rhASM concentration was determined by measuring the absorbance at 280 nm. The product pool was then further diluted to the target volume to obtain the final bulk drug orlipase alpha concentration.

[0215] Optionally, the liquid composition obtained herein can be spray dried. The spray dried composition is suitable for long term storage. Spray drying can be performed according to methods known in the art. For example, the liquid composition can be forced through an atomizer or nozzle to disperse it as small droplets of controlled size into a hot air stream in a chamber, thereby allowing the liquid composition to be quickly dried into a powder. The dried powder can then be collected at the bottom of the drying chamber. Other drying methods for preparing powdered compositions are also contemplated. Example 3: Efficacy, safety, pharmacodynamics and pharmacokinetic studies of oripase alfa in patients with acid sphingomyelinase deficiency

[0216] The recombinant human oripase alpha formulation prepared in Example 2 was used in a clinical study to evaluate its efficacy. The study design is shown in Fig.13 Baseline patient and disease characteristics are provided in Table 6. Table 6. Baseline Patient and Disease Characteristics DL CO = diffusion capacity for carbon monoxide; MN = multiple of normal; SD = standard deviation; SRS = splenomegaly correlation score

[0217] The primary objectives of this study were to evaluate the efficacy of a formulation administered intravenously every 2 weeks for 52 weeks in adult patients with acid sphingomyelinase deficiency (ASMD) by assessing changes in: 1) spleen volume as measured by abdominal magnetic resonance imaging (MRI) (and for the United States [US] only, combined with the patient's relative perception of spleen volume as measured by the Splenomegaly Related Score [SRS]); and 2) infiltrative lung disease, as measured by the diffusing capacity of the lung for carbon monoxide (DLCO) as measured by pulmonary function testing. Primary endpoint - prediction of DL CO % Change

[0218] like Fig.14 The results shown indicate that at baseline, the mean predicted DL CO % were similar and reflected moderate disease; the mean improvement from baseline to week 52 was 22% in the orlipase alfa group compared with 3% in the placebo group; the difference between the groups at week 52 was 19% (p<0.0004), and improvements were observed as early as week 26; the study was declared positive; and at week 52, mean predicted FVC % also improved in the orlipase alfa group but not in the placebo group.

[0219] Lung imaging studies have also demonstrated improvements in ASMD-mediated interstitial lung disease. Illustrative high-resolution computed tomography images from a patient treated with oriplasmase alfa ( Fig.15) showed the clearance of "ground-glass" shadows caused by sphingomyelin-filled macrophages. HRCT ground-glass appearance scores and interstitial lung disease scores in both lungs showed mean improvement in oripase alfa-treated patients, but not in placebo-treated patients ( Fig.16 ). Primary endpoint - spleen response

[0220] Spleen volume decreased in all patients treated with oripase alfa but not in those treated with placebo ( Fig.17 , left panel). The mean baseline spleen volume in the placebo group was 11.2 MN (multiples of normal), while the mean spleen volume in the orlipase group was 11.7 MN, indicating moderate splenomegaly. In orlipase alfa-treated patients, significant reductions in spleen volume were observed up to 6 months of treatment, with the greatest reductions observed in patients with the most severe baseline splenomegaly. At Week 52, a statistically significant 39% reduction in spleen size was demonstrated in orlipase alfa-treated patients, compared to a 0.5% increase in placebo patients (p<0.0001). In addition, 17 of 18 orlipase alfa-treated patients (94%) had a ≥30% reduction in spleen volume, compared to 0 / 18 placebo patients. The greatest reductions were observed in orlipase alfa-treated patients who had the largest spleens at baseline.

