Pharmaceutical recombinant human acid sphingomyelinase compositions and methods

Abstract

Disclosed herein are compositions comprising recombinant acid sphingomyelinase (rASM) having a desired purity, specific activity and / or rASM isoform. Methods for producing and purifying such compositions, including chromatography steps, are also provided. Additionally, methods for modulating rASM specific activity in a composition, and methods for modulating rASM isoform in a composition are provided. The methods disclosed herein may be particularly useful for the preparation of pharmaceutical compositions comprising rASM for treating acid sphingomyelinase deficiency (ASMD).

Description

[Technical field]

[0001] The present invention relates to compositions and methods for producing recombinant acid sphingomyelinase, such as recombinant human acid sphingomyelinase.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 321,636, filed March 18, 2022, the disclosure of which is incorporated by reference in its entirety.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The electronic copy of the Sequence Listing (created on March 15, 2023) is named 022548_WO042_SL.xml and is 32,066 bytes in size. [Background technology]

[0004] Acid sphingomyelinase deficiency (ASMD) is a rare, life-threatening lysosomal storage disorder. It is an autosomal recessive genetic disorder caused by mutations in the SMPD1 gene, which encodes the lysosomal enzyme acid sphingomyelinase (ASM) (Schuchman et al., Mol Genet Metab. (2017) l20(l-2):27-33). ASMD patients are unable to metabolize sphingomyelin, which results in its accumulation in lysosomes in multiple organs, leading to visceral disease and neurodegeneration in severe cases. ASMD patients have increased cholesterol and other lipids in the spleen, liver, lungs, and bone marrow.

[0005] Olipidase alpha is a recombinant human acid sphingomyelinase that can significantly improve the severe symptoms of ASMD in both adult and pediatric patients. However, there remains a need to produce pharmaceutical compositions containing olipidase alpha with a desired purity and consistent specific activity on a commercial scale. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides methods for purifying recombinant acid sphingomyelinase (rASM). In some embodiments, the methods include (i) subjecting a protein mixture comprising rASM and host cell proteins (HCPs) to cation exchange (CEX) chromatography; or subjecting a protein mixture comprising rASM and HCPs to immobilized metal affinity chromatography (IMAC); or subjecting a protein mixture comprising rASM and HCPs to both CEX chromatography and IMAC. In some embodiments, the methods further include (ii) recovering eluate from the CEX chromatography or IMAC, thereby obtaining a purified rASM preparation.

[0007] In some embodiments, the protein mixture is subjected to CEX chromatography and IMAC in parallel, and the eluate obtained from the CEX chromatography is subjected to IMAC.

[0008] In some embodiments, the rASM is recombinant human acid sphingomyelinase (rhASM).

[0009] In some embodiments, the protein mixture is obtained from Chinese Hamster Ovary (CHO) cells expressing rASM.

[0010] In some embodiments, the rASM comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0011] 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).

[0012] In some embodiments, the IMAC is a chelating resin.

[0013] In some embodiments, IMAC is performed with zinc, copper, or nickel.

[0014] In some embodiments, 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, the first optimal pH and the first optimal salt concentration being predetermined depending on the starting specific activity of the resin and the protein mixture.

[0015] 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, and under the second optimal pH and the second optimal salt concentration, rASM that binds to the CEX chromatography column after the washing step is removed from the column.

[0016] In some embodiments, the CEX wash buffer is selected from Table 2a and the CEX elution buffer is selected from Table 2b.

[0017] In some embodiments, IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, the third optimal pH and the third optimal salt concentration being predetermined depending on the starting specific activity of the resin and the protein mixture.

[0018] 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, and rASM that binds to the IMAC column after the washing step under the fourth optimal pH and fourth optimal salt concentration is removed from the column.

[0019] In some embodiments, the IMAC wash buffer is selected from Table 3 and the IMAC elution buffer is selected from Table 4.

[0020] In some embodiments, the purified rASM preparation has a specific activity of about 5-50 U / mg. In some embodiments, the purified rASM preparation has a specific activity of about 10-45 U / mg. In some embodiments, the purified rASM preparation has a specific activity of about 10-20 U / mg. The specific activity of the purified rASM preparation is determined according to Example 4.

[0021] In some embodiments, the resulting rASM preparation has a purity of 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%.

[0022] In some embodiments, the resulting rASM preparation has host cell protein (HCP) levels of 1.0 μg / mg or less, 2.0 μg / mg or less, 3.0 μg / mg or less, 4.0 μg / mg or less, or 5.0 μg / mg or less.

[0023] In some embodiments, purified rASM preparations contain, in total, no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of rASM isoforms having modifications of the total rASM population.

[0024] In some embodiments, the rASM isoform having a modification comprises one or more modifications selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

[0025] In some embodiments, the protein mixture is produced in a bioreactor having a production scale of at least 100 L.

[0026] In some embodiments, the protein mixture is produced in a bioreactor having a production scale of at least 500 L.

[0027] In some embodiments, the method is carried out partially or completely under refrigerated conditions at 8±3° C. In some embodiments, the method is carried out partially or completely under ambient temperature.

[0028] Also provided herein is a method of modulating the relative amounts of isoforms of recombinant acid sphingomyelinase (rASM) in an initial rASM composition, the initial rASM 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. In some embodiments, the method comprises 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 comprises recovering eluate from the CEX chromatography or IMAC, thereby obtaining a purified rASM preparation.

[0029] In some embodiments, the initial rASM composition is subjected to CEX chromatography and IMAC in parallel, and the eluate obtained from the CEX chromatography is subjected to IMAC.

[0030] In some embodiments, the initial rASM composition is subjected to IMAC and CEX chromatography in parallel, and the eluate obtained from IMAC is subjected to CEX.

[0031] In some embodiments, the initial rASM composition is subjected to both CEX chromatography and IMAC separately, with one or more additional steps in between.

[0032] 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.

[0033] 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).

[0034] In some embodiments, the IMAC is a chelating resin column.

[0035] In some embodiments, IMAC is performed with zinc, copper, or nickel.

[0036] In some embodiments, 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, the first optimal pH and the first optimal salt concentration being predetermined depending on the starting specific activity of the resin and the protein mixture.

[0037] 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 all species that bind to the CEX chromatography column after the washing step under the second optimal pH and the second optimal salt concentration are removed from the column.

[0038] In some embodiments, the CEX wash buffer is selected from Table 2a and the CEX elution buffer is selected from Table 2b.

[0039] In some embodiments, IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, the third optimal pH and the third optimal salt concentration being predetermined depending on the starting specific activity of the resin and the protein mixture.

[0040] 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 all species that bind to the IMAC column after the washing step at the fourth optimal pH and fourth optimal salt concentration are removed from the column.

[0041] In some embodiments, the IMAC wash buffer is selected from Table 3 and the IMAC elution buffer is selected from Table 4.

[0042] In some embodiments, the resulting rASM preparation has a specific activity of about 5-50 U / mg. In some embodiments, the resulting rASM preparation has a specific activity of about 10-45 U / mg. In some embodiments, the resulting rASM preparation has a specific activity of about 10-20 U / mg. The specific activity of the purified rASM preparation is measured according to Example 4.

[0043] In some embodiments, the resulting rASM preparation has host cell protein (HCP) levels of 1.0 μg / mg or less, 2.0 μg / mg or less, 3.0 μg / mg or less, 4.0 μg / mg or less, or 5.0 μg / mg or less.

[0044] In some embodiments, purified rASM preparations contain, in total, no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of rASM isoforms having modifications of the total rASM population.

[0045] In some embodiments, the modification is selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

[0046] In some embodiments, the initial composition comprising rASM is produced in a bioreactor having a production scale of at least 100 L or at least 500 L.

[0047] In some embodiments, the method is carried out partially or completely under refrigerated conditions at 8±3° C. In some embodiments, the method is carried out partially or completely under ambient temperature.

[0048] Further provided is a method of modulating rASM specific activity in a liquid composition comprising an unmodified recombinant acid sphingomyelinase (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. 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 recovering eluate from the CEX chromatography or IMAC, thereby obtaining a purified rASM preparation.

[0049] In some embodiments, the liquid composition is subjected to CEX chromatography and IMAC in parallel and the eluate obtained from the CEX chromatography is subjected to IMAC, or the liquid composition is subjected to IMAC and CEX chromatography in parallel and the eluate obtained from the IMAC is subjected to CEX chromatography.

[0050] In some embodiments, the initial rASM composition is subjected to both CEX chromatography and IMAC separately, with one or more additional steps in between.

[0051] 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.

[0052] 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).

[0053] In some embodiments, the IMAC is a chelating resin column.

[0054] In some embodiments, IMAC is performed with zinc, copper, or nickel.

[0055] In some embodiments, 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, the first optimal pH and the first optimal salt concentration being predetermined depending on the starting specific activity of the resin and the protein mixture.

[0056] 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 all species that bind to the CEX chromatography column after the washing step under the second optimal pH and the second optimal salt concentration are removed from the column.

[0057] In some embodiments, the CEX wash buffer is selected from Table 2a and the CEX elution buffer is selected from Table 2b.

[0058] In some embodiments, IMAC comprises washing the IMAC column with at least one IMAC wash buffer having a third optimal pH and a third optimal salt concentration, the third optimal pH and the third optimal salt concentration being predetermined depending on the starting specific activity of the resin and the protein mixture.

[0059] 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 all species that bind to the IMAC column after the washing step at the fourth optimal pH and fourth optimal salt concentration are removed from the column.

[0060] In some embodiments, the resulting rASM preparation has a specific activity of about 5-50 U / mg. In some embodiments, the resulting rASM preparation has a specific activity of about 10-45 U / mg. In some embodiments, the resulting rASM preparation has a specific activity of about 10-20 U / mg. The specific activity of the purified rASM preparation is measured according to Example 4.

[0061] In some embodiments, the rASM in the preparation has a purity of 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%.

[0062] In some embodiments, the resulting rASM preparation has host cell protein (HCP) levels of 1.0 μg / mg or less, 2.0 μg / mg or less, 3.0 μg / mg or less, 4.0 μg / mg or less, or 5.0 μg / mg or less.

[0063] In some embodiments, purified rASM preparations contain, in total, no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of rASM isoforms having modifications of the total rASM population.

[0064] In some embodiments, the modification is selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation.

[0065] In some embodiments, a liquid composition comprising rASM is produced in a bioreactor having a production scale of at least 100 L or at least 500 L.

[0066] In some embodiments, the method is carried out partially or completely under refrigerated conditions at 8±3° C. In some embodiments, the method is carried out partially or completely under ambient temperature.

[0067] The disclosure also provides an rASM preparation comprising an unmodified recombinant acid sphingomyelinase (rASM) isoform and at least one rASM isoform species having one or more modifications selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation. In some embodiments, the unmodified rASM isoform is 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 is 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.

[0068] In some embodiments, all modified rASM isoforms together comprise less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, or less than 40% of the total rASM population in an rASM preparation.

[0069] In some embodiments, all modified rASM isoforms, in total, comprise less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less of the total rASM population in an rASM preparation.

[0070] In some embodiments, rASM isoforms having C-terminal cysteinylation represent 10% or less of the total rASM population in an rASM preparation, hi some embodiments, rASM isoforms having C-terminal cysteinylation represent 9% or less, 8% or less, 7% or less, 6% or less, 5% or less or less of the total rASM population in an rASM preparation.

[0071] In some embodiments, rASM isoforms with C-terminal S-glutathionylation represent 5% or less of the total rASM population in an rASM preparation. In some embodiments, rASM isoforms with C-terminal S-glutathionylation represent 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less, of the total rASM population in an rASM preparation.

[0072] In some embodiments, the rASM isoform having C-terminal dimerization represents no more than 0.2% of the total rASM population in an rASM preparation.

