COMPOSITIONS, THEIR USE IN THE TREATMENT OF ASMD, CONTAINERS AND MANUFACTURED ARTICLES

Description

1 / 36 COMPOSITIONS, THEIR USE IN THE TREATMENT OF ASMD, CONTAINERS AND ARTICLES OF MANUFACTURE Divided from BR112020023829-5, filed on 05 / 24 / 2019 BACKGROUND OF THE INVENTION

[001] Acid sphingomyelinase deficiency (ASMD) is a rare, life-threatening lysosomal storage disorder. It is an autosomal recessive genetic disease resulting from mutations in the SMPD1 gene that encodes the lysosomal enzyme acid sphingomyelinase (ASM) (Schuchman et al., Mol. Genet. Metab.120(1-2): 27-33 (2017)). ASMD patients are unable to metabolize sphingomyelin, which consequently accumulates in lysosomes in multiple organs, causing visceral disease and neurodegeneration in severe cases. ASMD patients have elevated cholesterol and other lipids in the spleen, liver, lungs, and bone marrow.

[002] Infantile neurovisceral ASMD (also known as Niemann-Pick disease type A or NPD A) is the most severe phenotype of the disease and is characterized by early onset and acute neuropathic form. NPD A results in failure to thrive, hepatosplenomegaly, and rapidly progressive neurodegeneration. Patients die in early childhood (McGovern et al., Neurology 66(2): 228-232 (2006)).

[003] Patients with chronic visceral ASMD (NPD B) and chronic neurovisceral ASMD (NPD A / B) have an onset that varies from childhood to adulthood (Wasserstein et al., Pediatrics 114(6): e672-677 (2004); Wasserstein et al., J. Pediatr. 149(4): 554-559 (2006)). NPD B patients are usually diagnosed in childhood, typically after the age of two. Most NPD B patients live into adulthood. NPD A / B patients are classified as having an intermediate form, with childhood neurological symptoms manifesting that may develop into disease. Petition 870260052183, dated 05 / 29 / 2026, page 10 / 134 2 / 36 neurodegenerative. Liver, lung, and hematological disease morbidity occurs in all patients with chronic ASMD and includes hepatosplenomegaly, liver dysfunction, pulmonary infiltration disease, and thrombocytopenia (McGovern et al., Genet. Med. 15(8): 618-623 (2013); McGovern et al., Orphanet J. Rare Dis. 12(1): 41 (2017)). Growth restriction during childhood and bone disorders such as low bone density are also common features of chronic ASMD (Wasserstein et al., J. Pediatr. 142(4): 424-428 (2003)). Lung and liver diseases are the main causes of death in these patients (McGovern et al., Pediatrics 122(2): e341-349 (2008); Cassiman et al., Mol. Genet. Metab. 118(3): 206-213 (2016)).

[004] Due to the high morbidity and mortality rates of ASMD, there remains an urgent need for effective treatment of this genetic disease. SUMMARY OF THE INVENTION

[005] The present invention provides recombinant human ASM (rhASM) compositions for the treatment of ASMD. In some embodiments, the compositions comprise rhASM, sodium phosphate, methionine, and sucrose (or trehalose). In certain embodiments, rhASM is olipudase alfa (SEQ ID NO: 2).

[006] In some embodiments, the composition is freeze-dried. A freeze-dried composition of the invention may comprise, for example: 4 to 7% by weight / weight of alpha lipopudase, 3 to 7% by weight / weight of sodium phosphate, 15 to 25% by weight / weight of L-methionine, and 65 to 75% by weight / weight of sucrose.

[007] In certain embodiments, a freeze-dried composition of the invention may comprise: 5.5% by weight of alpha lipopudase, Petition 870260052183, dated 05 / 29 / 2026, page 11 / 134 3 / 36 2.3% by weight of dibasic sodium phosphate heptahydrate, 2.6% by weight of monobasic sodium phosphate heptahydrate, 20.5% by weight / weight of L-methionine, and 68.6% by weight / weight of sucrose.

[008] In some embodiments, the composition is an aqueous liquid composition. The aqueous liquid composition may comprise, for example: 1 to 10 mg / ml of alpha lipopudase, 10 to 50 mM of sodium phosphate, 70 to 150 mM of L-methionine, and 1 to 10% by weight / volume of sucrose, wherein the composition has a pH of 5 to 8.

[009] In certain embodiments, an aqueous liquid composition of the invention may comprise: 3 to 5 mg / ml of alpha lipopudase, 10 to 30 mM of sodium phosphate, 80 to 120 mM of L-methionine, and 4 to 6 by weight / volume of sucrose, resulting in a pH of 6 to 7.

[0010] In a particular embodiment, an aqueous liquid composition of the invention may comprise: mg / ml of alpha olipudase, mM of sodium phosphate, 100 mM of L-methionine, and 5% by weight / volume of sucrose, resulting in a pH of 6.5.

[0011] In some embodiments, the aqueous liquid composition of Petition 870260052183, dated 05 / 29 / 2026, page 12 / 134 4 / 36 The invention may also comprise 0.005% by weight / volume of polysorbate 80.

[0012] The invention also provides a composition obtained by drying (e.g., freeze-drying or spray-drying) an aqueous liquid composition described in this document. The invention also provides a process for manufacturing a freeze-dried composition, which comprises freeze-drying an aqueous liquid composition described in this document.

[0013] In some embodiments, the invention provides a container containing a freeze-dried composition described herein. In certain embodiments, the freeze-dried composition in the container comprises, or essentially consists of: 21.2 mg of olipudase alfa, 9.0 mg of dibasic sodium phosphate heptahydrate, 10.0 mg of monobasic sodium phosphate heptahydrate, mg of L-methionine, and 265 mg of sucrose.

[0014] In some embodiments, the lyophilized composition is reconstituted in 5.1 ml of sterile water to obtain an aqueous liquid composition.

[0015] In certain embodiments, the lyophilized composition in the container comprises, or consists essentially of: 4.8 mg of olipudase alfa, 2.0 mg of dibasic sodium phosphate heptahydrate, 2.3 mg of monobasic sodium phosphate heptahydrate, 17.9 mg of L-methionine, and 60 mg of sucrose.

[0016] In some embodiments, the lyophilized composition is reconstituted in 1.1 ml of sterile water to obtain an aqueous liquid composition. Petition 870260052183, dated 05 / 29 / 2026, page 13 / 134 5 / 36

[0017] The invention also provides a manufactured article comprising 1) a container containing a lyophilized composition described in this document, and 2) a container containing, for example, sterile water, 0.9% sodium chloride, or phosphate-buffered saline solution to reconstitute the lyophilized composition.

[0018] The invention also provides a method of treating ASMD in a human patient, which comprises administering to the patient a composition described herein, wherein the composition is reconstituted in a liquid form prior to administration if it is a lyophilized composition.

[0019] The invention also provides a composition described herein for use in the treatment of ASMD in a human patient.

[0020] The invention also provides the use of a composition described herein for the manufacture of a medicament for the treatment of ASMD in a human patient.

[0021] In some modalities, the treatment of ASMD as described in this document is for Niemann-Pick disease type A / B or type B or for non-neurological manifestations of ASMD. BRIEF DESCRIPTION OF THE FIGURES

[0022] FIGURE 1A shows the stability of rhASM as measured by specific (enzymatic) activity after two weeks of storage at 30°C in a succinate, citrate, citrate / phosphate, or phosphate buffer at various pHs.