[0221] Splenomegaly-related scores were also calculated for oripase alfa-treated patients and placebo-treated patients and decreased in parallel in both groups ( Fig.17 , right panel). There was no correlation between the SRS and baseline or final spleen volume; therefore, symptoms measured by the SRS do not reflect physiological disease burden. The mean baseline SRS score in the placebo group was 28.1, and the mean baseline SRS score in the orlipase alfa group was 24.6. Both groups showed a reduction in SRS scores; however, after multiple adjustment, the LS mean change in SRS score from baseline to week 52 in the orlipase alfa group (-7.66) was not statistically different compared with the placebo group (-9.28); p = 0.6364. Of note, the SRS was adapted from the Myelofibrosis Trial and has not been previously validated in patients with ASMD. Secondary endpoint - liver response

[0222] Patients had moderate baseline hepatomegaly, with a mean liver volume of 1.6 MN in the placebo group and 1.4 MN in the orlipase alfa group, in addition to atherogenic lipid panel results and abnormal liver function test results.

[0223] The LS mean percent change in liver volume from baseline to Week 52 showed a greater reduction in the orlipase alfa group (31.67%) compared with the placebo group (1.42%, nominal p<0.0001). Fig.18 Similarly, baseline atherogenic lipid panel results improved in oripase alfa-treated patients but not in placebo-treated patients, with mean decreases in LDL cholesterol and triglycerides and increases in HDL cholesterol. Mean ALT and AST, as well as other liver function tests, also improved in oripase alfa-treated patients but not in placebo-treated patients. Secondary endpoint - platelet count

[0224] Mean platelet counts improved in oripase alfa-treated patients but not in placebo-treated patients (+16.8% vs. +2.5%, respectively, p=0.019), with clinical differences observed up to week 26. Exploratory endpoint - liver sphingomyelin

[0225] Hepatic sphingomyelin levels were monitored in oripase alfa-treated patients and placebo-treated patients. The mean percentage of tissue area occupied by sphingomyelin decreased from 29% to 2% after 52 weeks in oripase alfa-treated patients but did not change in placebo patients ( Fig.19 Histological analysis data from liver biopsies showed that sphingomyelin was significantly cleared from Kupffer cells and hepatocytes in patients treated with oripase alfa, but not in patients treated with placebo ( ). Fig. 20 ). Exploratory endpoints - biomarker response

[0226] At baseline, mean levels of both plasma biomarkers chitotriosidase and lysophospholipids were significantly elevated. Baseline values ​​for both groups were >14x ULN for chitotriosidase and >38x ULN for lysophospholipids. At week 52, mean chitotriosidase levels were reduced by 54% in the orlipase alfa group, compared with a 12% reduction in the placebo group. Mean lysophospholipids were reduced by 78% in the orlipase alfa group, compared with a 6% reduction in the placebo group, at week 52. Both showed significant reductions starting from the first week of treatment in orlipase alfa-treated patients, but not in placebo-treated patients ( Fig.21 ). Summarize

[0227] The overall safety and tolerability profile was good, and no new safety risks were identified in this trial. No patients died, and no orlipase alfa was permanently discontinued due to adverse events. All patients continued into the trial extension, except for one patient who discontinued during the primary analysis period due to poor compliance. Three orlipase alfa-treated patients had five serious adverse events, and four placebo patients had 11 serious adverse events, but none of these serious adverse events were considered to be treatment-related. Infusion-related reactions, as expected in patients starting enzyme replacement therapy, were mild or moderate and easily manageable. Four of the 18 orlipase alfa-treated patients developed treatment-induced anti-drug antibodies; two of these patients had transient antibodies, and the remaining two patients had persistent but low antibody titers. No patient developed neutralizing antibodies that interfered with cellular uptake of the enzyme.

[0228] The study was declared positive because it reached DL CO Endpoints, and the overall data demonstrate clinical benefit. In patients treated with rhASM produced by the method described herein, ASMD-related interstitial lung disease was improved. Spleen volume decreased with increased platelet counts, reflecting the correction of hypersplenism. Liver volume decreased due to the clearance of sphingomyelin, which is supported by histological evidence of a series of liver biopsies. Metabolic function was improved, as demonstrated by improvements in liver function test results and lipid test results. Symptoms measured by SRS do not reflect the disease burden measured by physiological measures (spleen volume). Based on this study, the benefit-risk profile of orlipase alpha in adults with ASMD is favorable.