[0073] In some embodiments, rASM isoforms having C-terminal S-dimerization represent 0.1% or less of the total rASM population in an rASM preparation.

[0074] In some embodiments, the rASM isoform having a C-terminal truncation represents no more than 8% of the total rASM population in an rASM preparation.

[0075] In some embodiments, rASM isoforms having C-terminal truncations represent 7% or less, 6% or less, 5% or less, 4% or less, 3% or less or less of the total rASM population in an rASM preparation.

[0076] 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.

[0077] In some embodiments, the rASM preparation has a specific activity of about 5-50 U / mg. In some embodiments, the rASM preparation has a specific activity of about 10-20 U / mg. The specific activity of the purified rASM preparation is measured according to Example 4.

[0078] In some embodiments, the rASM in the preparation has a purity of 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%.

[0079] In some embodiments, the resulting rASM preparation has host cell protein (HCP) levels of 1.0 μg / mg or less, 2.0 μg / mg or less, 3.0 μg / mg or less, 4.0 μg / mg or less, or 5.0 μg / mg or less.

[0080] In some embodiments, the rASM preparation was produced using the methods described herein.

[0081] Also provided are pharmaceutical compositions prepared by using the recombinant acid sphingomyelinase (rASM) preparations described herein.

[0082] Further provided is a method of treating acid sphingomyelinase deficiency in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein, in some embodiments, the method further comprises buffer exchanging the purified rASM. [Brief description of the drawings]

[0083] [Figure 1] Various isoforms of rhASM with and without modifications are shown. [Diagram 2](SEQ ID NOs: 13-20) Shows the structures near the C-terminus of rhASM with or without modification. The C-terminus state of rhASM was monitored by LC-MS of rhASM native Asp-N digest after MMTS labeling. Only the C-terminal amino acid of rhASM is shown. [Diagram 3] The specific activity of enriched monomers of rhASM and enriched dimers of rhASM are shown, with the percent purity of each enriched population indicated above each bar. [Figure 4] Specific activities of rhASM compositions containing various relative abundances of all C-terminal modifications are shown. [Diagram 5] HCP clearance (upper panel) or recovery (lower panel) under various salt (NaCl) and pH conditions in the CEX chromatography step are shown. [Figure 6] 1 shows contour plots of HCP clearance (left panel) and recovery (right panel) under various salt (NaCl) and pH conditions in the IMAC process. [Figure 7] HCP clearance (upper panel) or specific activity (lower panel) under various salt (NaCl) and pH conditions during the CEX chromatography step are shown. [Figure 8] Contour plots of HCP clearance (left panel) and specific activity (right panel) under various salt (NaCl) and pH conditions in the IMAC process are shown. [Figure 9] Representative specific activities of rhASM in the load material, wash fractions, and elution fractions of the CEX run are shown. Several wash conditions were tested as specified (45 mM sodium chloride, pH 6.3, pH 6.5, and pH 6.7). [Figure 10A] Specific activity of rhASM in the packing material, wash fraction, and elution fraction of the IMAC process is shown. [Figure 10B]Figure 1 shows the purity of rhASM in the load material, wash fractions, and elution fractions of the IMAC process. Several 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; aggressive: 10 mM sodium phosphate, 80 mM sodium chloride at pH 5.8). [Figure 11] Representative normalized abundances of rhASM isoforms in the loading material, wash fractions, and elution fractions of the CEX run are shown: 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-terminal truncated form 1. For details of these isoforms, see Table 1. [Figure 12] Representative normalized abundances of rhASM isoforms in the loading material, wash fractions, and elution fractions of an IMAC run are shown: 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-terminal truncated form 1. For details of these isoforms, see Table 1. [Figure 13] 1 shows a clinical trial using purified rhASM (rhapidase alpha) to treat ASMD patients. [Figure 14] Percent change in predicted DLCO% in patients treated with olivotidase alfa or placebo is shown. DLCO = diffusing capacity for carbon monoxide; FVC = forced vital capacity [Figure 15] High-resolution computed tomography (HRCT) scans of the lungs before (left panel) and after (right panel) oliprodase alpha treatment. Sphingomyelin-filled macrophages are seen as ground-glass opacities. [Figure 16]HSCT ground glass appearance scores (left panel) and interstitial lung disease scores (right panel) in both lungs are shown, which showed a mean improvement in oliprodase alfa-treated patients but not in placebo-treated patients. [Figure 17] The percent change in spleen volume (left panel) and change in splenomegaly-related scores (right panel) in oliprodase-alpha treated patients but not in placebo treated patients are shown. [Figure 18] Percent change in liver volume is shown. ALT: alanine aminotransferase; AST: aspartate aminotransferase; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; MN: multiple of normal. [Figure 19] 4 shows the percent tissue area occupied by sphingomyelin in tissues from patients treated with placebo or olidocidase alpha. [Figure 20] Representative toluidine blue stained images of liver biopsies in patients treated with placebo or oliprodase alfa are shown (sphingomyelin appears as a dark stain). [Figure 21] Normalized plasma chitotriosidase in both placebo- and oliprodase alfa-treated patients (left panel) and pre-dose plasma lysosphingomyelin levels in both populations are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] Before describing the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood.

[0086] As used herein, "about" when referring to a measurable value such as an amount, duration, and the like, is meant to encompass variations of +20% or +10%, including +5%, +1%, and +0.1%, from the stated value, where such variations are appropriate for performing the disclosed methods.

[0087] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. Polypeptides can be of natural (tissue-derived) origin, recombinant or naturally expressed from prokaryotic or eukaryotic cell preparations, or chemically produced via synthetic methods. The term applies 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 natural and non-natural amino acid polymers. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Non-natural residues are well described in the scientific and patent literature.

[0088] "Peptide" as used herein includes peptides that are conservative variations of those peptides specifically exemplified herein. As used herein, "conservative variation" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative variations include, but are not limited to, the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine, with another, or the replacement of one polar residue with another, such as, for example, the replacement of arginine with lysine, the replacement of glutamic acid with aspartic acid, or the replacement of glutamine with asparagine. Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine, and threonine. "Conservative variation" also includes the use of a substituted amino acid in place of the unsubstituted parent amino acid, provided that the antibody against the substituted polypeptide also immunoreacts with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the class of peptides of the present invention.

[0089] "Recombinant" as used with respect to a protein indicates that the protein is produced by the introduction of heterologous nucleic acid into a host cell.

[0090] As used herein, "loading" refers to a composition that is loaded onto a chromatographic material. A loading buffer is a buffer used to load a composition containing a product of interest onto a chromatographic material. The chromatographic material may be equilibrated with an equilibration buffer before loading the composition to be purified. In some instances, a wash buffer is used after loading the composition onto the chromatographic material and before eluting the polypeptide of interest from the solid phase. However, a portion of the product of interest, e.g., the polypeptide, may be removed from the chromatographic material by the wash buffer (i.e., in the flow-through).

[0091] As used herein, "elution" refers to removing a product, e.g., 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, an elution buffer has different physical properties than a loading buffer. For example, an elution buffer may have a different conductivity than a loading buffer or a different pH than a loading buffer. In some embodiments, an elution buffer has a lower conductivity than a loading buffer. In some embodiments, an elution buffer has a higher conductivity than a loading buffer. In some embodiments, an elution buffer has a lower pH than a loading buffer. In some embodiments, an elution buffer has a higher pH than a loading buffer. In some embodiments, an elution buffer has a different conductivity and a different pH than a loading buffer. An elution buffer may have any combination of higher or lower conductivity and higher or lower pH.

[0092] "Conductivity" refers to the ability of an aqueous solution to conduct electric current between two electrodes. In a solution, electric current flows by ion transport. Thus, as the amount of ions present in an aqueous solution increases, the solution has a higher conductivity. The basic unit of measurement for conductivity is the 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 electric current, the conductivity of a solution can be changed by altering the concentration of ions in the solution. For example, the concentration of buffers and / or the concentration of salts (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be changed to achieve a desired conductivity. Preferably, the salt concentrations of the various buffers are adjusted to achieve the desired conductivity.

[0093] A "host cell protein" (HCP) is a protein derived from the cell in which the polypeptide was produced. For example, CHOP is a host cell derived protein, i.e., a 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 of the methods described herein, the amount of HCP (e.g., CHOP) is reduced by greater than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. The amount of HCP can be reduced by about any of 10%-99%, 30%-95%, 30%-99%, 50%-95%, 50%-99%, 75%-99%, or 85%-99%. In some embodiments, the amount of HCPs 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 HCPs in a composition recovered from the purification step to the amount of HCPs in the composition prior to the purification step.

[0094] The present disclosure provides compositions comprising recombinant ASM, such as recombinant human ASM (rhASM). In some embodiments, the rhASM is olivodase alpha. The compositions of the present disclosure have excellent homogeneity and purity. In some embodiments, the compositions of the present invention are pharmaceutical compositions, i.e., compositions that are in, or can be prepared into, a form that allows the biological activity of the active ingredient to be effective without additional ingredients that are significantly toxic or cause undesirable side effects not associated with the active ingredient in the patient. The terms "pharmaceutical composition" and "pharmaceutical formulation" are used interchangeably herein. The pharmaceutical compositions of the present invention are useful for treating patients with ASM deficiency.

[0095] Drug substance consistency is an important consideration for the final step in the manufacture of drug substances / active pharmaceutical ingredients. It ensures that consistent potency is maintained from batch to batch, thereby ensuring quality. Studies are needed to ensure that the entire batch contents are uniform and consistent from batch to batch.

[0096] During the purification process, the conditions that resulted in good host cell protein clearance may affect the specific activity of the product. The inventors conducted research to identify the critical steps to ensure that the specific activity in the final product is well controlled in the purification process. As a result, the inventors discovered a way to control the proportion of rhASM isoforms (e.g., rhASM isoforms with C-terminal cysteine ​​modifications, including cysteinylation, S-glutathionylation, dimerization, and C-terminal truncation) in the rhASM product by adjusting the relative amounts of unmodified and modified rhASM isoforms, thereby controlling the specific activity of the final product. This finding provides a robust and effective control of product quality and process performance.

[0097] Recombinant Human Acid Sphingomyelinase ASM is an enzyme that catalyzes the degradation of sphingomyelin to ceramide and phosphorylcholine. "Recombinant human ASM" refers to human ASM, with or without specific amino acid modifications relative to the wild-type sequence, prepared by recombinant means. For example, recombinant human ASM can be expressed in cultured mammalian host cells (such as COS, CHO, HeLa, 3T3, 293T, NSO, SP2 / 0, or Hut 78 cells) or animals transgenic for the human ASM coding sequence.

[0098] In some embodiments, the recombinant human ASM is oributidase alpha. Oributidase alpha is the glycoform alpha of human ASM (EC-3.1.4.12) produced in CHO cells. Mature oributidase alpha is a 570 amino acid polypeptide that retains the enzymatic and lysosomal targeting activity of the native human protein. The amino acid sequence of oributidase alpha, including its leader sequence (residues 1-57), is shown below as SEQ ID NO:1, with the leader sequence shown in italics and bold. The mature oributidase alpha sequence (SEQ ID NO:2, spanning residues 58-627 of SEQ ID NO:1) is without the leader sequence. [ka] [ka]

[0099] In other embodiments, human ASM useful in the invention is 99%, 98%, 97%, 96% or 95% identical in amino acid sequence to oligopeptidase alpha. For example, the human ASM in the composition may have the sequence set forth in U.S. Patent No. 6,541,218, the entire disclosure of which is incorporated herein by reference. The sequence (SEQ ID NO:3) is shown below, with the leader sequence (residues 1-59) in italics and bold, and the mature protein (SEQ ID NO:4, spanning residues 60-629 of SEQ ID NO:3) lacking the leader sequence. [ka] [ka]

[0100] The human ASM in the composition may also be identical in amino acid sequence to the human ASM disclosed in the UNIPLOT database as sequence P17405-1 or a polymorphic variant thereof. The P17405-1 sequence is shown below (SEQ ID NO:5), with the leader sequence (residues 1-59) shown in italics and bold, and the mature protein (SEQ ID NO:6, which spans residues 60-629 of SEQ ID NO:5) lacking the leader sequence. [ka] [ka]

[0101] rhASM DNA, diagnostic methods and rhASM protein are covered by US Pat. Nos. 5,773,278; 5,686,240, and 6,541,218, each of which is incorporated herein by reference.

