Formulations comprising recombinant acid α-glucosidase

Abstract

To provide formulations to store and transport recombinant enzymes, such as rhGAA, which preserve enzyme activity and concentration.SOLUTION: Provided are pharmaceutical formulations comprising a recombinant acid α-glucosidase, wherein the recombinant acid α-glucosidase is expressed in Chinese hamster ovary (CHO) cells and comprises an increased content of N-glycan units bearing one or two mannose-6-phosphate residues when compared to a content of N-glycan units bearing one or two mannose-6-phosphate residues of alglucosidase alfa; at least one buffer selected from the group consisting of a citrate, a phosphate and combinations thereof; and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, wherein the formulation has a pH of from about 5.0 to about 7.0. Also provided are methods of treating Pompe disease using these pharmaceutical formulations.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] Principles and embodiments of the present invention relate generally to recombinant acid α-glucosidase and formulations, including liquid formulations in particular. [Background technology]

[0002] Pompe disease, also known as acid maltase deficiency or type II glycogen storage disease, is one of several lysosomal storage diseases. Lysosomal storage diseases are a group of autosomal recessive disorders characterized by the accumulation of cellular glycosphingolipids, glycogen, or mucopolysaccharides in intracellular compartments called lysosomes. Individuals with these diseases carry mutated genes that code for enzymes that are defective in catalyzing the hydrolysis of one or more of these substances, which accumulate in the lysosomes. Other examples of lysosomal disorders include Gaucher disease, G M1 -Gangliosidoses, fucosidoses, mucopolysaccharidoses, Hurler syndrome, Niemann-Pick A and B diseases, and Fabry disease. Pompe disease is also classified as a neuromuscular or metabolic myopathy.

[0003] Pompe disease is estimated to occur in approximately 1 in 40,000 births and is caused by mutations in the GAA gene, which encodes the enzyme lysosomal α-glucosidase (EC:3.2.1.20), also commonly known as acid α-glucosidase. Acid α-glucosidase is involved in the metabolism of glycogen, a branched polysaccharide that is the primary storage form of glucose in animals, by catalyzing its hydrolysis to glucose within the lysosomes. Because individuals affected with Pompe disease produce defective acid α-glucosidase, which is inactive or a mutant with reduced activity, glycogen breakdown occurs slowly or not at all, and glycogen accumulates in lysosomes in various tissues, especially in striated muscle, resulting in a wide range of clinical symptoms, including progressive muscle weakness and respiratory failure. Tissues such as cardiac and skeletal muscles are particularly affected.

[0004] Pompe disease varies greatly by the degree of enzyme deficiency, severity and age of onset, with over 500 different mutations in the GAA gene identified, many of which cause disease symptoms of varying severity. The disease has been classified into broad types: early-onset or infantile-onset and late-onset. Earlier onset and lower enzyme activity are generally associated with a more severe clinical course. Pediatric Pompe disease is the most severe and results from complete or near-complete acid α-glucosidase deficiency, with symptoms including severe lack of muscle tone, weakness, liver and cardiac enlargement, and cardiomyopathy. The tongue may be enlarged and protruding, and swallowing may become difficult. Most affected children die of respiratory or cardiac complications by the age of 2 years. Late-onset Pompe disease can present at any age above 12 months and is characterized by the absence of cardiac involvement and a more favorable short-term prognosis. Symptoms are associated with progressive skeletal muscle dysfunction, with generalized muscle weakness in the trunk, proximal limbs, and diaphragm, and respiratory muscle wasting. Adult patients may have no significant symptoms or motor limitations. Prognosis generally depends on the extent of respiratory muscle involvement. Most subjects affected by Pompe disease will gradually progress to wheelchair use and physical debilitation requiring assisted ventilation, with early death often occurring due to respiratory failure.

[0005] Current treatment options for Pompe disease include enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (rhGAA). Conventional rhGAA products are known as Alglucosidase Alpha, Myozyme®, or Lumizyme® from Genzyme, Inc. ERT is a chronic treatment required for the patient's lifetime and involves administering the replacement enzyme by intravenous infusion. The replacement enzyme is then transported in the circulation and enters the lysosomes in cells, where it acts to break down stored glycogen, compensating for the missing activity of the endogenous defective mutant enzyme, thus alleviating the symptoms of the disease.

[0006] Methods for preparing, storing, transporting, and administering alternative enzymes such as rhGAA to patients are challenging. Enzymes used in ERT are generally relatively complex and delicate, making the selection of accompanying buffers, excipients, etc. important. If the enzymes are not properly stored, large quantities may be required, which can make treatment ineffective and inefficient.

[0007] Some conventional rhGAA products are provided to patients as a lyophilized (freeze-dried) powder in a single-use vial that does not contain preservatives. The rhGAA must then be reconstituted in the vial, then diluted, and administered intravenously. Although lyophilization helps preserve the enzyme after manufacture until it is ready to be administered to the patient, this process itself can damage the enzyme. Therefore, great care must be taken in the selection of ingredients in rhGAA formulations to help preserve protein concentration and activity.

[0008] Furthermore, recombinant enzymes are often structurally different from wild-type enzymes. Even if the amino acids of a recombinant enzyme are identical to its wild-type counterpart, there may be differences in the carbohydrate chemistry. Thus, when a new recombinant enzyme is discovered, a formulation for the enzyme must be developed specifically for the newly discovered enzyme chemistry.

[0009] Thus, there is a continuing need for formulations for storing and transporting recombinant enzymes, such as rhGAA, that preserve enzyme activity and concentration. Summary of the Invention [Means for solving the problem]

[0010] One aspect of the invention relates to a pharmaceutical formulation. In embodiment 1, the formulation comprises: (a) a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and containing an increased content of N-glycan units with one or two mannose-6-phosphate residues compared to the content of N-glycan units with one or two mannose-6-phosphate residues of alglucosidase alpha; (b) at least one buffering agent selected from the group consisting of citrate, phosphate, and combinations thereof; and (c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof. and the pH of the formulation is about 5.0 to about 7.0.