[0023] FIGURE 1B shows the stability of rhASM as measured by the percentage of high molecular weight species (% of HMWS) after one week of storage at 30°C in a succinate, citrate, citrate / phosphate, or phosphate buffer at various pHs. HMWS were determined by size exclusion chromatography (SEC). Petition 870260052183, dated 05 / 29 / 2026, page 14 / 134 6 / 36

[0024] FIGURE 1C shows the stability of rhASM as measured by thermal stability in a citrate / phosphate or phosphate buffer at various pHs. Thermal stability was determined by differential scanning calorimetry.

[0025] FIGURE 2A shows the specific activity of rhASM over time in a phosphate buffer at 10 mM, 20 mM, 50 mM, or 100 mM with a pH of 6.5 at 30°C.

[0026] FIGURE 2B shows the physical stability of rhASM over time as measured by % HMWS in a phosphate buffer at 10 mM, 20 mM, 50 mM, or 100 mM with a pH of 6.5 at 30°C.

[0027] FIGURE 3A shows the effects of 5% wt / v of mannitol, sucrose, or trehalose on the specific (enzymatic) activity of 4 mg / ml rhASM before lyophilization (liquid) and after lyophilization (lyo).

[0028] FIGURE 3B shows the effects of 5% w / v of mannitol, sucrose and trehalose on the physical stability of 4 mg / ml rhASM as measured by % HMWS before lyophilization (liquid) and after lyophilization (lyo).

[0029] FIGURE 4A shows the specific activity of rhASM over time at 5°C. rhASM was lyophilized from a solution containing 5% mannitol, 5% sucrose or 3% mannitol and 3% sucrose (all concentrations by weight / volume).

[0030] FIGURE 4B shows the physical stability of rhASM over time at 5°C as measured by the % HMWS. The rhASM was lyophilized from a solution containing 5% mannitol, 5% sucrose or 3% mannitol and 2% sucrose (all concentrations by weight / volume).

[0031] FIGURE 5A shows the specific activity of rhASM over time at 5°C. rhASM was lyophilized from a solution containing 5% sucrose with or without 100 mM methionine (all Petition 870260052183, dated 05 / 29 / 2026, page 15 / 134 7 / 36 concentrations by weight / volume).

[0032] FIGURE 5B shows the physical stability of rhASM over time at 5°C as measured by the % HMWS. rhASM is lyophilized from a solution containing 5% by weight / volume sucrose with or without 100 mM methionine.

[0033] FIGURE 6 shows the effects of pH, protein concentration, methionine concentration and sucrose concentration on dimer percentage over time in liquid rhASM compositions at 2 to 8°C.

[0034] FIGURE 7 shows the effects of pH, protein concentration, methionine concentration, and sucrose concentration on rhASM specific activity over time in liquid compositions at 2 to 8°C.

[0035] FIGURE 8 shows the % of HMWS over time at 2 to 8°C in liquid rhASM compositions with varying pH, protein concentration, methionine concentration, and sucrose concentration. Formulation numbers are indicated to the right of the graph.

[0036] FIGURE 9 shows the % of aggregation over time at 2 to 8°C in liquid rhASM compositions with varying pH, protein concentration, methionine concentration, and sucrose concentration. Formulation numbers are indicated to the right of the graph.

[0037] FIGURE 10 shows the % dimer, % aggregation and specific activity at 25°C in liquid rhASM compositions at various pHs. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention provides compositions comprising a recombinant human ASM, for example, olipudase alfa, and one or more pharmaceutically acceptable excipients. The compositions of the present invention have improved stability and Petition 870260052183, dated 05 / 29 / 2026, page 16 / 134 8 / 36 longer shelf life compared to other compositions. In some embodiments, the compositions of the invention are pharmaceutical compositions, that is, compositions that are in such a form, or can be prepared to assume such a form, so as to allow the biological activity of the active ingredient to be effective while not containing any additional ingredient that is significantly toxic or else causes undesirable side effects unrelated to the active ingredient in patients. The terms pharmaceutical composition and pharmaceutical preparation are used interchangeably in this document. The pharmaceutical compositions of the present invention are useful in the treatment of patients with ASM deficiency as described further below. Recombinant Human Acid Sphingomyelinase

[0039] ASM is an enzyme that catalyzes the breakdown of sphingomyelin into ceramide and phosphorylcholine. Recombinant human ASM refers to human ASM, with or without certain amino acid modifications relative to a wild-type sequence, that is prepared by recombinant means. For example, a recombinant human ASM can be expressed in cultured mammalian host cells (e.g., COS, CHO, HeLa, 3T3, 293T, NS0, SP2 / 0, or HuT 78 cells and others) or in transgenic animals for a human ASM coding sequence.

[0040] In some embodiments, recombinant human ASM is olipudase alfa. Olipudase alfa is the alpha glycoform of a human ASM (EC-3.1.4.12) produced in CHO cells. Mature olipudase alfa is a 570-amino acid polypeptide that retains the enzymatic and lysosomal target activity of the native human protein. The amino acid sequence of olipudase alfa, including its leader sequence (residues 1 to 57), is shown below as SEQ ID NO: 1, where the leader sequence is indicated in italics and bold. The mature olipudase alfa sequence Petition 870260052183, dated 05 / 29 / 2026, p. 17 / 134 9 / 36 (SEQ ID NO: 2, which transposes residues 58 to 627 from SEQ ID NO: 1) does not have the leader sequence. MARYGASLRQ LALALSDSRV GWGNLTCPIC LKIAPPAVCQ STCGHWDIFS FLTDLHWDHD YWGEYSKCDL DVWHQTRQDQ PVNSFPPPFI IGGFYALSPY LQWLVGELQA VARYENTLAA PSATTYIGLN QANIPGAIPH DMQLFQTFWF SCPRSGREQG LWAPAEA HPL KGLFTAINLG SIVHLFEDDM SWNISLPTVP YLEGTDPDCA PLRTLESLLS LRALTTVTAL EGNHSSRWLY PGLRLISLNM AEDRGDKVHI QFFGHTHVDE PGYRVYQIDG WQLLYRARET LYHKGHPPSE QDGTAGAPGL SPQGHPARLH LKKEPNVARV VEVWRRSVLS KPPPKPPSPP DPLCCRRGSG GLGPAGPFDM VRKFLGPVPV EAMAKAWEPW NFCSRENFWL IGHIPPGHCL FEVFYDEETL NYSGSSHVVL YGLPNTLPTA PCGTPCRLAT LWMGLALALA RIVPRLRDVF GSVAIKLCNL PSEACGLLLG APGAPVSRIL LPPASRPGAG VYWTGDIPAH YPAVGNHEST LPAEALRTLR LINSTDPAGQ KSWSWNYYRI SRPLAVAFLA DHETYILNLT WHNLVYRMRG LCAQLSARAD SPALCRHLMP DGSLPEAQSL WPRPLFC (SEQ ID NO: 1)