[0102] Recombinant human ASM (rhASM) produced in a host cell can exist as a mixture of one or more isoforms. In some embodiments, the isoforms are depicted in Figures 1 and 2, as also summarized in Table 1 below. The C-terminal status and relative proportions of C-terminal modified species can be determined by LC-MS analysis.

[0103] [Table 1]

[0104] Cell culture method Compositions and methods for the expression of recombinant ASM in host cells, such as Chinese hamster ovary cells, are described in U.S. Pat. No. 5,773,278, the entirety of which is incorporated herein by reference. To express biologically active ASM, the coding sequence of the enzyme, a functional equivalent, or a modified sequence can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for transcription and translation of the inserted coding sequence in a suitable host cell. Host cell expression systems that have the cellular machinery 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 to express biologically active enzymes that are properly folded and processed. When administered to humans, such expression products should exhibit appropriate tissue targeting and should not elicit adverse immunological responses.

[0105] Methods well known to those skilled in the art can be used to construct expression vectors containing the ASM coding sequence and appropriate transcriptional / translational 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).

[0106] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use for the expressed ASM protein. For example, when large amounts of ASM are produced, a vector that induces the expression of a high level of a fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. (1983) 2: 1791); pIN vector (Inouye & Inouye, Nucleic Acids Res. (1985) 13: 3101-9; Van Heeke & Schuster, J Biol Chem. (1989) 264: 5503-9), in which the ASM coding sequence can be ligated in frame with the lac Z coding region into the vector to produce a hybrid AS-lac Z protein.

[0107] A variety of eukaryotic host expression systems can be utilized to express the ASM coding sequence. Prokaryotic systems offer distinct advantages of ease of operation and low cost of scale-up, but their main drawback in the expression of ASM is the lack of proper post-translational modification of expressed mammalian proteins. Eukaryotic systems, preferably mammalian expression systems, allow for proper modification. Eukaryotic cells that have the cellular machinery to properly process the primary transcript, e.g., glycosylation, phosphorylation, and favorable secretion of the gene product, should be used as host cells for the expression of ASM. Mammalian cell lines are preferred. Such host cell lines may include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, -293, WI38, and the like.

[0108] For long-term, high-yield production of recombinant proteins, stable expression can be used. For example, after introduction of foreign DNA, engineered cells may be grown in enriched medium for 1-2 days and then switched to selective medium. Rather than using an expression vector containing a viral origin of replication, host cells can be transformed with ATN or DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. The herpes simplex virus thymidine kinase (Wigler et al., Cell (1977) 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc Natl Acad Sci. USA (1962) 48:2026), and adenine phosphoribosyltransferase (Lowy et al., Cell (1980) 22:817) genes can be used in tk cells, hgprt cells, or aprt cells, respectively. Antimetabolite resistance can also be used as the basis for selection of 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 & 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 the hygromycin gene (Santerre et al., Gene (1984) 30:147).Recently, additional selectable genes have been described: trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine (Hartman & Mulligan, Proc Natl Acad Sci. USA (1988) 85:8047); and the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed. (1987)).

[0109] 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) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the ASM coding sequence.

[0110] In yeast, a number of vectors containing constitutive or inducible promoters may be used. For an overview, see Current Protocols in Molecular Biology,Vol.2,1988,Ed.Ausubel et al.,Greene Publish.Assoc.& Wiley Interscience,Ch.13;Grant et al.,1987,Expression and Secretion Vectors for Yeast,in Methods in Enzymology,Eds. Wu & Grossman, 31987, Acad. Press, NY, Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., DC, Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, NY, Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast See Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II. For complementation assays in yeast, ASM cDNAs can be cloned into yeast episomal plasmids (YEp) that replicate autonomously in yeast due to the presence of 2μ circular yeast. The cDNAs can be cloned behind either constitutive yeast promoters such as ADH or LEU2 or inducible promoters such as GAL (Cloning in Yeast, Chpt. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. D M Glover, 1986, IRL Press, Wash., DC). Constructs can include the 5' and 3' untranslated regions of the cognate ASM mRNA or those corresponding to the yeast gene. YEp plasmids transform with high efficiency and the plasmids are very stable.Alternatively, vectors which facilitate integration of foreign DNA sequences into the yeast chromosome can be used.

[0111] When a plant expression vector is used, expression of the ASM coding sequence can be driven by any of a number 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); or heat shock promoters such as soybean hsp17.5-E or hsp17.3-B (Gurley et al., Mol Cell Biol. (1986) 6:559-565) can be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors; direct DNA transformation; microinjection, electroporation, etc. For reviews of such techniques, see, e.g., Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

[0112] 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 to express foreign genes. The virus grows in Spodoptera frugiperda cells. ASM sequences can be cloned into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the coding sequence results in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (see, e.g., Smith et al., J Viol. (1983) 46:584; Smith, U.S. Pat. No. 4,215,051).

[0113] Methods for purifying and modulating rASM specific activity and isoforms The present disclosure provides the unexpected discovery that the specific activity of rASM (e.g., rhASM) and the ratio of rASM isoforms in a composition comprising rASM can be adjusted by varying the purification process. Using this innovation, the specific activity of a batch of rASM can be adjusted to a target range. This method of adjusting specific activity can be easier to implement without significantly affecting process performance compared to other possible methods (e.g., altering the cell culture process).

[0114] Accordingly, the following methods are provided in the present disclosure: I. Methods 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, e.g., CHO cell proteins. In some embodiments, the method comprises subjecting the protein mixture comprising rASM and host cell proteins (HCPs) to cation exchange (CEX) chromatography as described herein. In some embodiments, the method further comprises collecting eluate from the CEX chromatography, thereby obtaining a purified rASM preparation. In some embodiments, the method comprises subjecting the protein mixture comprising rASM and HCPs to immobilized metal affinity chromatography (IMAC) as described herein. In some embodiments, the method further comprises collecting eluate from the IMAC chromatography, thereby obtaining a purified rASM preparation. In further embodiments, the method comprises subjecting the protein mixture comprising rASM and HCPs to both CEX chromatography and IMAC, whether separately or in parallel. The order of CEX and IMAC is interchangeable. For example, in some embodiments, the method includes (i) subjecting a protein mixture comprising rASM and host cell proteins (HCPs) to CEX chromatography as described herein. In some embodiments, the method further includes (ii) subjecting the eluate obtained from the CEX chromatography to IMAC, either directly or indirectly, as described herein. As used herein, the term "directly" means that the eluate obtained from the CEX chromatography is directly subjected to IMAC without being treated in a separate step, while the term "indirectly" means that the eluate obtained from the CEX chromatography is treated by one or more additional steps before being subjected to IMAC. For example, the eluate obtained from the CEX chromatography undergoes a separate purification step (e.g., a separate purification column) before being subjected to IMAC. In some embodiments, the method further includes recovering the eluate from the IMAC, thereby obtaining a purified rASM preparation.Optionally, the order of CEX chromatography and IMAC may be interchanged. For example, in some embodiments, the method includes (i) subjecting a protein mixture comprising rASM and HCPs to IMAC as described herein. In some embodiments, the method further includes (ii) subjecting the eluate obtained from IMAC directly or indirectly to CEX chromatography as described herein. As used herein, the term "directly" means that the eluate obtained from IMAC is directly subjected to CEX chromatography without being treated in a separate step, while the term "indirectly" means that the eluate obtained from IMAC is treated by one or more additional steps before being subjected to CEX chromatography. For example, the eluate obtained from IMAC undergoes another purification step (e.g., another purification column) before being subjected to CEX chromatography. In some embodiments, the method further includes recovering the eluate from CEX chromatography, thereby obtaining a purified rASM preparation. In some embodiments, one or more additional purification steps may be included before and / or after subjecting the sample to CEX chromatography and / or IMAC to remove impurities (e.g., HCPs) from the sample. Such purification processes are described in U.S. Pat. Nos. 8,796,419, 9,481,706, 10,259,842, and PCT Publication Nos. WO 2008 / 085988 A1 and WO 2019 / 121846 A1, each of which is incorporated by reference in its entirety.

[0115] II. A method of modulating the relative amount of an isoform of recombinant acid sphingomyelinase (rASM) in an initial rASM composition. The relative amount of an rASM isoform in a composition is equal to the percentage of the normalized abundance of the isoform compared to the total rASM abundance when all rASM isoforms are combined. For example, if the normalized abundance of unmodified rASM in a composition is 9 million, while the total rASM abundance of all rASM isoforms in the composition combined together is 10 million, then the relative amount of unmodified rASM in the composition is 90%. The initial rASM composition may include 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. In some embodiments, the method includes subjecting the initial rASM composition to CEX chromatography as described herein. In some embodiments, the method further comprises recovering eluate from the CEX chromatography, thereby obtaining an rASM preparation having adjusted relative amounts of isoforms of rASM. In some embodiments, the method comprises subjecting the initial rASM composition to immobilized metal affinity chromatography (IMAC) as described herein. In some embodiments, the method further comprises recovering eluate from the IMAC chromatography, thereby obtaining an rASM preparation having adjusted relative amounts of isoforms of rASM. In further embodiments, the method comprises subjecting the initial rASM composition to both CEX chromatography and IMAC, whether separately or in parallel. The order of CEX and IMAC is interchangeable. For example, in some embodiments, the method comprises (i) subjecting the initial composition comprising rASM to cation exchange (CEX) chromatography as described herein. In some embodiments, the method further comprises (ii) recovering eluate from the cation exchange (CEX) chromatography, thereby obtaining an rASM preparation having adjusted relative amounts of isoforms of rASM.In some embodiments, the method further comprises subjecting the eluate obtained from the CEX chromatography to IMAC, either directly or indirectly. In some embodiments, the method further comprises recovering the eluate from the IMAC, thereby obtaining an rASM preparation having a controlled relative amount of isoforms of rASM. Optionally, the order of CEX chromatography and IMAC may be interchanged. For example, in some embodiments, the method further comprises (i) subjecting an initial composition comprising rASM to IMAC as described herein. In some embodiments, the method further comprises (ii) recovering the eluate from the IMAC, thereby obtaining an rASM preparation having a controlled relative amount of isoforms of rASM. In some embodiments, the method further comprises subjecting the eluate obtained from IMAC, either directly or indirectly, to CEX chromatography. In some embodiments, the method further comprises recovering the eluate from the CEX chromatography, thereby obtaining an rASM preparation having a controlled relative amount of isoforms of rASM. In some embodiments, such methods increase the relative amount of unmodified rASM isoforms in the composition while decreasing the relative amount of at least one modified rASM isoform selected from the group consisting of C-terminal cysteinylation, S-glutathionylation, dimerization, and truncation, as described herein.In some embodiments, the relative amount of unmodified rASM isoform in the resulting composition compared to that in the initial rASM composition is 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 2.1%, 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%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6.0%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.5%, at least 6.6%, at least 6.7%, at 0.3%, 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 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.