[0011] Embodiment 2 includes a modification to the pharmaceutical formulation described in embodiment 1, where the recombinant acid α-glucosidase is present at a concentration of about 5 mg / mL to about 50 mg / mL.

[0012] Embodiment 3 includes a modification to the pharmaceutical formulation of embodiment 1 or 2, wherein the recombinant acid α-glucosidase is present at a concentration of about 15 mg / mL.

[0013] Embodiment 4 involves a modification to the pharmaceutical formulation described in any of embodiments 1-3, wherein the pH of the formulation is from about 5.5 to about 7.0.

[0014] Embodiment 5 involves a modification to the pharmaceutical formulation described in any of embodiments 1-4, wherein the pH of the formulation is about 6.0.

[0015] Embodiment 6 includes a modification to the pharmaceutical formulation of any of embodiments 1-5, where at least one buffering agent includes citrate.

[0016] Embodiment 7 includes a modification to the pharmaceutical formulation of any of embodiments 1-6, wherein at least one buffering agent comprises a potassium, sodium, or ammonium salt.

[0017] Embodiment 8 includes a modification to the pharmaceutical formulation of any of embodiments 1-7, where at least one buffering agent includes sodium citrate.

[0018] Embodiment 9 includes a modification to the pharmaceutical formulation of any of embodiments 1-8, wherein at least one buffering agent is present at a concentration of about 10 mM to about 100 mM.

[0019] Embodiment 10 includes a modification to the pharmaceutical formulation of any of embodiments 1-9, wherein at least one buffering agent is present at a concentration of about 25 mM.

[0020] Embodiment 11 includes a modification to the pharmaceutical formulation of any of embodiments 1-10, wherein trehalose, sucrose, glycine, or combinations thereof are excluded.

[0021] Embodiment 12 includes a modification to the pharmaceutical formulation of any of embodiments 1-11, where at least one excipient is mannitol present in a concentration of about 10 mg / mL to about 50 mg / mL.

[0022] Embodiment 13 includes a modification to the pharmaceutical formulation of any of embodiments 1-12, wherein at least one excipient is polysorbate 80 present in a concentration of about 0.2 mg / mL to about 0.5 mg / mL.

[0023] Embodiment 14 includes a modification to the pharmaceutical formulation of any of embodiments 1-13, wherein mannitol is present in a concentration of about 20 mg / mL and polysorbate 80 is present in a concentration of about 0.5 mg / mL.

[0024] Embodiment 15 includes the modification to the pharmaceutical formulation of any of embodiments 1-14, further including (d) water.

[0025] Embodiment 16 includes the modification to the pharmaceutical formulation of any of embodiments 1-15, further including: (d) an alkalizing agent; and / or (e) an acidifying agent, wherein the alkalizing agent and acidifying agent are present in an amount to maintain the pH of the pharmaceutical formulation at about 5.0 to about 6.0.

[0026] Embodiment 17 includes a modification to the pharmaceutical formulation of any of embodiments 1-16, wherein at least 30% of the recombinant human acid α-glucosidase contains one or more N-glycan units having one or two mannose-6-phosphate residues.

[0027] Embodiment 18 includes a modification to the pharmaceutical formulation of any of embodiments 1-17, wherein the recombinant human acid α-glucosidase contains an average of 0.5-7.0 moles of N-glycan units having one or two mannose-6-phosphate residues per mole of recombinant human acid α-glucosidase.

[0028] Embodiment 19 includes a modification to the pharmaceutical formulation of any of embodiments 1-18, wherein the recombinant human acid alpha-glucosidase contains, on average, at least 3 moles of mannose-6-phosphate residues per mole of recombinant human acid alpha-glucosidase and at least 4 moles of sialic acid residues per mole of recombinant human acid alpha-glucosidase.

[0029]

[0036] Embodiment 20 includes modifications to the pharmaceutical formulation of any of embodiments 1-19, wherein the recombinant human acid α-glucosidase comprises seven potential N-glycosylation sites, at least 50% of the recombinant human acid α-glucosidase comprises an N-glycan unit having two mannose-6-phosphate residues at a first site, at least 30% of the recombinant human acid α-glucosidase comprises an N-glycan unit having one mannose-6-phosphate residue at a second site, at least 30% of the recombinant human acid α-glucosidase comprises an N-glycan unit having two mannose-6-phosphate residues at a fourth site, and at least 20% of the recombinant human acid α-glucosidase comprises an N-glycan unit having one mannose-6-phosphate residue at a fourth site.

[0030]

[0081] Embodiment 21 includes a modification to the pharmaceutical formulation of any of embodiments 1-20, wherein the pharmaceutical formulation comprises: (a) Recombinant acid α-glucosidase; (b1) sodium citrate; (b2) citric acid monohydrate; (c1) Mannitol; (c2) polysorbate 80; (d) water; (e) optionally, an acidifying agent; and (f) optionally an alkalizing agent; and the pH of the formulation is about 5.0 to about 6.0.

[0031]

[0081] Embodiment 22 includes a modification to the pharmaceutical formulation of any of embodiments 1-21, wherein the pharmaceutical formulation comprises: (a) the recombinant acid α-glucosidase is present in a concentration of about 15 mg / mL; (b) a sodium citrate buffer, present at a concentration of about 25 mM; (c1) mannitol, present at a concentration of about 20 mg / mL; (c2) polysorbate 80, present at a concentration of about 0.5 mg / mL; and (d) water; (e) optionally, an acidifying agent; and (f) optionally an alkalizing agent; and the pH of the formulation is about 5.0 to about 6.0.