[0041] In other embodiments, the human ASM useful in the present invention is 99%, 98%, 97%, 96%, or 95% identical in amino acid sequence to olipudase alfa. For example, the human ASM in the composition may have the sequence shown in U.S. Patent 6,541,218, the disclosure of which is incorporated herein in its entirety. This sequence (SEQ ID NO: 3) is shown below, with the leader sequence (residues 1 to 59) indicated in italics and bold, where the mature protein (SEQ ID NO: 4, which transposes residues 60 to 629 of SEQ ID NO: 3) does not have the leader sequence. MPRYGASLRQ SCPRSGREQG QDGTAGAPGL LWMGLVLALA LALALALSDS RVLWAPAEA H PLSPQGHPAR LHRIVPRLRD VFGWGNLTCP ICKGLFTAIN LGLKKEPNVA RVGSVAIKLC Petition 870260052183, dated 05 / 29 / 2026, page 18 / 134 10 / 36 NLLKIAPPAV CQSIVHLFED LGSTCGHWDI FSSWNISLPT ILFLTDLHWD HDYLEGTDPD AGYWGEYSKC DLPLRTLESL AHDVWHQTRQ DQLRALTTVT ALVRKFLGPV PVYPAVGNHE LYEAMAKAWE PWLPAEALRT NMNFCSRENF WLLINSTDPA HIGH CLOCKSWNYY DEFEVFYDEE TLSRPLAVAF DGNYSRSSHV VLDHETYILN ETYGLPNTLP TAWHNLVYRM SEPCGTPCRL ATLCAQLSAR DMVEVWRRSV LSPSEACGLL VPKPPPKPPS PPAPGAPVSR CADPLCCRRG SGLPPASRPG LSGLGPAGPF DMVYWTGDIP SIPVNSFPPP FIEGNHSSRW LRIGGFYALS PYPGLRLISL GQLQWLVGEL QAAEDRGDKV RIVARYENTL AAQFFGHTHV LAPSATTYIG LNPGYRVYQI LTQANIPGAI PHWQLLYRAR RGDMQLFQTF WFLYHKGHPP ADSPALCRHL MPDGSLPEAQ SLWPRPLFC (SEQ ID NO: 3)

[0042] Human ASM in composition may also be identical in amino acid sequence to human ASM disclosed in the UNIPROT database as sequence P17405-1, or in its polymorphic variants. Sequence P17405-1 is shown below (SEQ ID NO: 5), with the leader sequence (residues 1 to 59) indicated in italics and bold, where the mature protein (SEQ ID NO: 6, which transposes residues 60 to 629 of SEQ ID NO: 5) does not have the leader sequence. MPRYGASLRQ LALALALSDS VFGWGNLTCP NLLKIAPPAV LGSTCGHWDI ILFLTDLHWD AGYWGEYSKC AHDVWHQTRQ STPVNSFPPP SCPRSGREQG RVLWAPAEA H ICKGLFTAIN CQSIVHLFED FSSWNISLPT HDYLEGTDPD DLPLRTLESL DQLRALTTVT FIEGNHSSRW QDGTAGAPGL PLSPQGHPAR LGLKKEPNVA DMVEVWRRSV VPKPPPKPPS CADPLCCRRG LSGLGPAGPF ALVRKFLGPV LYEAMAKAWE LWMGLVLALA LHRIVPRLRD RVGSVAIKLC LSPSEACGLL PPAPGAPVSR SGLPPASRPG DMVYWTGDIP PVYPAVGNHE PWLPAEALRT Petition: 870260052183, on May 29, 2026, p. 19 / 134 11 / 36 LRIGGFYALS GQLQWLVGEL RIVARYENTL LAPSATTYIG LTQANIPGAI RGDMQLFQTF PYPGLRLISL QAAEDRGDKV AAQFFGHTHV LNPGYRVYQI PHWQLLYRAR WFLYHKGHPP NMNFCSRENF HIIGHIPPGH DEFEVFYDEE DGNYSGSSHV ETYGLPNTLP SEPCGTPCRL WLLINSTDPA CLKSWSWNYY TLSRPLAVAF VLDHETYILN TAWHNLVYRM ATLCAQLSAR ADSPALCRHL MPDGSLPEAQ SLWPRPLFC (SEQ ID NO: 5) Recombinant Human Acid Sphingomyelinase Compositions

[0043] The compositions of the present invention contain a recombinant human ASM and demonstrate superior stability with respect to the enzyme. Stability or stability refers to the ability of an active ingredient in a composition to retain its physical stability, its chemical stability and / or its biological activity during storage, and / or when subjected to physical or chemical stress. Stability may be in the context of a selected temperature, for example, under refrigerated conditions (e.g., 2 to 8°C), or at room temperature (e.g., 23 to 25°C), for a selected period of time, for example, 16 weeks, 24 weeks, 36 weeks, four months, six months, one year, two years, three years, or longer.The stability of a protein can be measured in assays that are performed within a shorter time period, but whose results are indicative of stability in clinical settings. Such assays include freeze / thaw assays where a protein composition is subjected to one or more freeze / thaw cycles; or agitation assays where a protein composition is subjected to mechanical agitation treatment for a predetermined period. Protein stability can be determined by storing the protein composition at a designated storage temperature (such as 2 to 8°C) for a selected period of time. Petition 870260052183, dated 05 / 29 / 2026, page 20 / 134 12 / 36 and by analyzing its structural and functional attributes, such as the degree of dimerization or aggregation (e.g., as measured by size exclusion HPLC or protein gel), protein degradation (e.g., as measured by size exclusion HPLC or protein gel), composition color change, clarity of a liquid composition, enzymatic activity, glycan content and composition, receptor binding affinity, residual methionine oxidation, and composition biological activity.

[0044] The compositions of the present invention contain one or more pharmaceutically acceptable excipients. Excipient refers to an inert substance that is used as a diluent, a vehicle, a carrier, a preservative, a binder, or a stabilizing agent for the active ingredient(s) of a drug. For example, the compositions may contain a buffering agent, an isotonic agent, and / or a stabilizing agent such as an antioxidant. In some cases, an excipient may serve more than one of these purposes. In some embodiments, a composition of the invention contains a recombinant human ASM such as olipudase alfa, a buffering agent such as sodium phosphate or sodium citrate, a stabilizer such as L-methionine, and a non-reducing sugar such as sucrose or trehalose. The human ASM has improved stability due to the particular composition in the composition. The compositions of the invention may be aqueous liquid solutions or lyophilized preparations. Liquid Compositions

[0045] In some embodiments, the composition is an aqueous liquid composition comprising 1 to 10 mg / ml of rhASM (e.g., 3 to 5 mg / ml) (e.g., alpha lipopudase); 10 to 50 mM (e.g., 10 to 30 mM) of sodium phosphate; 70 to 150 mM (e.g., 80 to 120 mM) of methionine (e.g., L-methionine); and 1 to 10% by weight / volume (e.g., 4 to 6%) of sucrose or trehalose. The pH of Petition 870260052183, dated 05 / 29 / 2026, page 21 / 134 13 / 36 aqueous liquid composition can be from 5 to 8 (for example, from 6 to 7).

[0046] In some embodiments, the aqueous liquid composition does not comprise any detectable amount of mannitol, the most readily used crystalline excipient, because this could significantly increase the aggregation of human ASM during or after lyophilization of an aqueous liquid composition described herein.

[0047] In some embodiments, the aqueous liquid composition comprises 0.004 to 0.008%, 0.005 to 0.007%, or 0.005% by weight / volume of surfactant(s). Exemplary surfactants include nonionic detergents such as polysorbates (e.g., polysorbates 20 and 80) and poloxamers (e.g., poloxamer 188). In one particular embodiment, the aqueous liquid composition comprises 0.005% polysorbate 80. In some cases, the presence of surfactant may help to reduce turbidity in the liquid composition.

[0048] In some embodiments, the aqueous liquid composition comprises no more than 0.05, 0.01 or 0.005 mM of chelating agent(s), such as EDTA and EGTA; in an exemplary embodiment, the aqueous liquid composition does not comprise any detectable amount of chelating agent(s). In some cases, the presence of chelating agents at a concentration above, for example, 0.05 mM or 0.1 mM, may increase the aggregation of human ASM and decrease its stability, particularly after a prolonged storage period, for example, for 12 to 16 weeks, or under non-refrigerated conditions, for example, at 25°C.

[0049] In some embodiments, the aqueous liquid composition may contain from 0 to 50 ppm (e.g., 15 to 30 ppm) of zinc, which may, for example, be loaded from the manufacturing process or added externally.