[0229] This clinical study shows that the rhASM composition prepared by the method described in the present disclosure shows excellent safety and efficacy in treating ASMD patients. Example 4: In vitro activity determination of rhASM specific activity

[0230] This example describes a method for determining the activity (in U / mL) and specific activity (in U / mg) of recombinant human acid sphingomyelinase (rhASM) in a sample based on the hydrolysis of the synthetic substrate 2-(N-hexadecanoylamino)-4-nitrophenylphosphorylcholine (HDA-PC). The rate of hydrolysis of the synthetic substrate catalyzed by rhASM was measured as follows.

[0231] Approximately 1 μg / mL rhASM was incubated with 1 mM 2-(N-hexadecanoylamino)-4-nitrophenylphosphorylcholine in 50 mM sodium acetate, 0.1 mM zinc acetate, and 0.25 mg / mL bovine serum albumin (BSA) (pH 5.3) in a circulating water bath at 37.0°C for 15 minutes. Basically, 80 μL of substrate was added to 20 μL of enzyme to start the reaction. The reaction was stopped by adding 300 μL of 0.1 M glycine, 0.1 M NaOH, 50% ethanol solution, and the absorbance of the released 2-(N-hexadecanoylamino)-4-nitrophenol (HDA-NP) product was measured at 415 nm. One unit of activity is defined as the amount of enzyme required to hydrolyze 1 μmol of 2-(N-hexadecanoylamino-4-nitrophenyl)phosphocholine (HDA-PC) to 2-(N-hexadecanoylamino)-4-nitrophenol (HDA-NP) per minute under defined assay conditions. The specific activity (in U / mg) was calculated by dividing the enzyme activity result (in U / mL) by the corresponding rhASM protein concentration (in mg / mL). Equivalent programs and scope

[0232] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the embodiments described herein.The scope of the present disclosure is not intended to be limited by the above description, but rather is set forth in the claims appended hereto.

[0233] Unless the context clearly indicates the opposite or other meaning, articles such as "a", "an", and "the" may mean at least one. Unless the context clearly indicates the opposite or other meaning, a claim or specification containing "or" between two or more members of a group is deemed to satisfy the following conditions: there are one, more than one, or all of the group members. The disclosure of a group containing "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all of the group members are present. For the purpose of brevity, those embodiments are not separately set forth herein, but it should be understood that each of these embodiments is provided herein and can be specifically claimed or denied protection.

[0234] It should be understood that the present disclosure covers all variations, combinations and permutations in which at least one limitation, element, clause or descriptive term from at least one claim or from at least one relevant portion of the specification is introduced into another claim. For example, a claim that is dependent on another claim can be amended to include at least one limitation found in any other claim that is dependent on the same base claim. In addition, when a claim refers to a composition, it should be understood that any method of making or using the composition disclosed herein or according to methods known in the art (if any) is included unless it is clear to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0235] In the case where an element is presented in a list form (e.g., in the form of a Markush group), it should be understood that each possible subgroup of the element is also disclosed, and any element or element subgroup can be removed from the group. It should also be noted that the term "comprising" is intended to be open and allows the inclusion of other elements or steps. It should be understood that, in general, when an embodiment, product or method is referred to as including a specific element, feature or step, an embodiment, product or method consisting of or substantially consisting of such elements, features or steps is also provided. For the purpose of brevity, those embodiments are not separately set forth herein, but it should be understood that each of these embodiments is provided herein and can be specifically claimed or refused protection.

[0236] In the claims and throughout the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and "consisting of" shall be construed as open-ended, i.e., meaning including, but not limited to, "including." Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office's Manual of Patent Examining Procedures, Section 2111.03.