[0032] Another aspect of the present invention relates to a lyophilized pharmaceutical composition. Accordingly, embodiment 23 relates to a pharmaceutical composition comprising a formulation according to any one of embodiments 1 to 22 after lyophilization. Embodiment 24 relates to (a) a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and containing an increased content of N-glycan units with one or two mannose-6-phosphate residues compared to the content of N-glycan units with one or two mannose-6-phosphate residues of alglucosidase alpha; (b) a buffer selected from the group consisting of citrate, phosphate, and combinations thereof; and (c) at least one excipient selected from the group consisting of trehalose, mannitol, polysorbate 80, and combinations thereof; The present invention relates to a pharmaceutical composition comprising a lyophilized mixture comprising:

[0033] Another aspect of the invention relates to a method of treating Pompe disease. Thus, embodiment 25 comprises administering a pharmaceutical formulation according to embodiments 1-22 to a patient in need thereof. Embodiment 26 comprises a modification to the method according to embodiment 25, further comprising diluting the pharmaceutical formulation prior to administration to the patient. Embodiment 27 relates to a method of treating Pompe disease comprising reconstituting a pharmaceutical composition according to embodiment 23 or 24; and administering the reconstituted pharmaceutical composition to a patient in need thereof.

[0034] Another aspect of the present invention relates to a method for preparing said pharmaceutical formulation. Thus, embodiment 28 relates to a method for preparing the pharmaceutical formulation of any of embodiments 1 to 22, comprising adding at least one buffering agent, at least one excipient, and recombinant acid α-glucosidase to water to provide a solution; optionally adjusting the pH of said solution; and optionally adding additional water to said solution. Embodiment 29 includes the modification to the method of embodiment 28, further comprising filtering the solution. Embodiment 30 includes the modification to the method of embodiment 27 or 28, further comprising storing the solution.

[0035] Further features of the present invention will become apparent from the following specification and accompanying drawings: [Brief description of the drawings]