[0050] In a particular embodiment, the aqueous liquid composition Petition 870260052183, dated 05 / 29 / 2026, p. 22 / 134 14 / 36 comprises or consists essentially of 4 mg / ml of olipudase alfa, 20 mM sodium phosphate, 100 mM methionine, and 5% (by weight / volume) sucrose, and has a pH of 6.5. The term essentially consists of means that the composition does not contain other ingredients in detectable amounts nor may it contain only trace amounts of certain materials that are derived from the protein manufacturing process where such materials do not affect the biological activity of the enzyme or cause harm to human patients.

[0051] In some embodiments, the composition is an aqueous liquid composition comprising 1 to 20 mg / ml of rhASM (e.g., 10 mg / ml) (e.g., olipudase alfa) and 10 to 50 mM (e.g., 20 mM) of sodium phosphate. In certain embodiments, the aqueous liquid composition also comprises methionine (e.g., L-methionine) and sucrose or trehalose.In certain embodiments, the aqueous liquid composition also comprises 80 to 120 mM (e.g., 100 mM) of methionine and 4 to 6% by weight / volume (e.g., 5%) of sucrose. In particular embodiments, the aqueous liquid composition has a pH of 6.5.

[0052] In some embodiments, the composition is an aqueous liquid composition comprising 1 to 50 mg / ml of rhASM (e.g., 3, 8, 18, or 49 mg / ml) (e.g., alpha-lipudase) and 10 to 50 mM (e.g., 20 mM) of sodium phosphate. In certain embodiments, the aqueous liquid composition also comprises 1 to 15% (e.g., 5%, 6%, 7%, or 8%) of sucrose or trehalose. In certain embodiments, the aqueous liquid composition also comprises 80 to 120 mM (e.g., 100 mM) of methionine. In particular embodiments, the aqueous liquid composition has a pH of 6.5. The composition may comprise, for example, 3.8 mg / ml of rhASM, 20 mM of sodium phosphate, and 5% of sucrose; 18 mg / ml of rhASM, 20 mM sodium phosphate and 5% sucrose; or 49 mg / ml of Petition 870260052183, dated 05 / 29 / 2026, page 23 / 134 15 / 36 rhASM, 20 mM phosphate and 8% sucrose.

[0053] Aqueous liquid compositions can be prepared by mixing a human ASM produced by recombinant technology and subsequently purified from host cells with the excipients described herein in water, and by adjusting the resulting mixture to the desired pH. For example, the human ASM and the desired excipients can be added, or buffered in a sodium phosphate buffer with the desired sodium phosphate concentration and pH.

[0054] In some embodiments, the aqueous liquid composition can be prepared by reconstituting a lyophilized composition of the invention described in more detail below. Reconstitution can be done with a pharmaceutically acceptable liquid such as sterile water, saline solution (e.g., 0.9% sodium chloride), or phosphate-buffered saline solution. Freeze-dried Compositions

[0055] The present invention also provides freeze-dried compositions. Such compositions can be prepared by freeze-drying the aqueous liquid compositions described in this document. The freeze-dried compositions are suitable for long-term storage. Freeze-drying can be carried out according to methods known in the art. For example, a liquid composition can be cooled to a temperature below zero (degree Celsius) (e.g., from -5°C to -80°C) that allows freezing, and then placed in a low-pressure chamber (partial vacuum) to allow sublimation (primary drying) to occur; where desired, the temperature of the composition can be raised in a second drying stage (secondary drying) to remove further unwanted water molecules. In some embodiments, after completion of the freeze-drying process, a gas Petition 870260052183, dated 05 / 29 / 2026, p. 24 / 134 16 / 36 Inert substances such as nitrogen can be introduced into the container of the composition (e.g., a glass container) before the container is sealed.

[0056] In some embodiments, the present invention provides powder compositions, which can be prepared, for example, by spray drying of the aqueous liquid compositions described in this document. Spray-dried compositions are suitable for long-term storage. Spray drying can be carried out according to methods known in the art. For example, a liquid composition can be forced through an atomizer or spray nozzle to be dispersed as minute droplets of controlled size in a hot gas stream in a chamber, resulting in the rapid drying of the liquid composition as a powder. The dried powder can then be collected at the bottom of the drying chamber. Other drying methods for the preparation of powder compositions are also contemplated.

[0057] The authors of the present invention have unexpectedly found that sucrose (or trehalose) and methionine present in the quantities described herein provide superior results during lyophilization; the lyophilized products form elegant cakes while preserving the stability of human ASM during storage. The human ASM in the lyophilized compositions of the present invention can remain free from aggregation and biologically active for at least 4 months (for example, for at least 6 months or at least 12 months) under refrigerated conditions (for example, 0 to 10°C, 2 to 8°C, or 4°C).

[0058] In some embodiments, the composition of the invention is a lyophilized pharmaceutical composition comprising 4 to 50% of alpha-olipudase, 3 to 7% by weight of sodium phosphate and 45 to 90% of sucrose (all percentages by weight / weight). In certain embodiments Petition 870260052183, dated 05 / 29 / 2026, page 25 / 134 In 17 / 36 embodiments, the lyophilized composition comprises 5.5% alpha olipudase, 20.6% L-methionine, 2.3% dibasic sodium phosphate heptahydrate, 2.6% monobasic sodium phosphate monohydrate, and 69.0% sucrose (all percentages by weight / weight). In certain embodiments, the lyophilized composition comprises 6.6% alpha olipudase, 3.0% dibasic sodium phosphate heptahydrate, 3.3% monobasic sodium phosphate monohydrate, and 87.1% sucrose (all percentages by weight / weight). In certain embodiments, the freeze-dried composition comprises 25.2% alpha lipopudase, 2.4% dibasic sodium phosphate heptahydrate, 2.6% monobasic sodium phosphate monohydrate, and 69.9% sucrose (all percentages by weight / weight).In certain embodiments, the lyophilized composition comprises 47.8% alpha lipopudase, 1.7% dibasic sodium phosphate heptahydrate, 1.8% monobasic sodium phosphate monohydrate, and 48.8% sucrose (all percentages by weight / weight).

[0059] In some embodiments, the composition of the invention is a lyophilized pharmaceutical composition comprising 4 to 7% w / w of olipudase alfa, 15 to 25% w / w of L-methionine, 3 to 7% w / w of sodium phosphate and 65 to 75% w / w of sucrose (all percentages by weight). In a particular embodiment, the lyophilized composition comprises 5.5% olipudase alfa, 20.5% L-methionine, 2.3% dibasic sodium phosphate heptahydrate, 2.6% monobasic sodium phosphate monohydrate and 68.6% sucrose (all percentages by weight). In certain forms, the freeze-dried composition may also comprise, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% moisture.

[0060] In some embodiments, the invention provides a container containing a lyophilized pharmaceutical composition comprising Petition 870260052183, dated 05 / 29 / 2026, page 26 / 134 18 / 36 of 15 to 25 mg of alpha-olipudase, 75 to 85 mg of L-methionine, 15 to 25 mg of monobasic sodium phosphate monohydrate and 250 to 300 mg of sucrose. Before use, the composition can be reconstituted in 4 to 6 ml of sterile water.

[0061] In some embodiments, the container contains a lyophilized pharmaceutical composition comprising or consisting of 21.2 mg, 20.1 mg, 95.4 mg, or 259.7 mg of olipudase alfa; 9.0 mg of dibasic sodium phosphate heptahydrate; 10.0 mg of monobasic sodium phosphate heptahydrate; and 265 mg of sucrose. The lyophilized composition may optionally comprise 79.1 mg of L-methionine. The lyophilized pharmaceutical composition may optionally comprise 0 to 0.3 mg (e.g., 0.08 to 0.16 mg) of zinc, which may, for example, be loaded from the manufacturing process or added externally. In certain embodiments, the container may have an internally sterile nitrogen-charged atmosphere. In one particular embodiment, the lyophilized composition can be reconstituted in 5.1 ml of sterile water to obtain an alpha olipudase concentration of approximately 4.0 mg / ml, 3.8 mg / ml, 18 mg / ml, or 49 mg / ml, respectively.The reconstituted composition can also be diluted in a 0.9% sodium chloride solution to a specific volume based on the dose to be administered.