[0237] When giving a range, the endpoint is included. In addition, it should be understood that, unless otherwise specified or otherwise clearly defined according to the context and / or understanding of those of ordinary skill in the art, in the same embodiment, the value represented as a range can assume any specific value in the range, unless the context clearly specifies otherwise, otherwise accurate to one tenth of the unit of the lower limit of the range. For the purpose of simplicity, the value in each range is not separately set forth herein, but it should be understood that each of these values ​​is provided herein and can be specifically claimed or denied protection. It should also be understood that, unless otherwise specified or otherwise clearly defined according to the context and / or understanding of those of ordinary skill in the art, the value represented as a range can assume any sub-range within a given range, wherein the endpoint of the sub-range is represented with the same accuracy as follows: one tenth of the unit of the lower limit of the range.

[0238] In the case of websites, the URL address is provided in the form of non-browser executable code with the period of the corresponding web address in brackets. The actual web address does not include brackets.

[0239] In addition, it should be understood that any specific embodiment of the present disclosure may be explicitly excluded from any at least one claim. Where a range is given, any value within the range may be explicitly excluded from any at least one claim. Any embodiment, element, feature, application or aspect of the composition and / or method of the present disclosure may be excluded from any at least one claim. For the purpose of brevity, all embodiments excluding at least one element, feature, purpose or aspect are not explicitly set forth herein.

[0240] All publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned herein (e.g., in the Background, Summary, Detailed Description, Examples, and / or References sections) are hereby incorporated by reference in their entirety, as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated by reference herein. In the event of a conflict, the present application, including any definitions herein, will control.

Claims

1. A method for purifying recombinant acid sphingomyelinase (rASM), the method comprising the following steps: (i) subjecting a protein mixture comprising rASM and host cell proteins (HCP) to cation exchange (CEX) chromatography, or Subjecting the protein mixture containing rASM and HCP to immobilized metal affinity chromatography (IMAC), or subjecting the protein mixture comprising rASM and HCP to both CEX chromatography and IMAC; and (ii) collecting the eluate from the CEX chromatography or the IMAC to obtain a purified rASM preparation.

2. The method according to claim 1, wherein the protein mixture is subjected to CEX chromatography and IMAC in series, and the eluate obtained from the CEX chromatography is subjected to the IMAC.

3. The method according to claim 1, wherein the protein mixture is subjected to IMAC and CEX chromatography in series, and the eluate obtained from the IMAC is subjected to the CEX chromatography.

4. The method according to any one of claims 1 to 3, wherein the protein mixture is subjected to both the CEX chromatography and the IMAC separately with one or more further steps in between.

5. The method of claim 3, wherein before or after subjecting the protein mixture comprising rASM and HCP to the CEX chromatography or the IMAC, the mixture is subjected to one or more additional purification columns.

6. The method according to any one of claims 1 to 5, further comprising a step of inactivating and / or removing potential viral contaminants.

7. The method according to any one of claims 1 to 6, further comprising the step of concentrating the purified rASM.

8. The method according to any one of claims 1 to 7, wherein the rASM is recombinant human acid sphingomyelinase (rhASM).

9. The method according to any one of claims 1 to 8, wherein the protein mixture is obtained from Chinese Hamster Ovary (CHO) cells expressing the rASM.

10. The method according to any one of claims 1 to 9, wherein the rASM comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

11. The method according to any one of claims 1 to 10, wherein the cation exchange (CEX) chromatography comprises a resin selected from carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).

12. The method of any one of claims 1 to 11, wherein the IMAC is a chelating resin.

13. The method of claim 12, wherein the IMAC is performed with zinc, copper or nickel.

14. The method according to any one of claims 1 to 13, wherein the CEX chromatography comprises washing the CEX chromatography column with a CEX wash buffer having a first optimal pH and a first optimal salt concentration, wherein the first optimal pH and the first optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

15. The method of claim 14, wherein the CEX chromatography further comprises eluting the CEX chromatography column with a CEX elution buffer having a second optimal pH and a second optimal salt concentration, wherein rASM bound to the CEX chromatography column after the washing step is removed from the column at the second optimal pH and the second optimal salt concentration.