[0036] [Figure 1A] Non-phosphorylated high mannose glycans, mono-M6P glycans, and bis-M6P glycans are shown. [Figure 1B] The chemical structure of the M6P group is shown. [Figure 2A] Productive targeting of rhGAA via M6P-bearing glycans to target tissue (e.g., muscle tissue of subjects with Pompe disease) is described. [Figure 2B] This could be explained by non-productive drug clearance to non-target tissues (e.g., liver and spleen) or by binding of non-M6P glycans to non-target tissues. [Figure 3A]2A and 2B are graphs showing the results of CIMPR affinity chromatography of Lumizyme® and Myozyme®, respectively. The dashed line indicates the M6P elution gradient. Elution with M6P displaced GAA molecules bound via M6P-containing glycans to CIMPR. As shown in FIG. 2A, 78% of the GAA activity in Lumizyme® was eluted before the addition of M6P. FIG. 2B shows that 73% of the GAA Myozyme® activity was eluted before the addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme® was eluted with M6P. These figures show that most rhGAA in these two conventional rhGAA products lacks the glycans bearing M6P required to target CIMPR in target muscle tissues. [Figure 3B] 2A and 2B are graphs showing the results of CIMPR affinity chromatography of Lumizyme® and Myozyme®, respectively. The dashed line indicates the M6P elution gradient. Elution with M6P displaced GAA molecules bound via M6P-containing glycans to CIMPR. As shown in FIG. 2A, 78% of the GAA activity in Lumizyme® was eluted before the addition of M6P. FIG. 2B shows that 73% of the GAA Myozyme® activity was eluted before the addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme® was eluted with M6P. These figures show that most rhGAA in these two conventional rhGAA products lacks the glycans bearing M6P required to target CIMPR in target muscle tissues. [Figure 4] 1 shows a DNA construct for transforming CHO cells with DNA encoding rhGAA. CHO cells were transformed with a DNA construct encoding rhGAA. [Figure 5A] 5A and 5B are graphs showing the results of CIMPR affinity chromatography of Myozyme (registered trademark) and ATB200 rhGAA, respectively. As is clear from FIG. 5B, about 70% of rhGAA in ATB200 rhGAA contained M6P. [Figure 5B] 5A and 5B are graphs showing the results of CIMPR affinity chromatography of Myozyme (registered trademark) and ATB200 rhGAA, respectively. As is clear from FIG. 5B, about 70% of rhGAA in ATB200 rhGAA contained M6P. [Figure 6] FIG. 1 is a graph showing the results of CIMPR affinity chromatography of ATB200 rhGAA with and without capture on an anion exchange (AEX) column. [Figure 7] FIG. 1 is a graph showing the Polywax elution profiles of Lumizyme® and ATB200 rhGAA. [Figure 8] 1 is a table showing a summary of the N-glycan structures of Lumizyme® compared to three different preparations of ATB200 rhGAA identified as BP-rhGAA, ATB200-1 and ATB200-2. [Figure 9A] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9B] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9C] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9D] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9E] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9F] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9G] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9H] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 10A] FIG. 10A is a graph comparing the CIMPR binding affinity of ATB200 rhGAA (left trace) and Lumizyme (right trace). [Figure 10B] FIG. 10B is a table comparing the bis-M6P content of Lumizyme® and ATB200 rhGAA. [Figure 11A] FIG. 11A is a graph comparing ATB200 rhGAA activity (left trace) and Lumizyme® rhGAA activity (right trace) in normal fibroblasts at various GAA concentrations. [Figure 11B] FIG. 11B is a table comparing ATB200 rhGAA activity (left trace) and Lumizyme® rhGAA activity (right trace) in fibroblasts from a subject with Pompe disease at various GAA concentrations. [Figure 11C] FIG. 11C is a table comparing K uptake in fibroblasts from healthy subjects and subjects with Pompe disease. [Figure 12A] FIG. 12A is a graph showing the amount of glycogen versus dose of recombinant human acid α-glucosidase in mouse myocardium following contact with vehicle (negative control), 20 mg / ml alglucosidase alpha (Lumizyme®), or 5, 10 or 20 mg / kg ATB200. [Figure 12B] FIG. 12B is a graph showing the amount of glycogen versus dose of recombinant human acid α-glucosidase in mouse quadriceps muscle following contact with vehicle (negative control), 20 mg / ml alglucosidase alfa (Lumizyme®), or 5, 10 or 20 mg / kg ATB200. [Figure 12C] FIG. 12C is a graph showing the amount of glycogen versus dose of recombinant human acid α-glucosidase in mouse triceps muscle following contact with vehicle (negative control), 20 mg / ml alglucosidase alfa (Lumizyme®), or 5, 10 or 20 mg / kg ATB200. [Figure 13]1 is a table showing that the combination of ATB200 rhGAA with the chaperone miglustat provided significantly better glycogen clearance in GAA knockout mice than treatment with Lumizyme® or ATB200 rhGAA without the miglustat chaperone. [Figure 14] A series of electron micrographs of heart, septum and soleus muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat, showing levels of lysosome-associated membrane protein-1 (LAMP-1). [Figure 15] A series of electron micrographs of heart and soleus muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat, showing glycogen levels by staining with Periodic Acid Schiff's reagent (PAS). [Figure 16] Series of electron micrographs (1000x) of quadriceps muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, stained with methylene blue to show vacuoles (indicated by arrows). [Figure 17] A series of electron micrographs (40x) of quadriceps muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat, showing levels of the autophagy markers microtubule-associated protein 1A / 1B light chain 3 phosphatidylethanolamine conjugate (LC3A II) and p62, the insulin-dependent glucose transporter GLUT4 and the insulin-independent glucose transporter GLUT1. [Figure 18A] FIG. 13 is a graph showing wirehand and grip strength muscle data from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence of miglustat. [Figure 18B]FIG. 13 is a graph showing wirehand and grip strength muscle data from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence of miglustat. [Figure 19A] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence and absence of miglustat. [Figure 19B] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence and absence of miglustat. [Figure 19C] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence and absence of miglustat. [Figure 19D] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence and absence of miglustat. [Figure 19E] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence and absence of miglustat. [Figure 19F] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence and absence of miglustat. [Figure 19G]1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild type and Gaa knockout mice treated with vehicle, alglucosidase alpha and ATB200 in the presence and absence of miglustat. [Figure 20] A series of photomicrographs (100x and 200x) of vastus lateralis (VL) myofibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat, showing dystrophin signal. [Figure 21] The experimental design of an open-label, fixed sequential, dose-escalation, first-in-human, Phase 1 / 2 study to evaluate the safety, tolerability, PK, PD, and efficacy of intravenous infusion of ATB200 co-administered with oral miglustat in adults with Pompe disease is presented. [Figure 22A] FIG. 1 is a graph showing the concentration-time profile of GAA total protein in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 22B] FIG. 1 is a graph showing the concentration-time profile of GAA total protein in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 22C] 1 is a graph showing the AUC of GAA total protein in plasma in human subjects following administration of 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 22D] FIG. 1 is a graph showing the concentration-time profile of GAA total protein in plasma in two separate human subjects following administration of 20 mg / kg ATB200 and 260 mg miglustat. [Diagram 23]FIG. 1 is a graph showing the concentration-time profile of miglustat in plasma in human subjects following administration of 130 mg or 260 mg of miglustat. [Figure 24A] 1 is a graph showing the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK) and hexose tetrasaccharide (Hex4) levels in human patients following administration of increasing doses of ATB200 (5, 10 and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 24B] 1 is a graph showing the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK) and hexose tetrasaccharide (Hex4) levels in human patients following administration of increasing doses of ATB200 (5, 10 and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 24C] 1 is a graph showing the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK) and hexose tetrasaccharide (Hex4) levels in human patients following administration of increasing doses of ATB200 (5, 10 and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 24D] 1 is a graph showing the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK) and hexose tetrasaccharide (Hex4) levels in human patients following administration of increasing doses of ATB200 (5, 10 and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Before describing some exemplary embodiments of the invention, it should be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways. It will be understood that the embodiments listed below can be combined in other suitable combinations in accordance with the scope of the invention, not just those listed below.

[0038] Surprisingly, it has been discovered that careful selection of buffers and excipients can provide a formulation of recombinant GAA protein ATB200 that exhibits excellent stability and allows processes related to preparation, storage, transportation, reconstitution and administration of the formulation to be carried out while minimizing enzyme precipitation while maintaining enzyme activity and effectiveness.Thus, one aspect of the present invention relates to a formulation comprising rhGAA, a buffer, and at least one excipient.In one or more embodiments, the rhGAA comprises ATB200.In some embodiments, the formulation is a liquid formulation.Details regarding the various components of the formulation and various embodiments are described below.

[0039] definition The terms used herein generally have their ordinary meaning in the art, within the context of this invention and in the specific context in which each term is used. Certain terms are explained below or elsewhere in this specification to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

[0040] In this specification, for literal explanation or necessary implication, unless the context otherwise requires, the word "comprises" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify the presence of stated features, and do not exclude the presence or addition of further features in various embodiments of the invention.

[0041] As used herein, the term "Pompe disease" is intended to refer to a genetic lysosomal storage disease, also known as acid maltase deficiency, glycogenic storage disease type II (GSDII), and glycogenic disease type II, characterized by mutations in the GAA gene, which encodes the human acid α-glucosidase enzyme. The term includes, but is not limited to, early and late onset forms of Pompe disease, including, but not limited to, infantile, juvenile, and adult onset forms.