[0116] III. A method of modulating recombinant acid sphingomyelinase (rASM) specific activity in a 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. In some embodiments, the method comprises subjecting the liquid composition to cation exchange (CEX) chromatography as described herein. In some embodiments, the method further comprises (ii) recovering eluate from the cation exchange (CEX) chromatography, thereby obtaining an rASM preparation having a modulated specific activity. In some embodiments, the method comprises subjecting the liquid composition to immobilized metal affinity chromatography (IMAC) as described herein. In some embodiments, the method further comprises recovering eluate from the IMAC chromatography, thereby obtaining an rASM preparation having a modulated specific activity. In further embodiments, the method comprises subjecting the initial rASM composition to both CEX chromatography and IMAC, whether separately or in parallel. The order of CEX and IMAC is interchangeable. For example, in some embodiments, the method includes (i) subjecting the liquid composition to a cation exchange (CEX) chromatography as described herein. In some embodiments, the method further includes (ii) recovering an eluate from the cation exchange (CEX) chromatography, thereby obtaining an rASM preparation having an adjusted specific activity. In some embodiments, the method further includes subjecting the eluate obtained from the CEX chromatography directly or indirectly to IMAC. In some embodiments, the method further includes recovering an eluate from IMAC, thereby obtaining an rASM preparation having a further adjusted specific activity. Optionally, the order of CEX chromatography and IMAC can be interchanged. For example, in some embodiments, the method includes (i) subjecting the liquid composition to an IMAC as described herein. In some embodiments, the method further includes (ii) recovering an eluate from IMAC, thereby obtaining an rASM preparation having an adjusted specific activity.In some embodiments, the method further comprises subjecting the eluate obtained from IMAC directly or indirectly to CEX chromatography. In some embodiments, the method further comprises recovering the eluate from the CEX chromatography, thereby obtaining an rASM preparation having a further adjusted specific activity. In certain embodiments, the resulting rASM preparation has a reduced specific activity. In some embodiments, the specific activity in the resulting rASM preparation 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 of the resulting rASM preparation is about 5-50 U / mg, e.g., 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 of the resulting 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, or about 50 U / mg.

[0117] In some embodiments, the rASM of the disclosure may be produced within a host cell. For example, host cells expressing rASM and / or cell cultures containing rASM are harvested to produce a product. In the methods described herein, the harvested cells and / or cell cultures may be used as is or may be concentrated, as desired. In some embodiments, the product is concentrated. In some embodiments, the product is clarified by a suitable method (e.g., filtration) and then purified to produce a clarified product. In some embodiments, the product is lysed to produce a lysate.

[0118] In some embodiments, cation exchange (CEX) chromatography is used in the method 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 solid phase that is negatively charged and thus has free cations to exchange with cations in the aqueous solution that passes on or through the solid phase. The negatively charged ligand attached to the solid phase to form the cation exchange resin can be, for example, a carboxylate or a sulfonate. Commercially available cation exchange resins include, but are not limited to, carboxy-methyl-cellulose, sulfopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW™ or SP-SEPHAROSE HIGH PERFORMANCE™, from Pharmacia), and sulfonyl immobilized on agarose (e.g., S-SEPHAROSE FAST FLOW™, from Pharmacia). 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). 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 the rASM from the CEX chromatography membrane or column with an elution buffer; and (4) collecting the eluate comprising rASM. The term "equilibration buffer" refers to a buffer used to equilibrate the chromatography resin prior to loading a sample onto the chromatography. The term "wash buffer" refers to a buffer used to wash the chromatography resin after the sample has been loaded onto the chromatography. In some embodiments, the wash buffer and the equilibration buffer are the same or different. In some cases, the wash buffer and the load buffer can be the same."Washing" a chromatography medium means to include passing an appropriate buffer through or over the medium after the sample has been loaded onto the chromatography medium. An "elution buffer" is used to elute the target protein from the solid phase. The conductivity and / or pH of the elution buffer is usually such that the target protein is eluted from the chromatography resin. "Eluting" a molecule (e.g., a polypeptide of interest or an impurity) from a chromatography resin means removing the molecule by changing the solution conditions such that the buffer competes with the molecule of interest for binding to the chromatography resin, or by changing the solution conditions such that the binding interaction between the molecule of interest and the resin is weakened, dissociating the molecule of interest. A non-limiting example is eluting a molecule from an ion exchange resin by changing the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material. The term "eluting" as used herein refers to the solution containing the molecule of interest obtained by elution, as well as the flow-through fraction containing the target protein of interest obtained as a result of flow-through purification. In some embodiments, the term "eluting" refers to the elution pool from the binding and elution chromatography step.

[0119] In some embodiments, CEX chromatography 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.

[0120] In some embodiments, the CEX chromatography further comprises (2) washing the membrane or 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 according to the starting specific activity of the resin and the composition. In some embodiments, the wash buffer comprises a pH buffer system based on a phosphate, such as sodium phosphate. In some embodiments, the sodium phosphate concentration is about 5 to 100 mM, for example, 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.

[0121] In some embodiments, the wash buffer contains 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, for example, 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.

[0122] In some embodiments, the first optimum salt concentration and the first optimum pH are selected from the conditions in Table 2a below.

[0123] [Table 2]

[0124] [Table 3]

[0125] Generally, a higher pH and / or a higher salt concentration in the wash buffer results in higher purity (e.g., higher HCP clearance), but lower specific activity and lower recovery. Therefore, the CEX wash conditions can be selected from CEX W1 to CEX W250 depending on the target purity and specific activity. In some embodiments, the condition is selected from CEX W1 through CEX W25; in some embodiments, the condition is selected from CEX W26 through CEX W50; in some embodiments, the condition is selected from CEX W51 through CEX W75; in some embodiments, the condition is selected from CEX W76 through CEX W100; in some embodiments, the condition is selected from CEX W101 through CEX W125; in some embodiments, the condition is selected from CEX W126 through CEX W150; in some embodiments, the condition is selected from CEX W151 through CEX W175; in some embodiments, the condition is selected from CEX W176 through CEX W200; in some embodiments, the condition is selected from CEX W201 through CEX W225; in some embodiments, the condition is selected from CEX W226 through CEX W250. In some embodiments the condition is 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 condition is 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 condition is 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 condition is 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.

[0126] In some embodiments, the CEX chromatography further comprises (3) eluting the membrane or 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 as or close 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. Generally, 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 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250 mM. In some embodiments, the elution buffer comprises a phosphate-based pH buffer system, such as sodium phosphate. In some embodiments, the sodium phosphate concentration is about 5-100 mM, e.g., 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 concentration in the CEX wash buffer.

[0127] In some embodiments, the elution buffer comprises a salt at an optimal salt concentration. In some embodiments, the salt is sodium chloride.

[0128] 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.

[0129] [Table 4]

[0130] [Table 5]

[0131] In some embodiments, the condition is selected from CEX E1 through CEX E25; in some embodiments, the condition is selected from CEX E26 through CEX E50; in some embodiments, the condition is selected from CEX E51 through CEX E75; in some embodiments, the condition is selected from CEX E76 through CEX E100; in some embodiments, the condition is selected from CEX E101 through CEX E125; in some embodiments, the condition is selected from CEX E126 through CEX E150; in some embodiments, the condition is selected from CEX E151 through CEX E175; in some embodiments, the condition is selected from CEX E176 through CEX E200; in some embodiments, the condition is selected from CEX E201 through CEX E225; in some embodiments, the condition is selected from CEX E226 through CEX E250. In some embodiments the condition is selected from CEX E36, CEX E61, CEX E86, CEX E111, CEX E37, CEX E62, CEX E87, CEX E112, CEX E38, CEX E63, CEX E88, CEX E113, CEX E39, CEX E64, CEX E89, CEX E114, CEX E40, CEX E65, CEX E90, and CEX E115. In some embodiments the condition is 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 condition is 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 condition is 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.

[0132] In some embodiments, the CEX chromatography comprises any one of the following combinations of wash / elution conditions: (1) a CEX washing condition selected from CEX W1 to CEX W25, and a CEX elution condition selected from CEX E1 to CEX E25; (2) one of the CEX washing conditions CEX W1 to CEX W25, and one of the CEX elution conditions CEX E101 to CEX E125; (3) one of the CEX wash conditions CEX W1 to CEX W25, and one of the CEX elution conditions CEX E226 to CEX E250; (4) a CEX washing condition selected from CEX W101 to CEX W125, and a CEX elution condition selected from CEX E1 to CEX E25; (5) a CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; (6) a CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; (7) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; (8) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; (9) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250.

[0133] The CEX chromatography steps described herein can be carried out under refrigerated conditions (e.g., 8±3° C.) or at ambient temperature.

[0134] In some embodiments, the methods of the present disclosure further comprise immobilized metal affinity chromatography (IMAC) to reduce host cell proteins. The term "IMAC" as used herein refers to the binding of Zn to histidine or cysteine ​​in aqueous solution. 2+ , Cu 2+ , Ni 2+ , and Co 2+ IMAC refers to a solid phase based on the affinity of transition metal ions such as . Types of IMAC are described in Block et al., Methods in Enzymology (2009) 463:439-73. In further embodiments, the IMAC resin is charged with a divalent ion. In yet other embodiments, the divalent metal ion is nickel, copper, cobalt, or zinc. In more specific embodiments, the divalent metal ion is zinc.

[0135] In some embodiments, IMAC chromatography includes: (1) loading a liquid composition containing rASM onto an IMAC chromatography membrane or column; (2) washing the IMAC chromatography membrane or column with a wash buffer; (3) eluting the rASM from the IMAC chromatography membrane or column with an elution buffer; and (4) collecting an eluate containing rASM.

[0136] In some embodiments, the eluate obtained from CEX chromatography is subjected to IMAC chromatography in bind-elute mode.

[0137] In some embodiments, IMAC comprises (1) loading a composition comprising rASM onto an IMAC chromatography membrane or an IMAC chromatography column, hi some embodiments, the composition comprises an unmodified rASM isoform and at least one modified rASM isoform, as described herein.

[0138] In some embodiments, the IMAC chromatography further comprises (2) washing the membrane or column with a wash buffer having a third optimal pH and a third optimal salt concentration. The third optimal pH and the third optimal salt concentration are predetermined according to the starting specific activity of the resin and the composition. In some embodiments, the wash buffer comprises a pH buffer system based on a phosphate, such as sodium phosphate. In some embodiments, the sodium phosphate concentration is about 1 to 100 mM, for example, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 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 third optimum salt concentration and the third optimum pH are selected from the conditions in Table 3 below.

[0139] [Table 6]

[0140] [Table 7]

[0141] Generally, a lower pH and / or a higher salt concentration in the IMAC wash buffer results in higher purity (e.g., higher HCP clearance), but lower specific activity and lower recovery. Therefore, IMAC wash conditions can be selected from IMAC W1 to IMAC W250 depending on the target purity and specific activity. In some embodiments, the condition is selected from IMAC W1 through IMAC W25; in some embodiments, the condition is selected from IMAC W26 through IMAC W50; in some embodiments, the condition is selected from IMAC W51 through IMAC W75; in some embodiments, the condition is selected from IMAC W76 through IMAC W100; in some embodiments, the condition is selected from IMAC W101 through IMAC W125; in some embodiments, the condition is selected from IMAC W126 through IMAC W150; in some embodiments, the condition is selected from IMAC W151 through IMAC W175; in some embodiments, the condition is selected from IMAC W176 through IMAC W200; in some embodiments, the condition is selected from IMAC W201 through IMAC W225; in some embodiments, the condition is selected from IMAC W226 through IMAC W250. In some embodiments the condition is 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, IMAC W105.In some embodiments, the condition is 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 condition is 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 condition is 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.

[0142] Optionally, the IMAC chromatography includes an additional wash step (IMAC wash 2). In some embodiments, the second wash step includes using a lower pH than that used in the first IMAC wash step. In some embodiments, the second wash step includes using a pH that is 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 lower than that used in the first IMAC wash procedure. In some embodiments, the second wash step includes using salt conditions that are the same or close to those used in the first IMAC wash step. In some embodiments, the second wash step includes a higher salt concentration (e.g., NaCl) than the first wash. In some embodiments, the second wash step comprises a concentration of 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, Including sodium chloride concentrations of 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM or more.