[0062] In a particular embodiment, the container contains a lyophilized pharmaceutical composition comprising or consisting of 21.2 mg of olipudase alfa, 79 mg of L-methionine, 9.0 mg of dibasic sodium phosphate heptahydrate, 10.0 mg of monobasic sodium phosphate monohydrate, and 265 mg of sucrose. The lyophilized pharmaceutical composition may optionally comprise 0 to 0.3 mg (e.g., 0.08 to 0.16 mg) of zinc magnesium, which may, for example, be loaded from the manufacturing process or added externally. In certain embodiments, the composition Petition 870260052183, dated 05 / 29 / 2026, page 27 / 134 19 / 36 Lyophilized pharmaceutical composition is in the form of a cake or a lyophilized powder. In certain embodiments, the container may have an internally sterile nitrogen-charged atmosphere. In one particular embodiment, the lyophilized composition can be reconstituted in 5.1 ml of sterile water to obtain a concentration of approximately 4.0 mg / ml of olipudase alfa. The reconstituted composition can also be diluted in a 0.9% sodium chloride solution to a specific volume based on the dose to be administered.

[0063] In some embodiments, the invention provides a container containing a lyophilized pharmaceutical composition comprising 3 to 5 mg of magnesium alpha-olipudate, 15 to 17 mg of L-methionine, 3 to 5 mg of sodium phosphate and 50 to 60 mg of sucrose. Before use, the composition can be reconstituted in 0.8 to 1.2 ml of sterile water.

[0064] In a particular embodiment, the container contains a lyophilized pharmaceutical composition comprising or consisting of 4.8 mg of olipudase alfa, 17.9 mg of L-methionine, 2.0 mg of dibasic sodium phosphate heptahydrate, 2.3 mg of monobasic sodium phosphate monohydrate, and 60 mg of sucrose. In certain embodiments, the lyophilized pharmaceutical composition is in the form of a lyophilized cake or powder. The lyophilized composition may optionally comprise from 0 to 0.06 mg of zinc, which may, for example, be loaded from the manufacturing process or added externally. In certain embodiments, the container may have an internally sterile nitrogen-charged atmosphere. In a particular embodiment, the lyophilized composition may be reconstituted in 1.1 ml of sterile water to obtain a concentration of about 4.0 mg / ml of olipudase alfa.The reconstituted composition can also be diluted in a 0.9% sodium chloride solution to a specific volume based on the dose to be administered. Petition 870260052183, dated 05 / 29 / 2026, page 28 / 134 20 / 36 Manufactured Articles

[0065] The compositions of the invention may be provided in a manufactured article (e.g., a kit) that includes instructions for use and optionally other therapeutic agents for the treatment of ASM disorders. The pharmaceutically active ingredient in the articles (e.g., rhASM) may be provided in an amount that can be administered immediately according to the dosage regimens described herein. For example, a starter kit may include multiple containers of varying amounts of rhASM for use in a dose-escalation regimen.

[0066] For example, the manufactured article may include a container containing 15 to 25 mg of olipudase, 75 to 85 mg of L-methionine, 15 to 25 mg of sodium phosphate, and 250 to 300 mg of sucrose. In a particular embodiment, the article provides a lyophilized composition comprising 21.2 mg of olipudase alfa, 79 mg of methionine, 9.0 mg of dibasic sodium phosphate heptahydrate, 10.0 mg of monobasic sodium phosphate monohydrate, and 265 mg of sucrose.

[0067] By way of another example, the manufactured article may include a container containing 3 to 5 mg of olipudase alfa, 15 to 17 mg of L-methionine, 3 to 5 mg of sodium phosphate, and 50 to 60 mg of sucrose. In a particular embodiment, the article provides a lyophilized composition comprising 4.8 mg of olipudase alfa, 17.9 mg of L-methionine, 2.0 mg of dibasic sodium phosphate heptahydrate, 2.3 mg of monobasic sodium phosphate monohydrate, and 60 mg of sucrose.

[0068] In some embodiments, the manufactured article may also include a solution (e.g., sterile water, 0.9% sodium chloride, and / or phosphate-buffered saline solution) to reconstitute the lyophilized composition and / or to dilute the reconstituted composition before Petition 870260052183, dated 05 / 29 / 2026, page 29 / 134 21 / 36 of the administration to a patient. Use of Acid Sphingomyelinase Compositions

[0069] The pharmaceutical compositions of the invention can be administered parenterally to a patient in need thereof as an enzyme replacement therapy. Parenteral administration refers to a means of administration other than enteral and topical administration, generally by injection. Parenteral administration includes, without limitation, intravenous infusion or injection, and intramuscular, intradermal, intraperitoneal and subcutaneous injection. In a particular embodiment, the pharmaceutical composition is administered by intravenous infusion.

[0070] The appropriate dosage level of the pharmaceutical composition described herein may be determined based on a variety of factors, including the patient's age, weight, disease condition, general health and medical history, as well as the route and frequency of drug administration, the pharmacodynamics and pharmacokinetics of the ASM active ingredient in the drug, and any other drugs the patient may be taking concurrently. In some embodiments, a pharmaceutical composition described herein may be administered according to a dosage regimen described, for example, in U.S. Patent 9,655,954 (Schuchman et al.). For example, the patient may receive escalating doses of human ASM, with the dose intensity starting, for example, at 0.1 mg / kg or less, and ending at 3 mg / kg (maintenance dose) or less, depending on the patient's age and condition.In some modalities, the first one or two doses may be administered at a dose intensity of 0.03 mg / kg or 0.1 mg / kg for a pediatric patient, or 0.1 mg / kg for an adult patient; after the patient has received one or two doses at 0.03 and / or 0.1 mg / kg, the patient is then given additional doses. Petition 870260052183, dated 05 / 29 / 2026, page 30 / 134 22 / 36 subsequent sequential doses of 0.3 mg / kg, 0.3 mg / kg, 0.6 mg / kg, 0.6 mg / kg, 1.0 mg / kg, 2.0 mg / kg, and 3.0 mg / kg. In certain embodiments, some of these doses may be repeated (e.g., the 1.0 mg / kg and 2.0 mg / kg doses). For some patients, a dose intensity of 3.0 mg / kg is appropriate for maintenance doses, while for other patients, a lower dose intensity may be sufficient for maintenance. The intervals between successive doses may be two weeks, or shorter or longer than two weeks as determined to be appropriate by a clinician.

[0071] The invention provides a method of using a pharmaceutical composition described herein for the treatment of ASMD in a patient in need thereof, in a pharmaceutical composition described herein for use in the treatment of ASMD in a patient in need thereof, and the use of a pharmaceutical composition described herein for the manufacture of a medicament for the treatment of ASMD in a patient in need thereof. In some embodiments, the pharmaceutical composition may be a lyophilized composition, which may be reconstituted in a pharmaceutically acceptable liquid, such as sterile water, a 0.9% sodium chloride solution, or a phosphate-buffered saline solution.

[0072] Patients may be adults (e.g., patients 18 years of age or older, including geriatric patients who are 65 years of age or older).Patients may be pediatric patients (patients who are under 18 years of age, for example, patients who are newborns up to 6 years of age, who are 6 to 12 years of age, or who are 12 to 18 years of age). In some modalities, patients may have NPD A / B or NPD B. In some modalities, patients may have NPD A. Petition 870260052183, dated 05 / 29 / 2026, p. 31 / 134 In particular embodiments, the pharmaceutical composition is for the treatment of an adult or pediatric patient with chronic visceral ASMD (NPD B). In particular embodiments, the pharmaceutical composition is for the treatment of non-neurological manifestations of ASMD in an adult or pediatric patient. Illustrative Modalities

[0073] Other particular embodiments of the present invention are described as follows.