[0042] As used herein, the term "acid α-glucosidase" is intended to refer to a lysosomal enzyme that hydrolyzes the α-1,4 bonds between the D-glucose units of glycogen, maltose, and isomaltose. Alternative names include, but are not limited to, lysosomal α-glucosidase (EC:3.2.1.20); glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase, and exo-1,4-α-glucosidase. Human acid α-glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information (NCBI) Gene ID 2548), which is mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). The complete wild-type GAA amino acid sequence, as described in US Pat. No. 8,592,362, is set forth in SEQ ID NO:1 and has GenBank Accession No. AHE24104.1 (GI:568760974).

[0043] Over 500 mutations have now been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations that result in misfolding or misprocessing of the acid α-glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). Additionally, GAA mutations that affect maturation and processing of the enzyme include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE at amino acid residues 516-521 is required for activity of the acid α-glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to the acid α-glucosidase enzyme, and the italicized abbreviation "GAA" is intended to refer to the human gene encoding the human acid α-glucosidase enzyme. Thus, the abbreviation "rhGAA" is intended to refer to recombinant human acid α-glucosidase enzyme.

[0044] As used herein, the term "alglucosidase alfa" is intended to refer to recombinant human acid alpha-glucosidase identified as [199-Arginine, 223-Histidine] prepro-alpha-glucosidase (human); Chemical Abstracts Registry No. 420794-05-0. Alglucosidase alfa has been approved for commercial sale in the United States by Genzyme as of January 2016 under the trademarks Lumizyme® and Myozyme®.

[0045] As used herein, the term "ATB200" is intended to refer to recombinant human acid α-glucosidase as described in co-pending patent application PCT / US2015 / 053252, the disclosure of which is incorporated herein by reference.

[0046] As used herein, the term "glycan" is intended to refer to a polysaccharide chain covalently attached to an amino acid residue on a protein or polypeptide. As used herein, the term "N-glycan" or "N-linked glycan" is intended to refer to a polysaccharide chain attached to an amino acid residue on a protein or polypeptide by a covalent bond to the nitrogen atom of the amino acid residue. For example, an N-glycan may be covalently attached to the side chain nitrogen atom of an asparagine residue. A glycan may include one or several monosaccharide units, which may be covalently linked to form a linear or branched chain. In at least one embodiment, the N-glycan unit attached to ATB200 may include one or more monosaccharide units, each independently selected from N-acetylglucosamine, mannose, galactose, or sialic acid. The N-glycan unit on a protein may be determined by any suitable analytical technique, such as mass spectrometry. In some embodiments, N-glycan units can be determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using instruments such as a Thermo Scientific Orbitrap Velos Pro™ mass spectrometer, a Thermo Scientific Orbitrap Fusion Lumos Tribid™ mass spectrometer, or a Waters Xevo® G2-XS QTof mass spectrometer.

[0047] As used herein, the term "high mannose N-glycan" is intended to refer to an N-glycan having one to six or more mannose units. In at least one embodiment, a high mannose N-glycan unit contains a bis(N-acetylglucosamine) chain attached to an asparagine residue, which may further be attached to a branched polymannose chain. As used interchangeably herein, the term "M6P" or "mannose-6-phosphate" is intended to refer to a mannose unit that is phosphorylated at the 6-position, i.e., has a phosphate group attached to the hydroxyl group at the 6-position. In at least one embodiment, one or more mannose units of one or more N-glycan units are phosphorylated at the 6-position to form a mannose-6-phosphate unit.

[0048] As used herein, the term "complex N-glycan" is intended to refer to an N-glycan that includes one or more galactose and / or sialic acid units. In at least one embodiment, the complex N-glycan may be a high-mannose N-glycan in which one or more mannose units are further attached to one or more monosaccharide units, each independently selected from N-acetylglucosamine, galactose, and sialic acid.

[0049] As used herein, "therapeutically effective dose" and "effective amount" are intended to refer to the amount of acid alpha-glucosidase that produces a therapeutic response in a subject. The therapeutic response can be any response that a user (e.g., a clinician) recognizes as an effective response to treatment, including any surrogate clinical marker or symptom described herein and known in the art. Thus, in at least one embodiment, the therapeutic response can be an improvement or suppression of one or more symptoms or markers of Pompe disease, such as those known in the art. Symptoms or markers of Pompe disease include, but are not limited to, cardiomyopathy, cardiac hypertrophy, progressive muscle weakness especially in the trunk or legs, severe hypotension, macroglossia (and sometimes tongue protrusion), difficulty swallowing, sucking, and / or feeding, respiratory failure, hepatomegaly (moderate), facial muscle relaxation, areflexia, exercise intolerance, exertional dyspnea, orthopnea, sleep apnea, morning headache, somnolence, lordosis and / or scoliosis, decreased deep tendon reflexes, lower back pain, and failure to achieve developmental motor milestones.

[0050] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of non-natural purified enzymes into individuals with deficiencies of such enzymes. The administered protein can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of purified enzymes in individuals who otherwise require or would benefit from the administration of purified enzymes. In at least one embodiment, such individuals suffer from enzyme deficiencies. The introduced enzymes may be purified recombinant enzymes produced in vitro, or proteins purified from isolated tissues or fluids, such as placenta or animal milk, or from plants.

[0051] As used herein, the term "pharmaceutical acceptable" is intended to refer to molecular entities and compositions that are physiologically acceptable and typically do not produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, particularly humans.