[0143] In some embodiments, the second wash step involves using a wash buffer with the same or similar salt concentration as used in the first IMAC wash step.

[0144] In some embodiments, the IMAC chromatography further comprises (3) eluting the membrane or 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 a phosphate, such as sodium phosphate. In some embodiments, the sodium phosphate concentration is about 5 to 100 mM, for example, 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 concentration in the IMAC wash 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 wash buffer.

[0145] In some embodiments, the fourth optimal salt concentration and the fourth optimal pH are selected from the conditions in Table 4 below.

[0146] [Table 8]

[0147] [Table 9]

[0148] The IMAC elution conditions can be selected from IMAC E1 to IMAC E250 depending on the target purity and specific activity. In some embodiments, the condition is selected from IMAC E1 through IMAC E25; in some embodiments, the condition is selected from IMAC E26 through IMAC E50; in some embodiments, the condition is selected from IMAC E51 through IMAC E75; in some embodiments, the condition is selected from IMAC E76 through IMAC E100; in some embodiments, the condition is selected from IMAC E101 through IMAC E125; in some embodiments, the condition is selected from IMAC E126 through IMAC E150; in some embodiments, the condition is selected from IMAC E151 through IMAC E175; in some embodiments, the condition is selected from IMAC E176 through IMAC E200; in some embodiments, the condition is selected from IMAC E201 through IMAC E225; in some embodiments, the condition is selected from IMAC E226 through IMAC E250. In some embodiments, the condition is 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, IMAC E91, IMAC E116, IMAC E17, IMAC E42, IMAC E67, IMAC E92, IMAC E117, IMAC E18, IMAC E43, IMAC E68, IMAC E93, and IMAC E118.In some embodiments, the condition is selected from IMAC E138, IMAC E163, 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, IMAC E166, IMAC E191, IMAC E216, IMAC E241, IMAC E142, IMAC E167, IMAC E192, IMAC E217, and IMAC E242. In some embodiments, the condition is 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 condition is selected from IMAC E143, IMAC E168, IMAC E193, IMAC E218, IMAC E243, IMAC E144, IMAC E169, IMAC E194, 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, IMAC E197, IMAC E222, and IMAC E247.

[0149] In some embodiments, IMAC comprises any one of the following combinations of wash / elution conditions: (1) one of the IMAC washing conditions IMAC W1 to IMAC W25, and one of the IMAC elution conditions IMAC E51 to IMAC E75; (2) one of the IMAC wash conditions IMAC W1 to IMAC W25, and one of the IMAC elution conditions IMAC E126 to IMAC E150; (3) one of the IMAC wash conditions IMAC W1 to IMAC W25, and one of the IMAC elution conditions IMAC E226 to IMAC E250; (4) one of the IMAC wash conditions, IMAC W76 to IMAC W100, and one of the IMAC elution conditions, IMAC E51 to IMAC E75; (5) one of the IMAC washing conditions, IMAC W76 to IMAC W100, and one of the IMAC elution conditions, IMAC E126 to IMAC E150; (6) one of the IMAC washing conditions, IMAC W76 to IMAC W100, and one of the IMAC elution conditions, IMAC E226 to IMAC E250; (7) one of the IMAC washing conditions IMAC W176 to IMAC W200, and one of the IMAC elution conditions IMAC E51 to IMAC E75; (8) one of the IMAC washing conditions IMAC W176 to IMAC W200, and one of the IMAC elution conditions IMAC E126 to IMAC E150; (9) One of the IMAC washing conditions, IMAC W176 to IMAC W200, and one of the IMAC elution conditions, IMAC E226 to IMAC E250.

[0150] The IMAC processes described herein can be carried out either under refrigerated conditions (eg, 8±3° C.) or at ambient temperature.

[0151] In some embodiments, the methods described herein include both CEX chromatography and IMAC, either in parallel or separately, regardless of the order of CEX chromatography and IMAC, and the methods include any one of the following wash / elution conditions: (1) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E1 to CEX E25; a IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (2) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E1 to CEX E25; a IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (3) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (4) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (5) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (6) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (7) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (8) any one of CEX washing conditions of CEX W1 to CEX W25, any one of CEX elution conditions of CEX E1 to CEX E25, any one of CEX washing conditions of CEX W1 to CEX W25, any one of CEX elution conditions of CEX E1 to CEX E25, any one of IMAC EX washing conditions of IMAC W176 to IMAC W200, and any one of IMAC elution conditions of IMAC E126 to IMAC E150; (9) A CEX washing condition of any one of CEX W1 to CEX W25, a CEX elution condition of any one of CEX E1 to CEX E25, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0152] (10) A CEX washing condition selected from any one of CEX W1 to CEX W25, and a CEX elution condition selected from any one of CEX E101 to CEX E125; (11) One of the IMAC washing conditions, IMAC W1 to IMAC W25, and one of the IMAC elution conditions, IMAC E51 to IMAC E75; (12) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (13) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (14) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (15) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (16) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (17) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (18) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (19) A CEX washing condition of any one of CEX W1 to CEX W25, a CEX elution condition of any one of CEX E101 to CEX E125, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0153] (20) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; a IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (21) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (22) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; a IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (23) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (24) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (25) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (26) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (27) A CEX washing condition of any one of CEX W1 to CEX W25, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (28) A CEX washing condition of any one of CEX W1 to CEX W25, a CEX elution condition of any one of CEX E226 to CEX E250, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0154] (29) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (30) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (31) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (32) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (33) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (34) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (35) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (36) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (37) A CEX washing condition of any one of CEX W101 to CEX W125, a CEX elution condition of any one of CEX E1 to CEX E25, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0155] (38) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (39) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (40) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (41) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (42) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (43) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (44) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (45) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (46) A CEX washing condition of any one of CEX W101 to CEX W125, a CEX elution condition of any one of CEX E101 to CEX E125, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0156] (47) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (48) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (49) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (50) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (51) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (52) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (53) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (54) A CEX washing condition of any one of CEX W101 to CEX W125, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (55) A CEX washing condition of any one of CEX W101 to CEX W125, a CEX elution condition of any one of CEX E226 to CEX E250, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0157] (56) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (57) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (58) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (59) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (60) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (61) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (62) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (63) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E1 to CEX E25; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (64) A CEX washing condition of any one of CEX W226 to CEX W250, a CEX elution condition of any one of CEX E1 to CEX E25, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0158] (65) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (66) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (67) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (68) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (69) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (70) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (71) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (72) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (73) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E101 to CEX E125; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (74) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (75) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (76) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W1 to IMAC W25, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (77) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (78) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (79) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W76 to IMAC W100, and an IMAC elution condition of any one of IMAC E226 to IMAC E250; (80) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E51 to IMAC E75; (81) A CEX washing condition of any one of CEX W226 to CEX W250, and a CEX elution condition of any one of CEX E226 to CEX E250; an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E126 to IMAC E150; (82) A CEX washing condition of any one of CEX W226 to CEX W250, a CEX elution condition of any one of CEX E226 to CEX E250, an IMAC washing condition of any one of IMAC W176 to IMAC W200, and an IMAC elution condition of any one of IMAC E226 to IMAC E250.

[0159] Recombinant acid sphingomyelinase compositions 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 oliprodase 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 to any one of SEQ ID NOs: 1-6, or a mixture thereof.

[0160] In some embodiments, the compositions described herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions are formulated according to the methods described herein. In some embodiments, the compositions are liquid formulations. In some embodiments, the compositions are lyophilized formulations.

[0161] In some embodiments, the composition is an rASM preparation, such as an 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.

[0162] In some embodiments, the disclosure provides a container (e.g., a vial) containing a composition described herein.

[0163] In some embodiments, compositions of the disclosure contain rhASM and demonstrate excellent rhASM isoform homogeneity and purity with well-controlled specific activity.

[0164] In some embodiments, the rASM compositions of the present disclosure have 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. The purity of the compositions of the present disclosure may be determined by any suitable method known in the art. In some embodiments, the purity is determined by HPLC, such as RP-HPLC.

[0165] In some embodiments, the rASM compositions of the disclosure have unmodified rASM isoforms that are 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 are 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 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. In some embodiments, all modified rASM isoforms, in total, represent 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 an rASM preparation.

[0166] In some embodiments, rASM isoforms having C-terminal cysteinylation represent less than 50%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less of the total rASM population in an rASM preparation.

[0167] In some embodiments, rASM isoforms having C-terminal S-glutathionylation represent 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%, no more than 1% or less of the total rASM population in an rASM preparation. In some embodiments, rASM isoforms having C-terminal S-glutathionylation represent 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 an rASM preparation.

[0168] In some embodiments, rASM isoforms having C-terminal dimerization are 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, or 0.2% or less of the total rASM population in the rASM preparation. In some embodiments, rASM isoforms having C-terminal dimerization are 0.1% or less of the total rASM population in the rASM preparation. Isoforms having C-terminal dimerization include, but are not limited to, those listed in Table 1.

[0169] In some embodiments, rASM isoforms with C-terminal truncations represent 50% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less of the total rASM population in an rASM preparation. In some embodiments, rASM isoforms with C-terminal truncations represent 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less than the total rASM population in an rASM preparation. Isoforms with C-terminal truncations include, but are not limited to, those listed in Table 1.

[0170] 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, about 65 U / mg, about 66 U / mg, about 67 U / mg, about g, 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, or about 50 U / mg. In some embodiments, the specific activity is about 5 to 50 U / mg. In some embodiments, the specific activity is about 10 to 40 U / mg. In some embodiments, the specific activity is about 10 to 30 U / mg. In some embodiments, the specific activity is about 10 to 20 U / mg. In some embodiments, the specific activity of the resulting rASM preparation is about 5-50 U / mg, e.g., 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 of the resulting 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, or about 50 U / mg. The specific activity of the rASM composition may be determined by any suitable method known in the art. In some embodiments, the specific activity of the rASM composition is determined by an assay as described in Example 4.

[0171] The recombinant ASM compositions described herein have host cell protein (HCP) levels of 5.0 μg / mg or less. In some embodiments, the rASM compositions have HCP levels of 5.0 μg / mg or less, 4.5 μg / mg or less, 4.0 μg / mg or less, 3.5 μg / mg or less, 3.0 μg / mg or less, 2.5 μg / mg or less, 2.0 μg / mg or less, 1.5 μg / mg or less, 1.0 μg / mg or less, 0.9 μg / mg or less, 0.8 μg / mg or less, 0.7 μg / mg or less, 0.6 μg / mg or less, 0.5 μg / mg or less, or less.

[0172] In some embodiments, the rASM compositions of the 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 characteristics selected from the group consisting of: (1) having 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) having 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 in the rASM composition being unmodified rASM isoform; (3) having rASM isoforms that contain C-terminal cysteinylation that account for less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less of the total rASM population; (4) having rASM isoforms that contain C-terminal S-glutathionylation that account for less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less of the total rASM population; (5) having rASM isoforms that contain C-terminal dimerization that account for less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the total rASM population; (6) having rASM isoforms containing C-terminal truncations that account for less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3% or less of the total rASM population; (7) having 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) Having a host cell protein (HCP) level of 5.0 μg / mg or less, 4.5 μg / mg or less, 4.0 μg / mg or less, 3.5 μg / mg or less, 2.0 μg / mg or less, 1.5 μg / mg or less, 1.0 μg / mg or less, 0.5 μg / mg or less, or less.