16. The method according to any one of claims 1 to 15, wherein the IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, wherein the third optimal pH and the third optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

17. The method of claim 16, wherein the IMAC further comprises eluting the IMAC column with an IMAC elution buffer having a fourth optimal pH and a fourth optimal salt concentration, wherein rASM bound to the IMAC column after the washing step is removed from the column at the fourth optimal pH and the fourth optimal salt concentration.

18. The method of any one of claims 1 to 17, wherein the purified rASM preparation has a specific activity of about 5 to 50 U / mg.

19. The method of claim 18, wherein the purified rASM preparation has a specific activity of about 10 to 45 U / mg.

20. The method of claim 18, wherein the purified rASM preparation has a specific activity of about 10-20 U / mg.

21. The method of any one of claims 1 to 20, wherein the HCP level of the purified rASM preparation does not exceed 1.0 μg / mg.

22. The method of any one of claims 1 to 21, wherein the purified rASM preparation comprises rASM isoforms with modifications that total no more than 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% of the entire rASM population.

23. The method of claim 22, wherein the modified rASM isoform comprises one or more modifications selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

24. The method of any one of claims 1 to 23, wherein the protein mixture is produced in a bioreactor at a production scale of at least 100 L.

25. The method of claim 24, wherein the protein mixture is produced in a bioreactor at a production scale of at least 500 L.

26. A method for regulating the relative amounts of isoforms of recombinant acid sphingomyelinase (rASM) in an initial rASM composition, wherein the initial rASM composition comprises an unmodified rASM isoform and at least one rASM isoform having one or more modifications selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation, wherein the method comprises (i) subjecting the initial rASM composition to cation exchange (CEX) chromatography, or subjecting the initial rASM composition to immobilized metal affinity chromatography (IMAC), or subjecting the initial rASM composition to both CEX chromatography and IMAC; and (ii) collecting the eluate from the CEX chromatography or the IMAC to obtain a purified rASM preparation.

27. The method of claim 26, wherein the initial rASM composition is subjected to CEX chromatography and IMAC in series, and the eluate obtained from the CEX chromatography is subjected to the IMAC.

28. The method of claim 26, wherein the initial rASM composition is subjected to IMAC and CEX chromatography in series, and the eluate obtained from the IMAC is subjected to the CEX.

29. The method of claim 26, wherein the initial rASM composition is subjected to both the CEX chromatography and the IMAC separately with one or more additional steps in between.

30. The method of any one of claims 26 to 29, wherein the rASM is recombinant human acid sphingomyelinase (rhASM).

31. The method of any one of claims 26 to 30, wherein the rASM comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

32. The method of any one of claims 26 to 31, wherein the cation exchange chromatography comprises a resin selected from carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).

33. The method of any one of claims 26 to 32, wherein the IMAC is a chelating resin column.

34. The method of claim 33, wherein the IMAC is performed with zinc, copper or nickel.

35. The method of any one of claims 26 to 34, wherein the CEX chromatography comprises washing the CEX chromatography column with a CEX wash buffer having a first optimal pH and a first optimal salt concentration, wherein the first optimal pH and the first optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

36. The method of claim 35, wherein the CEX chromatography further comprises eluting the CEX chromatography column with a CEX elution buffer having a second optimal pH and a second optimal salt concentration, wherein at the second optimal pH and the second optimal salt concentration, all species bound on the CEX chromatography column after the washing step are removed from the column.

37. The method according to any one of claims 26 to 34, wherein the IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, wherein the third optimal pH and the third optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

38. The method of claim 37, wherein the IMAC further comprises eluting the IMAC column with an IMAC elution buffer having a fourth optimal pH and a fourth optimal salt concentration, wherein at the fourth optimal pH and the fourth optimal salt concentration, all species bound on the IMAC column after the washing step are removed from the column.

39. The method according to any one of claims 26 to 38, wherein the specific activity of the rASM preparation obtained is about 5 to 50 U / mg.