[0052] As used herein, the term "excipient" refers to a substance other than the active ingredient contained in the formulation, which is generally an inactive substance.Excipients can help transport active drugs to the site where the drug is intended to act, control the release of active drugs, or aid in solubilization, or perform various other functions.Examples of excipients include, but are not limited to, buffers, surfactants, antibacterial agents, antioxidants, bulking agents, stabilizers, isotonicity agents, etc.

[0053] As used herein, the term "buffer" refers to a solution containing both a weak acid and its conjugate weak base, the pH of which changes only slightly upon addition of alkali or acid. As further described below, in some embodiments, the buffer used in the pharmaceutical formulation is a citrate and / or phosphate buffer.

[0054] As used herein, the term "subject" or "patient" is intended to refer to a human or non-human animal. In at least one embodiment, the subject is a mammal. In at least one embodiment, the subject is a human.

[0055] As used herein, the terms "about" and "approximately" are intended to refer to an acceptable degree of error for the measured quantity, given the nature or precision of the measurement. For example, the degree of error can be indicated by the number of significant figures provided for a measurement, as understood in the art, including but not limited to a variation of ±1 of the most accurate significant figure reported for a measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, particularly in biological systems, the terms "about" and "approximately" can mean values ​​within an order of magnitude, preferably within 5 times, and more preferably within 2 times of a given value. Numerical values ​​described herein are approximate unless otherwise specified, and the terms "about" or "approximately" mean that they can be inferred when not explicitly stated.

[0056] Throughout this specification, the terms "one embodiment," "particular embodiment," "various embodiments," "one or more embodiments," or "embodiments" mean that a particular feature, structure, material, or property described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of "one or more embodiments," "particular embodiment," "various embodiments," "in one embodiment," or "in an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Furthermore, the particular features, structures, materials, or properties may be combined in any suitable manner in one or more embodiments.

[0057] ATB200 rhGAA The formulation contains ATB200, a recombinant GAA (rhGAA) protein suitable for use in enzyme replacement therapy. Details regarding the structure and production of ATB200 and variants are provided below.

[0058] In at least one embodiment, ATB200 is expressed in Chinese hamster ovary (CHO) cells and contains an increased content of N-glycan units with one or more mannose-6-phosphate residues compared to the content of N-glycan units with one or more mannose-6-phosphate residues of alglucosidase alpha. There are seven potential N-linked glycosylation sites in rhGAA. Because each glycosylation site is heterogeneous in the types of N-linked oligosaccharides (N-glycans) present, rhGAA consists of a complex mixture of proteins and N-glycans with various binding affinities to M6P receptors and other carbohydrate receptors. High mannose containing rhGAA with N-glycans with one M6P group (mono-M6P) binds to CIMPR with low affinity (about 6,000 nM), while rhGAA with two M6P groups on the same N-glycan (bis-M6P) binds with high affinity (about 2 nM). Representative structures of non-phosphorylated, mono-M6P and bis-M6P glycans are shown in Figure 1A. The mannose-6-P group is shown in Figure 1B. Once inside the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAA has low total levels of glycans with M6P and bis-M6P, thus resulting in poor delivery of rhGAA to the lysosomes of target muscle cells. Productive drug targeting of rhGAA is shown in Figure 2A. The majority of rhGAA molecules in these conventional products do not have phosphorylated N-glycans, thereby lacking affinity for CIMPR. Non-phosphorylated high mannose glycans can also be removed by the mannose receptor resulting in non-productive clearance of ERT (Figure 2B).

[0059] Other types of N-glycans, glycoconjugates, containing galactose and sialic acid are also present in rhGAA. Because complex N-glycans are not phosphorylated, they have no affinity for CIMPR. However, complex-type N-glycans with exposed galactose residues have moderate to high affinity for the asialoglycoprotein receptor on hepatocytes, resulting in rapid nonproductive clearance of rhGAA (Figure 2B).

[0060] In at least one embodiment, the acid α-glucosidase is a recombinant human acid α-glucosidase, referred to herein as ATB200, as described in co-pending International Patent Application PCT / US2015 / 053252. ATB200 is a high affinity (K D It binds to the cation-independent mannose-6-phosphate receptor (CIMPR) at ~2–4 nM and binds to Pompe fibroblasts and skeletal myoblasts (K 取り込み ATB200 has been characterized in vivo and has been shown to be efficiently internalized by alglucosidase alfa (t 1 / 2 ~60 min). 1 / 2 It has been shown that the kinetics of the endothelial cell cycle is approximately 45 minutes.

[0061] In one or more embodiments, the recombinant human acid alpha-glucosidase has the wild-type GAA amino acid sequence shown in SEQ ID NO:2, which corresponds to amino acid residues 57-952 and is identical to wild-type (WT) human GAA after natural intracellular proteolytic processing to remove the first 56 residues, including the signal peptide and precursor peptide. The ATB200 amino acid sequence has been confirmed by trypsin digestion followed by liquid chromatography / mass spectrometry as well as amino acid sequencing. In contrast, current care rhGAA enzyme replacement therapy (ERT) (commercially available alglucosidase alpha-containing products are Myozyme® in most countries and Lumizyme®, Genzyme, a Sanofi Company in the United States) differs from WT GAA in that it contains three amino acid residue substitutions: a histidine to arginine change at position 199, an arginine to histidine change at 223, and a valine to isoleucine change at 780.

[0062] In at least one embodiment, recombinant human acid α-glucosidase undergoes post-translational and / or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, asparagine residues may undergo deamination to aspartic acid. As yet another example, aspartic acid may undergo isomerization to isoaspartic acid. Thus, in some embodiments, the enzyme is initially expressed as having the amino acid sequence set forth in SEQ ID NO:1, or SEQ ID NO:2, and the enzyme undergoes one or more of these post-translational and / or chemical modifications. Such modifications are also within the scope of the present disclosure.