[0173] In some embodiments, compositions comprising recombinant acid sphingomyelinase (rASM) described herein are produced by purifying rASM expressed in a host cell using the methods described herein. In particular, compositions comprising rASM described herein are produced by a process that includes the purification methods described herein. In further embodiments, compositions comprising rASM described herein are purified using CEX chromatography as described herein. In further embodiments, compositions comprising rASM described herein are purified using CEX chromatography and / or IMAC as described herein.

[0174] Formulated pharmaceutical compositions containing purified rhASM The disclosure also provides pharmaceutical compositions comprising purified rASM (e.g., rhASM) as described herein. In some embodiments, the pharmaceutical compositions are made by formulating an rASM preparation as described herein. In some embodiments, the formulation process does not change or does not substantially change the specific activity of rASM in the rASM preparation and / or the associated ratio of rASM isoforms in the preparation. For example, after the formulation 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 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold or more compared to the specific activity before the formulation process.

[0175] In some embodiments, the compositions of the present invention contain one or more pharma- ceutically acceptable excipients. "Excipient" refers to an inert substance used as a diluent, vehicle, carrier, preservative, binder, or stabilizer for the active ingredient of a drug. For example, the compositions may contain a stabilizer, such as a buffer, an isotonicity agent, and / or an antioxidant. In some cases, one agent may serve two or more of these purposes. In some embodiments, the compositions of the present invention contain recombinant human ASM, such as oliprodase alpha, 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. Human ASM has improved stability due to its specific configuration in the composition. The compositions of the present invention may be an aqueous liquid solution or a lyophilized preparation.

[0176] In some embodiments, the composition is an aqueous liquid composition comprising 1-10 mg / mL (e.g., 3-5 mg / mL) rhASM (e.g., oliprodase 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 may be 5-8 (e.g., 6-7).

[0177] In some embodiments, the aqueous liquid composition does not contain detectable amounts of mannitol, which is the most readily used crystalline excipient, as this can significantly increase aggregation of human ASM during or after lyophilization of the aqueous liquid compositions described herein.

[0178] In some embodiments, the aqueous liquid composition comprises 0.004-0.008%, 0.005-0.007%, or 0.005% w / v of a surfactant. Exemplary surfactants include non-ionic surfactants such as polysorbates (e.g., polysorbate 20 and 80) and poloxamers (e.g., poloxamer 188). In certain embodiments, the aqueous liquid composition comprises 0.005% polysorbate 80. In some cases, the presence of a surfactant can help reduce turbidity in the liquid composition.

[0179] In some embodiments, the aqueous liquid composition comprises no more than 0.05, 0.01, or 0.005 mM of a chelating agent, such as EDTA and EGTA; in exemplary embodiments, the aqueous liquid composition does not comprise detectable amounts of a chelating agent. In some cases, the presence of a chelating agent, e.g., at concentrations greater than 0.05 mM or 0.1 mM, may increase aggregation and decrease the stability of human ASM, especially after extended storage periods, e.g., after 12-16 weeks, or under non-refrigerated conditions, e.g., 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 may be added exogenously, for example.

[0180] In a particular embodiment, the aqueous liquid composition comprises or consists essentially of 4 mg / mL oliprodase 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 other components in detectable amounts, or may contain only trace amounts of certain materials from the protein production process where such materials do not affect the biological activity of the enzyme or are harmful to the human patient.

[0181] In some embodiments, the composition is an aqueous liquid composition comprising 1-20 mg / mL (e.g., 10 mg / mL) rhASM (e.g., olidocidase 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 certain embodiments, the aqueous liquid composition has a pH of 6.5.

[0182] 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., olidocidase 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 certain 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 sodium phosphate, and 8% sucrose.

[0183] Aqueous liquid compositions can be prepared by mixing human ASM, produced by recombinant techniques 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 or buffer exchanged into a sodium phosphate buffer having a desired sodium phosphate concentration and pH.

[0184] In some embodiments, aqueous liquid compositions may be prepared by reconstituting a lyophilized composition of the invention, as described in more detail below. Reconstitution may be performed with a pharma- ceutically acceptable liquid, such as sterile water, saline (e.g., 0.9% sodium chloride), or phosphate buffered saline.

[0185] The present invention also provides a lyophilized composition. Such a composition may be prepared by freeze-drying the aqueous liquid composition described herein. The lyophilized composition is suitable for long-term storage. Lyophilization may be performed according to methods known in the art. For example, the liquid composition may be cooled to subzero (Celsius) temperatures (e.g., −5° C. to −80° C.) to allow freezing, and then placed in a low-pressure (partial vacuum) chamber to allow sublimation (primary drying); if necessary, the temperature of the composition may be increased in a second stage of drying (secondary drying) to further remove unwanted water molecules. In some embodiments, an inert gas such as nitrogen may be introduced into the container of the composition (e.g., a glass vial) after completion of the lyophilization process and before the container is sealed.

[0186] In some embodiments, the present invention provides a powder composition that can be prepared, for example, by spray drying the aqueous liquid composition described herein. The spray-dried composition is suitable for long-term storage. Spray drying can be carried out according to methods known in the art. For example, the liquid composition can be extruded through an atomizer or spray nozzle to be dispersed in a hot gas stream in a chamber as droplets of controlled size, thereby rapidly drying the liquid composition into a powder. The dried powder can then be collected at the bottom of the drying chamber. Other drying methods for preparing the powder composition are also contemplated.

[0187] Sucrose (or trehalose) and methionine present in the amounts described herein provide excellent results during lyophilization; the lyophilized product forms an excellent cake while maintaining the stability of human ASM during storage. Human ASM in the lyophilized compositions of the invention can remain non-aggregated and biologically active for at least 4 months (e.g., at least 6 months or at least 12 months) under refrigerated conditions (e.g., 0-10° C., 2-8° C., or 4° C.).

[0188] In some embodiments, the composition of the present invention is a lyophilized pharmaceutical composition comprising 4-50% oliprodase alpha, 3-7% sodium phosphate, and 45-90% sucrose (all w / w%). In certain embodiments, the lyophilized composition comprises 5.5% oliprodase alpha, 20.6% L-methionine, 2.3% sodium phosphate dibasic heptahydrate, 2.6% sodium phosphate monobasic monohydrate, and 69.0% sucrose (all w / w%). In certain embodiments, the lyophilized composition comprises 6.6% oliprodase alpha, 3.0% sodium phosphate dibasic heptahydrate, 3.3% sodium phosphate monobasic monohydrate, and 87.1% sucrose (all w / w%). In a particular embodiment, the lyophilized composition comprises 25.2% oliprodase alpha, 2.4% sodium phosphate dibasic heptahydrate, 2.6% sodium phosphate monobasic monohydrate, and 69.9% sucrose (all w / w%). In a particular embodiment, the lyophilized composition comprises 47.8% oliprodase alpha, 1.7% sodium phosphate dibasic heptahydrate, 1.8% sodium phosphate monobasic monohydrate, and 48.8% sucrose (all w / w%).

[0189] In some embodiments, the composition of the present invention is a lyophilized pharmaceutical composition comprising 4-7% oliprodase alpha, 15-25% L-methionine, 3-7% sodium phosphate, and 65-75% sucrose (all w / w%). In certain embodiments, the lyophilized composition comprises 5.5% oliprodase alpha, 20.5% L-methionine, 2.3% sodium phosphate dibasic heptahydrate, 2.6% sodium phosphate monobasic monohydrate, and 68.6% sucrose (all w / w%). 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% water.

[0190] In some embodiments, the present invention provides a vial containing a lyophilized pharmaceutical composition comprising 15-25 mg of olivopridase alpha, 75-85 mg of L-methionine, 15-25 mg of sodium phosphate, and 250-300 mg of sucrose. Prior to use, the composition may be reconstituted in 4-6 mL of sterile water.

[0191] 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 oliprodase alpha; 9.0 mg sodium phosphate dibasic heptahydrate; 10.0 mg sodium phosphate monobasic monohydrate; and 265 mg sucrose. The lyophilized composition may optionally contain 79.1 mg L-methionine. The lyophilized pharmaceutical composition may optionally contain 0-0.3 mg (e.g., 0.08-0.16 mg) zinc, which may be carried over from the manufacturing process or added exogenously, for example. In certain embodiments, the vial may have an internal sterile nitrogen-filled atmosphere. In certain embodiments, the lyophilized composition is reconstituted with 5.1 mL of sterile water to obtain an oliprodase alpha concentration of about 4.0 mg / mL, 3.8 mg / mL, 18 mg / mL, or 49 mg / mL, respectively. The reconstituted composition may be further diluted to a specific volume in 0.9% sodium chloride solution based on the dose to be administered.

[0192] In certain embodiments, the vial contains a lyophilized pharmaceutical composition comprising or consisting of 21.2 mg of oliprodase alpha, 79 mg of L-methionine, 9.0 mg of sodium phosphate dibasic heptahydrate, 10.0 mg of sodium phosphate monobasic monohydrate, and 265 mg of sucrose. The lyophilized pharmaceutical composition may optionally contain 0-0.3 mg (e.g., 0.08-0.16 mg) of zinc, which may be carried over from the manufacturing process or added exogenously, 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 internal sterile nitrogen-filled atmosphere. In certain embodiments, the lyophilized composition is reconstituted in 5.1 mL of sterile water to obtain an oliprodase alpha concentration of about 4.0 mg / mL. The reconstituted composition may be further diluted to a specific volume in 0.9% sodium chloride solution based on the dose to be administered.

[0193] In some embodiments, the present invention provides a vial containing a lyophilized pharmaceutical composition comprising 3-5 mg of olivopridase alpha, 15-17 mg of L-methionine, 3-5 mg of sodium phosphate, and 50-60 mg of sucrose. Prior to use, the composition may be reconstituted in 0.8-1.2 mL of sterile water.

[0194] In certain embodiments, the vial contains a lyophilized pharmaceutical composition comprising or consisting of 4.8 mg oliprodase alpha, 17.9 mg L-methionine, 2.0 mg sodium phosphate dibasic heptahydrate, 2.3 mg sodium phosphate monobasic monohydrate, and 60 mg sucrose. In certain embodiments, the lyophilized pharmaceutical composition is in the form of a cake or lyophilized powder. The lyophilized composition may optionally contain 0-0.06 mg zinc, which may be carried over from the manufacturing process or added externally, for example. In certain embodiments, the vial may have an internal sterile nitrogen filled atmosphere. In certain embodiments, the lyophilized composition is reconstituted in 1.1 mL of sterile water to obtain an oliprodase alpha concentration of about 4.0 mg / mL. The reconstituted composition may be further diluted to a specific volume in 0.9% sodium chloride solution based on the dose to be administered.

[0195] product rASM purified by the methods described herein and / or a formulation comprising rASM purified by the methods described herein may be included in an article of manufacture. The article of manufacture may include a container containing the rASM and / or the rASM formulation. In certain embodiments, the article of manufacture includes (a) a container containing a composition comprising the rASM and / or rASM formulation described herein within the container; and (b) a package insert comprising instructions for administering the formulation to a subject.

[0196] The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds or contains the formulation and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a polypeptide. The label or package insert indicates the use of the composition in a subject and provides specific guidance regarding the dosage and interval of administration of the polypeptide and any other drugs. The article of manufacture can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the container is a syringe. In some embodiments, the syringe is further included within an injection device. In some embodiments, the injection device is an autoinjector.

[0197] "Package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information regarding indications, use, dosage, administration, contraindications, other therapeutic products that may be combined with the packaged product, and / or warnings regarding the use of such therapeutic products. EXAMPLES

[0198] The following examples are provided 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 this invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0199] Example 1: Effect of purification on rhASM specific activity During process characterization studies, an attempt was made to better understand the factors that affect rhASM specific activity. The purpose of such process characterization studies was to understand how the manufacturing process affects rhASM product specific activity and to find the critical steps sufficient to control the product specific activity.