[0074] 1. A composition comprising recombinant human acid sphingomyelinase, sodium phosphate, methionine and sucrose.

[0075] 2. The composition of embodiment 1, wherein the composition is a freeze-dried composition comprising: 4 to 7% by weight / weight of olupudase alfa (SEQ ID NO: 2) 3 to 7% by weight / weight of sodium phosphate, 15 to 25% by weight / weight of L-methionine, and 65 to 75% by weight / weight of sucrose.

[0076] 3. The composition of modality 2, which essentially consists of: 5.5% by weight of alpha lipopudase, 2.3% by weight of dibasic sodium phosphate heptahydrate, 2.6% by weight of monobasic sodium phosphate heptahydrate, 20.5% by weight of L-methionine, and 68.6% by weight / weight of sucrose.

[0077] 4. The composition of embodiment 1, wherein the composition is an aqueous liquid composition comprising: 1 to 10 mg / ml of alpha olipudase, 10 to 50 mM of sodium phosphate, Petition 870260052183, dated 05 / 29 / 2026, page 32 / 134 24 / 36 containing 70 to 150 mM of L-methionine, and 1 to 10% by weight / volume of sucrose, resulting in a pH of 5 to 8.

[0078] 5. The composition of embodiment 4, wherein the composition is an aqueous liquid composition comprising: 3 to 5 mg / ml of alpha lipopudase, 10 to 30 mM of sodium phosphate, 80 to 120 mM of L-methionine, and 4 to 6% by weight / volume of sucrose, resulting in a pH of 6 to 7.

[0079] 6. The composition of modality 4, which essentially consists of: mg / ml of alpha olipudase, mM of sodium phosphate, 100 mM of L-methionine, and 5% by weight / volume of sucrose, resulting in a pH of 6.5.

[0080] 7. The composition of any of the embodiments 4 to 6, which also comprises 0.005% by weight / volume of polysorbate 80.

[0081] 8. A composition obtained by lyophilization of the aqueous liquid composition according to any of the embodiments 4 to 7.

[0082] 9. A process for manufacturing a freeze-dried composition, which comprises: obtaining the aqueous liquid composition of any of the modalities 4 to 7, and the lyophilization of the aqueous liquid composition.

[0083] 10. A container which contains a freeze-dried composition consisting essentially of: 21.2 mg of olipudase alfa, Petition 870260052183, dated 05 / 29 / 2026, page 33 / 134 25 / 36 9.0 mg of dibasic sodium phosphate heptahydrate, 10.0 mg of monobasic sodium phosphate heptahydrate, 79 mg of L-methionine, and 265 mg of sucrose.

[0084] 11. An aqueous liquid composition, which is obtained by reconstituting a lyophilized composition consisting essentially of: 21.2 mg of olipudase alfa, 9.0 mg of dibasic sodium phosphate heptahydrate, 10.0 mg of monobasic sodium phosphate heptahydrate, mg of L-methionine, and 265 mg of sucrose in 5.1 ml of sterile water.

[0085] 12. A container which contains a freeze-dried composition consisting essentially of: 4.8 mg of olipudase alfa, 2.0 mg of dibasic sodium phosphate heptahydrate, 2.3 mg of monobasic sodium phosphate heptahydrate, 17.9 mg of L-methionine, and mg of sucrose.

[0086] 13. An aqueous liquid composition, which is obtained by reconstituting a lyophilized composition consisting essentially of: 4.8 mg of olipudase alfa, 2.0 mg of dibasic sodium phosphate heptahydrate, 2.3 mg of monobasic sodium phosphate heptahydrate, 17.9 mg of L-methionine, and mg of sucrose in 1.1 ml of sterile water.

[0087] 14. A manufactured article, which comprises the container Petition 870260052183, dated 05 / 29 / 2026, page 34 / 134 26 / 36 of modality 10 or 12 and a container containing sterile water, 0.9% sodium chloride, or a phosphate-buffered saline solution to reconstitute the lyophilized composition.

[0088] 15. A method of treating acid sphingomyelinase deficiency (ASMD) in a human patient, comprising administering to the patient the composition of any of the embodiments 1 to 8, 11 and 13, wherein the composition is reconstituted in a liquid form prior to administration if it is a lyophilized composition.

[0089] 16. A composition according to any of the embodiments 1 to 8, 11, and 13, which is to be used in the treatment of ASMD in a human patient.

[0090] 17. Use of a composition of any of the embodiments 1 to 8, 11 and 13, which serves for the manufacture of a medicament for the treatment of ASMD in a human patient.

[0091] 18. The method of modality 15, in which the composition serves for the use of modality 16, or the use of modality 17, in which ASMD is Niemann-Pick Disease type A / B or type B.

[0092] 19. The method, composition for use, or modality of use 18, in which the treatment is for non-neurological manifestations of ASMD.

[0093] All publications and other references mentioned in this document are incorporated by reference in their entirety. Although a number of documents are cited in this document, such citation does not constitute an admission that any of these documents forms part of the common general knowledge in the state of the art. Unless otherwise defined in this document, scientific and technical terms used in relation to the present invention shall have the meanings that are generally understood by those normally versed in the art. Petition 870260052183, dated 05 / 29 / 2026, p. 35 / 134 27 / 36 State of the Art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. In case of conflict, this descriptive report, including the definitions, shall prevail. Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry described herein are those well known and generally used in the state of the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as generally performed in the state of the art or as described herein.Furthermore, unless otherwise required by the context, singular terms will include plurals, and plural terms will include singulars. Throughout this descriptive report and in the modalities, the words have and include or variations such as has, having, includes or includes, will be understood as implying the inclusion of a given whole or group of wholes, but not the exclusion of any other whole or group of wholes.

[0094] In order that the present invention may be better understood, the following examples are presented. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. EXAMPLES Example 1: Formulation of Recombinant Human Acid Sphingomyelinase Petition 870260052183, dated 05 / 29 / 2026, p. 36 / 134 28 / 36

[0095] This example describes studies that evaluated the stability of various aqueous and lyophilized liquid compositions of olipudase alfa. Materials and methods Solution Turbidity

[0096] The opalescence of the solution was evaluated by a spectroscopic turbidity assay. The optical density in the 340–360 nm range was used to fit the ranges to previously established opalescence categories based on European Pharmacopoeia reference suspensions at specific NTU values. The analysis was performed on a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). Aggregation

[0097] Aggregation and dimer analysis were performed using SEC. Before being loaded into HPLC vessels, each sample was mixed by five cycles of gentle pipetting. SEC analysis was performed on an 1100 / 1200 series HPLC (Agilent, Santa Clara, CA) equipped with a TSK G3000SWXL gel analyzer (Tosoh Bioscience, Tokyo, Japan) and a compatible shielding column. The mobile phase used consisted of 20 mM sodium phosphate at pH 6, 200 mM sodium chloride adjusted to a flow rate of 0.5 ml min-1 for 35 minutes. Three injections of approximately 80 μg were performed for each sample. Detection was performed using UV absorbance at 280 nm.