40. The method of claim 39, wherein the specific activity of the rASM preparation obtained is about 10 to 45 U / mg.

41. The method of claim 39, wherein the specific activity of the rASM preparation obtained is about 10 to 20 U / mg.

42. The method of any one of claims 26 to 41, wherein the host cell protein (HCP) level of the rASM preparation obtained does not exceed 1.0 μg / mg.

43. The method of any one of claims 26 to 42, wherein the purified rASM preparation comprises rASM isoforms with modifications that total no more than 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% of the entire rASM population.

44. The method according to any one of claims 26 to 43, wherein the modification is selected from C-terminal cysteinylation, S-glutathionylation, dimerization and truncation.

45. The method of any one of claims 26 to 44, wherein the starting composition comprising rASM is produced in a bioreactor at a production scale of at least 100 L or at least 500 L.

46. ​​A method for modulating the specific activity of a recombinant acid sphingomyelinase (rASM) in a liquid composition, the liquid composition comprising an unmodified rASM isoform and at least one rASM isoform having one or more modifications selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation, wherein the method comprises: (i) subjecting the liquid composition to cation exchange (CEX) chromatography, or subjecting the liquid composition to immobilized metal affinity chromatography (IMAC), or subjecting the liquid composition to both CEX chromatography and IMAC; and (ii) collecting the eluate from the CEX chromatography or the IMAC to obtain a purified rASM preparation.

47. A method according to claim 46, wherein the liquid composition is subjected to CEX chromatography and IMAC in series and the eluate obtained from the CEX chromatography is subjected to the IMAC, or wherein the liquid composition is subjected to IMAC and CEX chromatography in series and the eluate obtained from the IMAC is subjected to the CEX chromatography.

48. The method of claim 46, wherein the initial rASM composition is subjected to both the CEX chromatography and the IMAC separately with one or more additional steps in between.

49. The method of any one of claims 46 to 48, wherein the rASM is recombinant human acid sphingomyelinase (rhASM).

50. The method of any one of claims 46 to 49, wherein the rASM comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

51. The method of any one of claims 46 to 50, wherein the cation exchange (CEX) chromatography comprises a resin selected from carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).

52. The method of any one of claims 46 to 51, wherein the IMAC is a chelating resin column.

53. The method of claim 52, wherein the IMAC is performed with zinc, copper or nickel.

54. The method of any one of claims 46 to 53, wherein the CEX chromatography comprises washing the CEX chromatography column with a CEX wash buffer having a first optimal pH and a first optimal salt concentration, wherein the first optimal pH and the first optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

55. The method of claim 54, wherein the CEX chromatography further comprises eluting the CEX chromatography column with a CEX elution buffer having a second optimal pH and a second optimal salt concentration, wherein at the second optimal pH and the second optimal salt concentration, all species bound to the CEX chromatography column after the washing step are removed from the column.

56. The method of any one of claims 46 to 55, wherein the IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, wherein the third optimal pH and the third optimal salt concentration are predetermined based on the starting specific activity of the resin and the protein mixture.

57. The method of claim 56, wherein the IMAC further comprises eluting the IMAC column with an IMAC elution buffer having a fourth optimal pH and a fourth optimal salt concentration, wherein at the fourth optimal pH and the fourth optimal salt concentration, all species bound to the IMAC column after the washing step are removed from the column.

58. The method according to any one of claims 46 to 57, wherein the specific activity of the rASM preparation obtained is about 5 to 50 U / mg.

59. The method of claim 58, wherein the specific activity of the rASM preparation obtained is about 10 to 45 U / mg.

60. The method of claim 58, wherein the rASM preparation obtained has a specific activity of about 10 to 20 U / mg, optionally wherein the purity of the rASM in the preparation is at least 98%.

61. The method of any one of claims 46 to 60, wherein the host cell protein (HCP) level of the rASM preparation obtained does not exceed 5.0 μg / mg.