[0200] Several different isoforms of rhASM are present in the ASM population produced in the bioreactor. Details of the rhASM isoforms are listed in Table 1. Testing confirmed that the dimerized form of rhASM has a higher specific activity compared to the monomeric form of rhASM (Figure 3). Also, the isoform of olivopidase alpha chemically modified at the C-terminal cysteine ​​(C570) has a higher specific activity than the unmodified form, likely due to the association between C570 and the active site. This testing further demonstrates the correlation between the abundance of the C-terminal modified form and the increased specific activity (Figure 4). Essentially, if there is a higher abundance of modified rhASM in a composition, the composition will have a higher specific activity.

[0201] Samples containing rhASM were subjected to cation exchange (CEX) chromatography. rhASM binds to the resin while impurities flow through. Further impurity reduction was achieved by post-loading washes, and then oligopeptidase alpha was eluted from the column by increasing the salt concentration compared to the wash buffer.

[0202] The cation exchange (CEX) chromatography eluate was further purified by immobilized metal affinity chromatography (IMAC). The resin was loaded with metal ions. The cation exchange (CEX) chromatography eluate was adjusted to the target pH and loaded onto an equilibrated IMAC column. The rhASM was then eluted with high salt elution buffer.

[0203] The immobilized metal affinity chromatography (IMAC) eluates were collected, combined and concentrated, and the resulting bulk sample was then formulated as described in Example 2.

[0204] It was found that the purity of the rhASM preparation (measured by RP-HPLC) was well controlled in the CEX chromatography and / or IMAC steps. As shown in Figure 5, in the CEX chromatography step, higher HCP clearance was achieved at higher pH and higher salt concentration, but higher HCP clearance resulted in lower recovery yield. Similarly, in the IMAC step, higher HCP clearance was achieved at lower pH and higher salt concentration, but higher HCP clearance also resulted in lower yield, as shown in Figure 6. Recovery was measured by both A280 absorbance and activity level. HCP levels were measured by both Octet® assay and ELISA.

[0205] Unexpectedly, the conditions that provided superior HCP clearance had a detrimental effect on specific activity. As shown in Figure 7, higher HCP clearance was achieved at higher pH, higher salt conditions in the CEX chromatography step, but the resulting rhASM preparations had lower specific activity. Similarly, as shown in Figure 8, higher HCP clearance was achieved at lower pH, higher salt conditions in the IMAC step, but the resulting rhASM preparations had lower specific activity.

[0206] We hypothesized that the highly active rhASM isoforms bound weakly to the chromatography column and were lost during the washing steps of the chromatography process.

[0207] To test this hypothesis, the loading material (e.g., product pool-rhASM sample prior to the CEX chromatography step), the wash fractions obtained from the CEX chromatography step, and the elution fractions obtained from the CEX chromatography step were analyzed to determine their specific activities in vitro as described in Example 4. Indeed, the wash fractions recovered from the CEX run showed higher specific activities than the initial loading material and the elution fractions (Figure 9, at the specified wash conditions). Several other salt concentration / pH wash conditions were also tested, yielding similar results.

[0208] For the IMAC step, the loading material (e.g., product pool-rhASM sample prior to the IMAC chromatography step), the wash fractions obtained from the IMAC step, and the elution fractions obtained from the IMAC step were also analyzed to determine their specific activities in vitro. Similarly, the wash fractions recovered from the IMAC procedure showed higher specific activities than the initial loading material and the elution fractions (Figure 10A, at the specified wash conditions). Figure 10B shows that the IMAC procedure further improved the purity of rhASM in the elution fractions. Purities of 98% or greater or even 99% were achieved. Several other salt concentration / pH conditions were also tested, yielding similar results.

[0209] To further verify, the loading material, wash fractions, and elution fractions of the CEX and IMAC runs were subjected to mass spectrometry to quantify the unmodified and modified isoforms. The free cysteine ​​residues of rhASM were labeled before analyzing the ASM isoform progeny. The relative abundance of the unmodified and modified isoforms is shown in Figure 11 (CEX run) and Figure 12 (IMAC run). Indeed, in both runs, the rhASM isoform with the C-terminal modification was enriched in the wash fraction.

[0210] It was also observed that the relative abundance of unmodified and modified rhASM isoforms changed in both the wash and elution fractions through the CEX procedure. LC-MS analysis showed that the relative abundance of unmodified rhASM isoforms in the elution fraction was enriched compared to the loading material. The combined modified rhASM isoforms in the elution population was lower compared to the loading material. Thus, the CEX procedure can regulate the ratio between unmodified and modified rhASM isoforms in the rhASM product.

[0211] In summary, optimization of two bind-elute chromatography steps in the manufacturing process of oliprodase alfa resulted in unexpected effects on specific activity and rhASM isoform population. Specific activity was observed to decrease across both runs, with high specific activity observed in the wash fraction for both runs. Mass spectrometry identified weakly bound "activated" rhASM isoforms that were enriched in the wash fraction and decreased in the elution fraction. Analytical results from both studies suggest that the specific activity in the eluate of either the CEX or IMAC run can be reduced by selectively removing the highly active C-terminal modified species during the wash step. Furthermore, the runs can adjust the ratio between unmodified and modified rhASM isoforms in the rhASM preparation, thereby improving the homogeneity of the rhASM population in the final product. The methods described herein allow for adjustment of the specific activity and ratio of unmodified and modified rhASM isoforms during the manufacturing process, thereby obtaining rhASM preparations with the desired specific activity.

[0212] Example 2: Recombinant Human Acid Sphingomyelinase Preparations Formulation steps are performed to achieve final oligopeptide alfa and excipient concentrations in the drug substance.

[0213] Formulation is carried out according to the method described in WO2019 / 227029A1, the entirety of which is incorporated herein by reference. The rhASM preparation obtained from the purification process in Example 1 was filtered to remove viral contamination and concentrated to increase protein concentration before formulation. The formulated bulk was mixed and the rhASM concentration was determined by measuring absorbance at 280 nm. The product pool was then further diluted to target volume to obtain the final drug substance olivodase alfa concentration.

[0214] Optionally, the liquid composition obtained herein can be spray-dried. The spray-dried composition is suitable for long-term storage. Spray-drying can be carried out according to methods known in the art. For example, the liquid composition can be extruded through an atomizer or spray nozzle to be dispersed in a hot gas stream in a chamber as droplets of controlled size, thereby rapidly drying the liquid composition into a powder. The dried powder can then be collected at the bottom of the drying chamber. Other drying methods for preparing powder compositions are also contemplated.

[0215] Example 3: Study of the efficacy, safety, pharmacodynamics and pharmacokinetics of oliprodase alfa in patients with acid sphingomyelinase deficiency The recombinant human oliprosidase alfa preparation prepared in Example 2 was used in a clinical trial to evaluate its efficacy. The study design is shown in Figure 13. Baseline patient and disease characteristics are shown in Table 6.

[0216] [Table 10]

[0217] The primary objective of this study was to evaluate the efficacy of a formulation administered intravenously once 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 in the United States [US] only, along with patient perception related to spleen volume as measured by the splenomegaly related score [SRS]); and 2) infiltrative lung disease as measured by pulmonary function testing, diffusing capacity for carbon monoxide (DLCO).

[0218] Primary endpoint - predicted DL CO % Change Percent The results shown in FIG. 14 show that at baseline, the mean % predicted DL COwere similar, reflecting moderate disease in both groups; the mean improvement from baseline to week 52 was 22% for the oliprodase alfa group compared with 3% for the placebo group; the difference between the groups was 19% at week 52 (p<0.0004), with improvements seen as early as week 26; the test was judged positive; and mean predicted FVC% also showed improvement at week 52 in the oliprodase alfa group but not in the placebo group.

[0219] Lung imaging also showed improvement in ASMD-mediated interstitial lung disease. An exemplary high-resolution computed tomography image from an oliprodase alpha-treated patient ( FIG. 15 ) shows clearance of the “ground glass” opacity caused by sphingomyelin-filled macrophages. HRCT ground glass appearance score and interstitial lung disease score in both lungs showed a mean improvement in oliprodase alpha-treated patients but not in placebo-treated patients ( FIG. 16 ).

[0220] Primary endpoint - Splenic response Spleen volume was reduced in all oripudase alfa-treated patients but not in placebo-treated patients (Figure 17, left panel). Mean baseline spleen volume was 11.2 MN (multiple of normal) in the placebo group and 11.7 MN in the oripudase group, indicating moderate splenomegaly. A significant reduction in spleen volume was seen in oripudase alfa-treated patients over 6 months of treatment, with the greatest reduction seen in patients with the worst baseline splenomegaly. A statistically significant 39% reduction in spleen size in oripudase alfa-treated patients was demonstrated at week 52, compared with a 0.5% increase in placebo patients (p<0.0001). Additionally, 17 of 18 oripudase-alfa-treated patients (94%) had a ≥30% reduction in spleen volume compared with 0 / 18 placebo patients. The greatest reduction was seen in oripudase alfa-treated patients with the largest spleens at baseline.

[0221] Splenomegaly-related scores were also calculated in oliprodase alfa- and placebo-treated patients, which decreased in parallel in both groups (Figure 17, right panel). There was no correlation between SRS and baseline or final spleen volume; therefore, symptoms measured by SRS did not reflect physiological disease burden. The mean baseline SRS scores were 28.1 in placebo and 24.6 in oliprodase alfa. Both groups showed a decrease in SRS scores; however, the LS mean change in SRS score from baseline to week 52 was not statistically different in the oliprodase alfa group (-7.66) compared to the placebo group (-9.28) after multiplicity adjustment; p = 0.6364. Of note, the SRS was adapted from a myelofibrosis study and has not been previously validated in ASMD patients.

[0222] Secondary endpoints - Hepatic response Patients had moderate baseline hepatomegaly, with a mean liver volume of 1.6 MN in the placebo group and 1.4 MN in the oliprodase alfa group, in addition to an atherogenic lipid profile and abnormal liver function tests.

[0223] LS mean percentage change in liver volume from baseline to week 52 showed a greater reduction in the oliprodase-alpha group (31.67%) compared to the placebo group (1.42%; nominal p<0.0001) (Figure 18). Similarly, baseline atherogenic lipid profiles improved in oliprodase-alpha treated patients but not in placebo patients, with mean reductions in LDL cholesterol and triglycerides and increases in HDL cholesterol. Mean ALT and AST and other liver function tests also improved in oliprodase-alpha treated patients but not in placebo treated patients.

[0224] Secondary endpoint - Platelet count Mean platelet counts improved in patients treated with oliprodase alfa but not in those treated with placebo (+16.8% vs. +2.5%, respectively, p=0.019), with clinical differences seen through 26 weeks.

[0225] Exploratory Endpoints – Hepatic Sphingomyelin Hepatic sphingomyelin levels were monitored in oliprodase alpha-treated and placebo-treated patients. In oliprodase alpha-treated patients, the mean tissue area percentage occupied by sphingomyelin decreased from 29% to 2% after 52 weeks, but remained unchanged in placebo patients (Figure 19). Histological analysis of liver biopsy data showed substantial clearance of sphingomyelin in Kupffer cells and hepatocytes in oliprodase alpha-treated patients, but not in placebo-treated patients, as demonstrated by representative liver biopsy images (Figure 20).

[0226] Exploratory Endpoints - Biomarker Response The mean levels of the plasma biomarkers chitotriosidase and lysosphingomyelin were both significantly higher at baseline. Baseline values ​​in both groups were >14xULN for chitotriosidase and >38xULN for lysosphingomyelin. Mean chitotriosidase levels declined by 54% in the oliprodase alpha group versus 12% in placebo at week 52. Mean lysosphingomyelin declined by 78% in the oliprodase alpha group versus 6% in placebo at week 52. Both showed substantial declines from the first few weeks of treatment in oliprodase-alpha treated patients but not in placebo treated patients (Figure 21).