[0063] In at least one embodiment, the recombinant human acid α-glucosidase has the wild-type GAA amino acid sequence set forth in SEQ ID NO:1, as described in U.S. Patent No. 8,592,362, and has GenBank Accession No. AHE24104.1 (GI:568760974). In at least one embodiment, the recombinant human acid α-glucosidase is glucosidase alpha, the human acid α-glucosidase enzyme encoded by the most prevalent of nine observed haplotypes of the GAA gene.

[0064] In at least one embodiment, the recombinant human acid alpha-glucosidase is initially expressed as having the full-length 952 amino acid sequence of wild-type GAA as set forth in SEQ ID NO:1, and the recombinant human acid alpha-glucosidase undergoes intracellular processing that removes some of the amino acids, for example the first 56 amino acids. Thus, the recombinant human acid alpha-glucosidase secreted by the host cell may have a shorter amino acid sequence than the recombinant human acid alpha-glucosidase initially expressed in the cell. In at least one embodiment, the shorter protein may have the amino acid sequence as set forth in SEQ ID NO:2, which differs from SEQ ID NO:1 only in that the first 56 amino acids, including the signal peptide and precursor peptide, are removed, thus resulting in a protein with 896 amino acids. Other changes in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and / or insertions relative to the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the rhGAA product comprises a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0065] In at least one embodiment, recombinant human acid α-glucosidase undergoes post-translational and / or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues can undergo oxidation. As another example, N-terminal glutamine can form pyroglutamic acid. As another example, asparagine residues can undergo deamination to aspartic acid. As yet another example, aspartic acid residues can undergo isomerization to isoaspartic acid. As yet another example, unpaired cysteine ​​residues in the protein can form disulfide bonds with free glutathione and / or cysteine. Thus, in some embodiments, the enzyme is initially expressed as having the amino acid sequence set forth in SEQ ID NO: 1, or SEQ ID NO: 2, and the enzyme undergoes one or more of these post-translational and / or chemical modifications. Such modified forms are also within the scope of the present disclosure.

[0066] Preferably, 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10 or 5% or less of the total recombinant human acid α-glucosidase molecules lack an N-glycan unit having one or more mannose-6-phosphate residues or lack the ability to bind to the cation-independent mannose-6-phosphate receptor (CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, <100% or more of the recombinant human acid α-glucosidase molecules contain at least one N-glycan unit having one or more mannose-6-phosphate residues or have the ability to bind to CIMPR.

[0067] A recombinant human acid α-glucosidase molecule may have one, two, three or four mannose-6-phosphate (M6P) groups on its glycan. For example, only one N-glycan in a recombinant human acid α-glucosidase molecule may have M6P (mono-phosphorylation), a single N-glycan may have two M6P groups (bis-phosphorylation), or two different N-glycans in the same recombinant human acid α-glucosidase molecule may each have a single M6P group. A recombinant human acid α-glucosidase molecule may also have an N-glycan that does not have an M6P group. In another embodiment, the N-glycans contain, on average, more than 3 moles / mole of M6P and more than 4 moles / mole of sialic acid, such that the human acid α-glucosidase contains, on average, at least 3 moles of mannose-6-phosphate residues per mole of recombinant human acid α-glucosidase and at least 4 moles of sialic acid per mole of recombinant human acid α-glucosidase. On average, at least about 3, 4, 5, 6, 7, 8, 9 or 10% of the total glycans in the recombinant human acid α-glucosidase may be in the form of mono-M6P glycans, for example, about 6.25% of the total glycans may have a single M6P group, on average, at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans in the recombinant human acid α-glucosidase are in the form of bis-M6P glycans, and on average, less than 25% of the total recombinant human acid α-glucosidase does not contain phosphorylated glycans that are bound to CIMPR.

[0068] The recombinant human acid α-glucosidase may have an average content of M6P-bearing N-glycans ranging from 0.5 to 7.0 moles / mole recombinant human acid α-glucosidase, or any intermediate subrange including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0 moles / mole recombinant human acid α-glucosidase. The recombinant human acid α-glucosidase may be fractionated to provide recombinant human acid α-glucosidase preparations having different average numbers of M6P-containing or bis-M6P-containing glycans, allowing further customization of the recombinant human acid α-glucosidase for targeting to lysosomes of target tissues by selecting specific fractions or by selectively combining different fractions.

[0069] In some embodiments, the recombinant human acid α-glucosidase has an average of 2.0 to 8.0 moles of M6P per mole of recombinant human acid α-glucosidase, including all intermediate values ​​and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 moles M6P / mole recombinant human acid α-glucosidase.

[0070] Up to 60% of the N-glycans in the recombinant human acid α-glucosidase may be fully sialylated, for example up to 10%, 20%, 30%, 40%, 50% or 60% of the N-glycans may be fully sialylated. In some embodiments, 4-20% of the total N-glycans are fully sialylated. In other embodiments, no more than 5%, 10%, 20% or 30% of the N-glycans in the recombinant human acid α-glucosidase have sialic acid and terminal galactose residues (Gal). This range includes all intermediate values ​​and subranges, for example, 7-30% of the total N-glycans in the recombinant human acid α-glucosidase can carry sialic acid and terminal galactose. In still other embodiments, no more than 5, 10, 15, 16, 17, 18, 19 or 20% of the N-glycans in the recombinant human acid α-glucosidase have only terminal galactose and no sialic acid. This range includes all intermediate values ​​and subranges, for example, 8-19% of the total N-glycans in the recombinant human acid α-glucosidase in the composition may have only terminal galactose and no sialic acid.