62. The method of any one of claims 46 to 61, wherein the purified rASM preparation comprises rASM isoforms with modifications that total no more than 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% of the entire rASM population.

63. The method of claim 62, wherein the modification is selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

64. The method of any one of claims 46 to 63, wherein the liquid composition comprising rASM is produced in a bioreactor at a production scale of at least 100 L or at least 500 L.

65. A recombinant acid sphingomyelinase (rASM) preparation, comprising an unmodified rASM isoform and at least one rASM isoform species having one or more modifications selected from C-terminal cysteinylation, S-glutathionylation, dimerization and truncation, wherein the unmodified rASM isoform accounts for at least 50%, 55%, 60%, 65%, 70%, 75% or 80% of the total rASM population in the rASM preparation.

66. The rASM preparation of claim 65, wherein the unmodified rASM isoform accounts for at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more of the total rASM population in the rASM preparation.

67. The rASM preparation of claim 65 or claim 66, wherein all modified rASM isoforms together comprise no more than 40% of the total rASM population in the rASM preparation.

68. The rASM preparation of claim 67, wherein all modified rASM isoforms collectively account for no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5% or less of the total rASM population in the rASM preparation.

69. The rASM preparation according to any one of claims 65 to 68, wherein the rASM isoform with C-terminal cysteinylation accounts for no more than 10% of the total rASM population in the rASM preparation.

70. The rASM preparation of claim 69, wherein the rASM isoform having C-terminal cysteinylation comprises no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5% or less of the total rASM population in the rASM preparation.

71. The rASM preparation according to any one of claims 65 to 70, wherein the rASM isoform with C-terminal S-glutathionylation accounts for no more than 5% of the total rASM population in the rASM preparation.

72. The rASM preparation of claim 71, wherein the rASM isoform having C-terminal S-glutathionylation accounts for no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% or less of the total rASM population in the rASM preparation.

73. The rASM preparation of any one of claims 65 to 72, wherein the rASM isoform with C-terminal dimerization accounts for no more than 0.2% of the total rASM population in the rASM preparation.

74. The rASM preparation of claim 73, wherein the rASM isoform with C-terminal S-dimerization accounts for no more than 0.1% of the total rASM population in the rASM preparation.

75. The rASM preparation of any one of claims 65 to 74, wherein the rASM isoforms having C-terminal truncations account for no more than 8% of the total rASM population in the rASM preparation.

76. The rASM preparation of claim 75, wherein the rASM isoform with a C-terminal truncation accounts for no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3% or less of the total rASM population in the rASM preparation.

77. The rASM preparation of any one of claims 65 to 76, wherein the rASM preparation has a purity of at least 95%.

78. The rASM preparation of any one of claims 65 to 77, wherein the rASM preparation has a purity of at least 96%, at least 97%, at least 98%, at least 99% or more.

79. The rASM preparation of any one of claims 65 to 78, wherein the specific activity of the rASM preparation is about 5 to 50 U / mg.

80. The rASM preparation of claim 79, wherein the rASM preparation has a specific activity of about 10 to 20 U / mg.

81. The rASM preparation of any one of claims 65 to 80, wherein the host cell protein (HCP) level of the rASM preparation does not exceed 5.0 μg / mg.

82. The rASM preparation of any one of claims 65 to 82, wherein the rASM preparation is manufactured using the method of any one of claims 1 to 25.

83. A pharmaceutical composition prepared by using the recombinant acid sphingomyelinase (rASM) preparation according to any one of claims 65 to 82.

84. A method of treating acid sphingomyelinase deficiency in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition according to any one of claims 65 to 82.

85. The method of any one of claims 1 to 64, further comprising the step of subjecting the purified rASM to buffer exchange.

86. The method of any one of claims 1 to 64, wherein the method is performed partially or completely under refrigeration conditions at 8°C ± 3°C.

87. The method of any one of claims 1 to 64, wherein the method is carried out partially or completely at ambient temperature.

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