[0098] The level of the high molecular weight species related to alpha olipudase (HMWS) was determined using SDS-PAGE under non-reducing conditions followed by staining with Coomassie blue. The alpha olipudase reference standard is included in each gel. Alpha olipudase samples were mixed with sample buffer and loaded onto a 4-20% Tris-Glycine gradient gel together Petition 870260052183, dated 05 / 29 / 2026, p. 37 / 134 29 / 36 with molecular weight markers. After electrophoresis on a 125 V target for approximately 2 hours, the gels were stained with Coomassie blue and destained in methanol, acetic acid, and HPLC-grade water. Densitometric analysis was performed to obtain quantitative results on the percentage of HMWS bands relative to all observed bands. Enzyme Activity

[0099] rhASM samples were diluted 2000:1 in 1.2 ml library tubes. The hydrolysis rate of 2-(N-hexadecanoylamino)-4-nitrophenylphosphorylcholine (HDA-PC) at 37°C catalyzed by rhASM was measured in this procedure. The released chromophore was measured by absorbance at 415 nm using a SpectraMax Plus 384 microplate spectrophotometer. One unit of rhASM activity is defined as the amount of enzyme that generates 1 μmol of 2-(N-hexadecanoylamino)-4-nitrophenol per minute from HDA-PC under the specified assay conditions. Protein Concentration

[00100] The protein concentration of the rhASM samples was determined by absorbance at 280 nm. Samples were diluted in duplicate to 1:10 and 1:20 using compatible buffers. Absorbance at 280 nm was determined on a SpectraMax Plus 384 microplate spectrophotometer. pH

[00101] The pH analysis of the sample was performed using a Thermo Electron Microprobe pH meter (Thermo Scientific, Beverly, MA). A Thermo Orion 8203BN PerHecT Ross Semi micro probe (Thermo Scientific) was used. Differential Scanning Calorimetry

[00102] Differential scanning calorimetry (DSC) analysis was performed using a CAP-VP-DSC microcalorimeter (MicroCal-GE) Petition 870260052183, dated 05 / 29 / 2026, page 38 / 134 30 / 36 Samples were diluted to 0.4 mg / ml with compatible buffer. Samples were processed at a scan rate of 200°C / h from 15 to 100°C. Data analysis was performed on Origin 7.0 (OriginLab Corp., Northampton, MA) equipped with DSC analysis summation (MicroCal-GE Healthcare). Results Buffer and pH assessment

[00103] A variety of buffer pH and buffer species were evaluated for their effects on rhASM stability. Assays were performed by incubating 4 mg / ml of olipudase alfa in 20 mM buffer at 30°C for two weeks and evaluating the physical and functional stability of the enzyme (FIGURES 1A to 1C).

[00104] Significant instability, both physical and functional, was observed in alpha olipudase below pH 6.0. Enzyme activity decreased precipitously below pH 6.0, but remained relatively constant at pH 6.0 or higher (FIGURE 1A). The propensity for aggregation was at a minimum between pH 5.5 and pH 6.5. Rapid increases in aggregation were observed below pH 5.5, and gradual increases in aggregation were seen above pH 6.5 (FIGURE 1B). At comparable pH values, stability was higher in the phosphate buffer compared to the citrate / phosphate buffer. These stability trends were consistent with data obtained from samples stored at refrigeration temperatures. The gradually increasing aggregation rates observed above pH 6.5 were corroborated by DSC analysis, which showed decreased thermal stability at a higher pH in the phosphate buffer (FIGURE 1C).Based on these data, sodium phosphate buffer with a pH of approximately 6.5 was identified as an appropriate buffering system for the formulation of rhASM.

[00105] Next, the impact of ionic resistance, or Petition 870260052183, dated 05 / 29 / 2026, page 39 / 134 The stability of olipudase alfa was investigated by varying the concentration of sodium phosphate from 10 mM to 100 mM. As shown in FIGURES 2A and 2B, ionic resistance at low buffer concentrations (10 mM, 20 mM, or 50 mM) had little effect on the enzymatic activity and physical stability of olipudase alfa. The stability of olipudase alfa in sodium phosphate, at pH 6.5, was relatively consistent for buffer concentrations of 50 mM or less. However, there was a marked decrease in stability when the sodium phosphate concentration was increased to 100 mM. This concentration resulted in a rapid increase in the aggregated species and a concomitant reduction in activity (FIGURES 2A and 2B). The data at 30°C were consistent with those seen during the storage of olipudase alfa under refrigerated conditions.A sodium phosphate concentration of 20 mM was selected as a buffer concentration that, while low, still ensured sufficient buffering capacity. Excipient Evaluation

[00106] A variety of pharmaceutically acceptable excipients were evaluated for their impact on the stability of olipudase alfa in liquid and lyophilized compositions. The impact of known stabilizers acting through a preferential exclusion mechanism on liquid stability was investigated first (see, for example, Timasheff, Proc Acad Nat Sci USA. 99: 9721-9726 (2002); and Lee et al., J. Biol. Chem. 256: 7193-7201 (1981)). Sucrose, trehalose, and propylene glycol were added to the basic formulation of 20 mM sodium phosphate, 0.005% PS80, pH 6.5 and evaluated for their impact on the enzymatic activity and physical stability of 4 mg / ml olipudase alfa. The data showed that the presence of any of these preferential exclusion stabilizers did not enhance the stability of olipudase alfa in the liquid state at 5°C or 30°C (data Petition 870260052183, dated 05 / 29 / 2026, p. 40 / 134 32 / 36 not shown). Although there was a loss of enzymatic activity and physical stability under all conditions studied, sucrose appeared slightly more favorable than trehalose and propylene glycol.

[00107] Next, the impact of sucrose and trehalose on the stability of olipudase alpha during freeze-drying was investigated. Mannitol was also investigated, as it is a bulking agent commonly used in freeze-drying (FIGURES 3A and 3B). The three polyols were included at 5% w / v in the liquid compositions, which were subsequently freeze-dried. The data in FIGURE 3A show that the addition of mannitol decreased the enzymatic activity of olipudase alpha after freeze-drying. The data in FIGURE 3B show that the addition of mannitol resulted in a large increase in protein aggregation after freeze-drying. The increases in aggregation in the presence of sucrose and trehalose were minimal. Sucrose was chosen as a cryoprotectant at a concentration of 5% w / v in the liquid compositions, which could then be freeze-dried.

[00108] The stability of a lyophilized formulation of olipudase alfa over time in the presence of mannitol was also investigated. Olipudase alfa was freeze-dried from liquid compositions containing 20 mM sodium phosphate buffer (pH 6.5), 0.005% PS80, and (i) 5% w / v mannitol, (ii) 5% w / v sucrose, or (iii) 3% mannitol and 2% sucrose. The data show that mannitol resulted not only in immediate but also continuous increases in protein aggregation during freeze-drying when used alone, but this also occurred when used in combination with sucrose over a six-month period (FIGURES 3B and 4B). The enzymatic activity in Petition 870260052183, dated 05 / 29 / 2026, page 41 / 134 33 / 36 six months was also lower in the presence of mannitol than in the absence of mannitol (FIGURE 4B). Therefore, mannitol was considered an unsuitable bulking agent for lyophilized rhASM formulations.

[00109] Since mannitol proved detrimental to the stability of rhASM during freeze-drying, methionine was evaluated as a potential bulking agent. The enzymatic activity and physical stability during liquid storage of olipudase alfa in the presence of L-methionine were evaluated, and it was found that the addition of 100 mM L-methionine neither improved nor negatively impacted the liquid stability of olipudase alfa at 5°C. As shown in FIGURES 5A and 5B, lyophilized compositions prepared from liquid compositions containing 100 mM L-methionine resulted in a stable lyophilized configuration for olipudase alfa. No change in activity or aggregation was observed during the course of six months of storage at 5°C. Furthermore, cakes obtained from freeze-drying with alpha lipopudase in the presence of methionine and sucrose had an improved appearance compared to those freeze-dried with sucrose alone.