[0227] overview The overall safety and tolerability profile was favorable, and no new safety risks were identified in the study. No patients died and there were no permanent discontinuations of oliprodase alfa due to adverse events. All patients continued in the study extension, except for one patient who discontinued during the primary analysis period due to poor compliance. Three oliprodase alfa-treated patients had five serious adverse events and four placebo patients had 11 serious adverse events, but the serious adverse events were not considered related to treatment. Expected infusion-related reactions in patients initiating enzyme replacement therapy were mild or moderate and easily managed. Four of 18 oliprodase 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 patients developed neutralizing antibodies that interfered with cellular uptake of the enzyme.

[0228] This test is DL CO The endpoint was met and the overall data demonstrated clinical benefit, so it is considered positive. ASMD-associated interstitial lung disease improved in patients treated with rhASM produced by the methods described herein. Spleen volume was reduced, accompanied by an increase in platelet count, reflecting improvement in hypersplenism. Liver volume was reduced due to clearance of sphingomyelin, which was supported by histological evidence from serial liver biopsies. Metabolic function was improved, as evidenced by improved liver function tests and lipid profile. Symptoms measured by SRS did not reflect disease burden as measured by physiological measures (spleen volume). The benefit-risk profile of oliprodase alfa in adults with ASMD is favorable based on this study.

[0229] Clinical trials suggest that rhASM compositions prepared by the methods described in this disclosure have demonstrated superior safety and efficacy in treating ASMD patients.

[0230] Example 4: rhASM specific activity in in vitro activity assay This example describes a method to determine 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) at pH 5.3 in a circulating water bath at 37.0° C. for 15 minutes. Essentially, 80 μL of substrate was added to 20 μL of enzyme to initiate the reaction. The reaction was stopped by the addition of 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 was defined as the amount of enzyme required to hydrolyze 1 μmol of 2-(N-hexadecanoylamino-4-nitrophenyl)phosphorylcholine (HDA-PC) to 2-(N-hexadecanoylamino-4-nitrophenol (HDA-NP) per minute under the defined assay conditions. 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).

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

[0233] Articles such as "a," "an," and "the" may mean at least one, unless stated to the contrary or otherwise clear from the context. A claim or description containing "or" between two or more members of a group is considered to be satisfied when one, more than one, or all of the members of the group are present, unless stated to the contrary or otherwise clear from the context. 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 two or more members of the group are present, and embodiments in which all of the group members are present. For the sake of brevity, the embodiments are not individually described herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0234] It should be understood that the present disclosure encompasses all variations, combinations, and substitutions in which at least one limitation, element, clause, or descriptive term from at least one of the claims or at least one relevant portion of this specification is introduced into another claim. For example, a claim that is dependent on another claim may be modified to include at least one of the limitations found in any other claim that is dependent on the same base claim. Furthermore, when a claim describes a composition, it should be understood that the claim includes the method of making or using the composition according to the method of making or using disclosed herein or the method known in the art, if any, unless otherwise indicated or unless it is obvious to a person skilled in the art that a contradiction or inconsistency would occur.

[0235] When elements are presented as a list, e.g., in Markush group format, it is understood that all possible subgroups of the elements are also disclosed, and that any element or subgroup of elements can be deleted from the group. It is also noted that the term "comprising" is intended to be open, allowing for the inclusion of additional elements or steps. In general, when an embodiment, product, or method is indicated as comprising a particular element, feature, or step, it is understood that an embodiment, product, or method that comprises or consists essentially of such element, feature, or step is also provided. For the sake of brevity, the embodiments are not individually described herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

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

[0237] When ranges are stated, the endpoints are included. Furthermore, unless otherwise stated or otherwise clear from the context and / or the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can take any particular value within the stated range in some embodiments, up to one tenth of the unit of the lower limit of that range, unless otherwise clearly indicated by the context. For the sake of brevity, the values ​​within each range are not individually set forth herein, but it will be understood that each of these values ​​is provided herein and can be specifically claimed or disclaimed. It should also be understood that unless otherwise stated or otherwise clear from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges can take any subrange within the given range, with the endpoints of the subrange being expressed to the same degree of precision as one tenth of the unit of the lower limit of that range.

[0238] Where websites are provided, URL addresses are provided as non-browser executable code, including the respective web address range within brackets. The actual web addresses do not include the brackets.

[0239] Furthermore, it should be understood that any particular embodiment of the present disclosure may be expressly excluded from at least one of the claims. When a range is stated, any value within that range may be expressly excluded from at least one of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the present disclosure may be excluded from any at least one of the claims. For the sake of brevity, not all of the embodiments in which at least one element, feature, purpose, or aspect is excluded are explicitly described herein.

[0240] All publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned herein, for example in the Background, Summary, Detailed Description, Examples, and / or References sections, are incorporated herein by reference as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. 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), (i) Subject a protein mixture containing rASM and host cell protein (HCP) to cation exchange (CEX) chromatography, or A protein mixture containing rASM and HCP is subjected to immobilized metal affinity chromatography (IMAC), or A protein mixture containing rASM and HCP is subjected to both CEX chromatography and IMAC, and (ii) Collect the eluate from the CEX chromatography or IMAC and obtain a purified rASM preparation therefrom. Includes, A method wherein the rASM is recombinant human acid sphingomyelinase (rhASM).

2. A method for adjusting the relative amount of recombinant acid sphingomyelinase (rASM) isoforms 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 cysteineization, S-glutathioneization, dimerization, and cleavage, and the method is (i) Subject the initial rASM composition to cation exchange (CEX) chromatography, or The initial rASM composition is subjected to immobilized metal affinity chromatography (IMAC), or The initial rASM composition is subjected to both CEX chromatography and IMAC, and (ii) Collect the eluate from the CEX chromatography or IMAC and obtain a purified rASM preparation therefrom. Includes, A method wherein the rASM is recombinant human acid sphingomyelinase (rhASM).

3. A method for adjusting the specific activity of rASM in a liquid composition comprising an unmodified recombinant acid sphingomyelinase (rASM) isoform and at least one rASM isoform having one or more modifications selected from the group consisting of C-terminal cysteineization, S-glutathioneization, dimerization, and cleavage, (i) Subjecting the liquid composition to cation exchange (CEX) chromatography, or The liquid composition is subjected to immobilized metal affinity chromatography (IMAC), or The liquid composition is subjected to both CEX chromatography and IMAC, and (ii) Recover the eluate from the CEX chromatography or IMAC and obtain a purified rASM preparation therefrom. Includes, A method wherein the rASM is recombinant human acid sphingomyelinase (rhASM).

4. The protein mixture, initial rASM composition, or liquid composition is i) CEX chromatography and IMAC are performed in parallel, and the eluate obtained from the CEX chromatography is subjected to the IMAC; ii) Subjected to IMAC and CEX chromatography in parallel, wherein the eluate obtained from IMAC is subjected to CEX chromatography; or iii) Subjected separately to both CEX chromatography and IMAC, with one or more additional steps in between, The method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 3, wherein the protein mixture comprising rASM and HCP, the initial rASM composition, or the liquid composition is subjected to one or more additional purification columns before or after the mixture or composition is subjected to the CEX chromatography or the IMAC.

6. (i) Inactivating and / or removing potential viral contaminants; (ii) Concentrating the purified rASM; and / or (iii) Perform buffer exchange in the generated rASM. Includes, The method according to any one of claims 1 to 3, wherein the method is optionally carried out partially or completely under refrigerated conditions of 8 ± 3°C or at ambient temperature.

7. The method according to any one of claims 1 to 3, wherein the protein mixture, initial rASM composition, or liquid composition is obtained from Chinese hamster ovary (CHO) cells expressing the rASM, and optionally the rASM comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

8. The method according to any one of claims 1 to 3, wherein the CEX chromatography comprises a resin selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S).

9. The method according to any one of claims 1 to 3, wherein the IMAC comprises a chelate resin, and optionally the IMAC is made using zinc, copper, or nickel.

10. The CEX chromatography has a first optimal pH and a first optimal salt concentration. The process includes washing the CEX chromatography column with CEX washing buffer, wherein the first optimal pH and the first optimal salt concentration are predetermined according to the initial specific activity of the resin and protein mixture, the initial rASM composition, or the liquid composition. The method according to any one of claims 1 to 3, optionally further comprising eluting the CEX chromatography column with a CEX elution buffer having a second optimal pH and a second optimal salt concentration, wherein the rASM bound to the CEX chromatography column after washing is removed from the column under the second optimal pH and second optimal salt concentration.

11. The IMAC includes washing the IMAC column with at least one IMAC washing 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 according to the initial specific activity of the resin and protein mixture, the initial rASM composition, or the liquid composition. The method according to any one of claims 1 to 3, optionally further comprising eluting the IMAC column with an IMAC elution buffer having a fourth optimal pH and a fourth optimal salt concentration, wherein the rASM bound to the IMAC column after washing is removed from the column under the fourth optimal pH and the fourth optimal salt concentration.

12. (i) The purified rASM preparation has a specific activity of about 5 to 50 U / mg, optionally about 10 to 45 U / mg, and even more optionally about 10 to 20 U / mg; (ii) The purified rASM preparation has an HCP level of 1.0 μg / mg or less or 5.0 μg / mg or less; and / or (iii) The method according to any one of claims 1 to 3, wherein the purified rASM preparation comprises, in total, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or less of the total rASM population of modified rASM isoforms, and optionally, the modified rASM isoforms comprise one or more modifications selected from the group consisting of C-terminal cysteine, S-glutathione, dimerization, and cleavage.

13. The method according to any one of claims 1 to 3, wherein the protein mixture, initial rASM composition, or liquid composition is produced in a bioreactor having a production capacity of at least 100 L or at least 500 L.

14. A recombinant acid sphingomyelinase (rASM) preparation comprising an unmodified recombinant acid sphingomyelinase (rASM) isoform and at least one rASM isoform species having one or more modifications selected from the group consisting of C-terminal cysteineization, S-glutathioneization, dimerization, and cleavage, wherein the unmodified rASM isoform constitutes at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the total rASM population in the rASM preparation, and the rASM is recombinant human acid sphingomyelinase (rhASM).

15. The rASM preparation according to claim 14, wherein the unmodified rASM isoform constitutes 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.

16. All modified rASM isoforms together constitute 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, and 7% of the total rASM population in the rASM preparation. The rASM preparation according to claim 14 or 15, wherein the amount is 6% or less, 5% or less, or less than that.

17. (i) The rASM isoform having a C-terminal cysteine ​​is present in an amount of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, or less of the total rASM population in the rASM preparation; (ii) The rASM isoform having C-terminal S-glutathione is present in an amount of 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less of the total rASM population in the rASM preparation; (iii) The rASM isoform having C-terminal dimerization accounts for 0.2% or less of the total rASM population in the rASM preparation; (iv) The rASM isoform having a C-terminal S-dimerization accounts for 0.1% or less of the total rASM population in the rASM preparation; and / or (v) The rASM preparation according to claim 14 or 15, wherein the rASM isoform having a C-terminal cleavage accounts for 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less or less of the total rASM population in the rASM preparation.

18. The rASM preparation according to claim 14 or 15, wherein the rASM preparation has a purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher.

19. (i) The rASM preparation has a specific activity of about 5 to 50 U / mg, optionally about 10 to 20 U / mg; (ii) The rASM preparation has a host cell protein (HCP) level of 5.0 μg / mg or less; (iii) The rASM preparation according to claim 14 or 15, wherein the rASM preparation is manufactured using the method described in claim 1.

20. A pharmaceutical composition prepared by using the rASM preparation described in claim 14 or 15.

21. The pharmaceutical composition according to claim 20 for treating acid sphingomyelinase deficiency in subjects who require treatment for acid sphingomyelinase deficiency.

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