[00110] The identification of an appropriate amount of methionine for lyophilization was performed by X-ray diffraction (XRD) of lyophilized olipudase alfa cakes from liquid compositions containing 20 mM sodium phosphate (pH 6.5), 5 wt / v% sucrose, and higher amounts of L-methionine (data not shown). A sample containing no methionine was completely amorphous. There was some evidence of crystallinity in samples containing 33 mM methionine, and more in samples containing 66 mM and 100 mM methionine. Lyophilization of olipudase alfa at methionine levels below 100 mM (e.g., 10 mM and 33 mM) resulted in containers that appeared wavy and crumbled. Petition 870260052183, dated 05 / 29 / 2026, page 42 / 134 34 / 36 (shrunk). Thus, 100 mM methionine was chosen as a bulking agent for the lyophilized rhASM formulations. Example 2: Robustness of Alpha Olipudase Compositions

[00111] To evaluate the robustness of compositions containing alpha-lipudase, sucrose, and L-methionine, each of these components was adjusted at five different levels relative to the low, medium, central (control), medium-high, and high control points in a 20 mM sodium phosphate buffer (Table 1). Table 1 - Excipient Levels for Evaluating the Robustness of Olipudase Alfa Formulation Components Control and Medium Medium, Low Central Medium, High Elevated Sucrose (% by weight / volume) 5 4 4.33 5 5.67 6 L-methionine (mM) 100 80 86.51 100 113.49 120 Olipudase (mg / ml) 4 3 3.33 4 4.67 5 pH 6.5 6 6.16 6.5 6.84 7

[00112] A total of 26 liquid formulation variants were generated (Table 2). Formulations 2 and 7 represent control formulations, or central points. The remaining 24 formulation variants represent various conditions around the central points. All 26 variants were stored at 2 to 8°C (24 weeks or up to 12 months) and at 25°C (16 weeks) to monitor their stability. Table 2 Formulation of Alpha Olipudase for Robustness Assessment Formulation Sucrose (%) Methionine (mM) Olipudase (mg / ml) pH 1 5.00 100.00 3.00 6.50 2 5.00 100.00 4.00 6.50 3 5.00 80.00 4.00 6.50 4 5.00 100.00 5.00 6.50 5 5.00 120.00 4.00 6.50 6 6.00 100.00 4.00 6.50 7 5.00 100.00 4.00 6.50 8 4.00 100.00 4.00 6.50 9 5.67 113.49 4.67 6.16 10 5.67 113.49 3.33 6.16 11 4.33 113.49 3.33 6.16 12 4.33 113.49 4.67 6.16 13 4.33 86.51 4.67 6.16 Petition 870260052183, dated 05 / 29 / 2026, page 43 / 134 35 / 36 14 4,33 86,51 3,33 6,16 15 5,67 86,51 3,33 6,16 16 5,67 86,51 4,67 6,16 17 5,67 113,49 3,33 6,84 18 5,67 86,51 4,67 6,84 19 5,67 113,49 4,67 6,84 20 4,33 86,51 3,33 6,84 21 4,33 113,49 3,33 6,84 22 4,33 86,51 4,67 6,84 23 4,33 113,49 4,67 6,84 24 5,67 86,51 3,33 6,84 25 5,00 100,00 4,00 7,00 26 5,00 100,00 4,00 6,00

[00113] At the end of the test periods, the following parameters should indicate the stability of the compositions: (1) clear colorless appearance; (2) pH 6.0 to 7.0; (3) not more than 3.0% aggregate; (4) 3.5 to 4.5 mg / ml of protein; (5) not more than 15% HMWS; and (6) specific activity from 11 to 42 ( / mg. FIGURES 6 to 8 show the effects of different factors on % dimer, specific activity, and % HMWS, respectively, in the 26 formulation variants after 24 weeks of storage at 2 to 8°C. The data show that there was no significant difference between the variants with respect to % dimer, specific activity, or % HMWS and thus that all 24 variants along with the two control formulations were stable at 2 to 8°C for 24 weeks.

[00114] The lack of significant effects of pH and varying ingredient concentrations on dimer percentage and specific activity was also observed for some variants across 36 weeks. FIGURE 9 shows that all variants have comparable aggregation at 36 weeks.

[00115] At an accelerated temperature of 25°C, there was also little effect of the excipients on stability over 16 weeks in the tested ranges. The most prominent effect was observed in the variants with different pH (FIGURE 10). There was greater stability for alpha olipudase at higher pH (from 6.8 to 7.0), and more aggregation. Petition 870260052183, dated 05 / 29 / 2026, p. 44 / 134 36 / 36 and dimer at lower pH (6.5 or less).

[00116] This study shows that the variant formulations at 2 to 8°C were robust and stable in the selected component ranges. Petition 870260052183, dated 05 / 29 / 2026, p. 45 / 134

Claims

1 / 3 CLAIMS 1. A container containing a lyophilized composition, wherein the lyophilized composition is for use in the treatment of acid sphingomyelinase deficiency (ASMD) in a human patient, characterized in that it contains a lyophilized composition consisting essentially of: 20 mg of olipudase alfa, 5 mg of dibasic sodium phosphate, 8 mg of monobasic sodium phosphate, 75 mg of L-methionine, and 250 mg of sucrose.

2. Container according to claim 1, characterized in that the lyophilized composition comprises no more than 0.5% moisture, and optionally comprises 0.3% moisture.

3. Aqueous liquid composition for the treatment of acid sphingomyelinase deficiency (ASMD) in a human patient, characterized in that it is obtained by reconstituting a lyophilized composition consisting essentially of approximately: 20 mg of olipudase alfa, 5 mg of dibasic sodium phosphate, 8 mg of monobasic sodium phosphate, 75 mg of L-methionine, and 250 mg of sucrose, in 5.1 ml of sterile water.

4. Container containing a lyophilized composition, wherein the lyophilized composition is for the treatment of acid sphingomyelinase deficiency (ASMD) in a human patient, characterized in that it consists essentially of: 4 mg of olipudase alfa, Petition 870260052183, dated 05 / 29 / 2026, page 46 / 134 2 / 3 0.9 mg of dibasic sodium phosphate, 1.6 mg of monobasic sodium phosphate, 15 mg of L-methionine, and 50 mg of sucrose.

5. Container, according to claim 4, characterized in that the lyophilized composition comprises no more than 0.5% moisture, and optionally comprises 0.3% moisture.

6. Aqueous liquid composition for the treatment of acid sphingomyelinase deficiency (ASMD) in a human patient, characterized in that it is obtained by reconstituting a lyophilized composition consisting essentially of approximately: 4 mg of olipudase alfa, 0.9 mg of dibasic sodium phosphate, 1.6 mg of monobasic sodium phosphate, 15 mg of L-methionine, and 50 mg of sucrose, in 1.1 ml of sterile water.

7. Article of manufacture for the treatment of acid sphingomyelinase deficiency (ASMD) in a human patient, characterized in that it comprises the container, as defined in claim 1, 2, 4 or 5, and a container containing sterile water, 0.9% sodium chloride, or phosphate-buffered saline solution to reconstitute the lyophilized composition.

8. A manufactured article according to claim 7, characterized in that the freeze-dried composition comprises no more than 0.5% moisture, and optionally comprises 0.3% moisture.

9. Use of a container or composition, as defined in any of claims 1 to 6, characterized by being used in the manufacture of a medicament for the treatment of acid sphingomyelinase deficiency (ASMD) in a human patient.

10. Use according to claim 9, characterized in that ASMD is Niemann-Pick disease type A / B or type B.

11. Use according to claim 10, characterized in that the treatment is for non-neurological manifestations of ASMD. Petition 870260052183, dated 05 / 29 / 2026, p. 48 / 134

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