[0071] In other embodiments of the invention, 40, 45, 50, 55-60% of the total N-glycans in the recombinant human acid α-glucosidase are complex type N-glycans; or 1, 2, 3, 4, 5, 6, 7% or less of the total N-glycans in the recombinant human acid α-glucosidase are hybrid type N-glycans; 5, 10 or 15% or less of the high mannose type N-glycans in the recombinant human acid α-glucosidase are non-phosphorylated; at least 5% or 10% of the high mannose type N-glycans in the recombinant human acid α-glucosidase are phosphorylated mono-M6P; and / or at least 1 or 2% of the high mannose type N-glycans in the recombinant human acid α-glucosidase are phosphorylated bis-M6P. These values ​​include all intermediate values ​​and subranges. The recombinant human acid α-glucosidase may meet one or more of the above content ranges.

[0072] In some embodiments, the recombinant human acid α-glucosidase has an average of 2.0 to 8.0 moles of sialic acid residues per mole of recombinant human acid α-glucosidase. This range includes all intermediate values ​​and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 mole residues / mole recombinant human acid α-glucosidase. Without wishing to be bound by theory, it is believed that the presence of N-glycan units with sialic acid residues may prevent non-productive clearance of the recombinant human acid α-glucosidase by the asialoglycoprotein acceptor.

[0073] In one or more embodiments, rhGAA has M6P and / or sialic acid units at certain N-glycosylation sites of recombinant human lysosomal proteins. For example, there are seven potential N-linked glycosylation sites in rhGAA. These potential glycosylation sites are present at the following positions in SEQ ID NO:2: N84, N177, N334, N414, N596, N826 and N869. Similarly, with respect to the full-length amino acid sequence of SEQ ID NO:1, these potential glycosylation sites are present at the following positions: N140, N233, N390, N470, N652, N882 and N925. Other variants of rhGAA may have similar glycosylation sites depending on the position of the asparagine residue. In general, the sequence ASN-X-SER or ASN-X-THR in a protein amino acid sequence indicates a potential glycosylation site, with the exception that X cannot be HIS or PRO.

[0074] In various embodiments, rhGAA has a specific N-glycosylation profile. In one or more embodiments, at least 20% of rhGAA is phosphorylated at the first N-glycosylation site (e.g., N84 of SEQ ID NO:2 and N140 of SEQ ID NO:1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of rhGAA can be phosphorylated at the first N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA has a mono-M6P unit at the first N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA has a bis-M6P unit at the first N-glycosylation site.

[0075] In one or more embodiments, at least 20% of the rhGAA is phosphorylated at the second N-glycosylation site (e.g., N177 of SEQ ID NO:2 and N223 of SEQ ID NO:1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA may be phosphorylated at the second N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA has mono-M6P units at the second N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA has bis-M6P units at the second N-glycosylation site. In one or more embodiments, at least 5% of rhGAA is phosphorylated at the third N-glycosylation site (e.g., N334 of SEQ ID NO:2 and N390 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA is phosphorylated at the third N-glycosylation site. For example, the third N-glycosylation site may have a mixture of non-phosphorylated high mannose glycans, di-, tri-, and tetra-antennary complex glycans, and hybrid glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of rhGAA is sialylated at the third N-glycosylation site.

[0076] In one or more embodiments, at least 20% of the rhGAA is phosphorylated at the fourth N-glycosylation site (e.g., N414 of SEQ ID NO:2 and N470 of SEQ ID NO:1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA may be phosphorylated at the fourth N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA has a mono-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA has a bis-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20% or 25% of the rhGAA is sialylated at the fourth N-glycosylation site.

[0077] In one or more embodiments, at least 5% of rhGAA is phosphorylated at the fifth N-glycosylation site (e.g., N596 of SEQ ID NO:2 and N692 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA is phosphorylated at the fifth N-glycosylation site. For example, the fifth N-glycosylation site may have a fucosylated di-antennary complex glycan as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA is sialylated at the fifth N-glycosylation site.

[0078] In one or more embodiments, at least 5% of rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 of SEQ ID NO:2 and N882 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site may have a mixture of di-, tri-, and tetra-antennary complex glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA is sialylated at the sixth N-glycosylation site.

[0079] In one or more embodiments, at least 5% of rhGAA is phosphorylated at the seventh N-glycosylation site (e.g., N869 of SEQ ID NO:2 and N925 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20% or 25% of rhGAA is phosphorylated at the seventh N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60% or 65% of rhGAA has any glycan at the seventh N-glycosylation site. In some embodiments, at least 30%, 35% or 40% of rhGAA has a glycan at the seventh N-glycosylation site.

[0080] In various embodiments, rhGAA has an average fucose content of 0-5 moles per mole rhGAA, a GlcNAc content of 10-30 moles per mole rhGAA, a galactose content of 5-20 moles per mole rhGAA, a mannose content of 10-40 moles per mole rhGAA, an M6P content of 2-8 moles per mole rhGAA, and a sialic acid content of 2-8 moles per mole rhGAA. In various embodiments, rhGAA has an average fucose content of 2-3 moles per mole rhGAA, a GlcNAc content of 20-25 moles per mole rhGAA, a galactose content of 8-12 moles per mole rhGAA, a mannose content of 22-27 moles per mole rhGAA, an M6P content of 3-5 moles per mole rhGAA, and a sialic acid content of 4-7 moles per mole rhGAA.

[0081] Recombinant human acid α-glucosidase is preferably produced by Chinese Hamster Ovary (CHO) cells, such as the CHO cell line GA-ATB200 or ATB200-001-X5-14, or by subcultures or derivatives of such CHO cell cultures. DNA constructs expressing allelic variants of acid α-glucosidase or other variant acid α-glucosidase amino acid sequences, such as those that are at least 90%, 95% or 99% identical to SEQ ID NO: 1, can be constructed and expressed in CHO cells. These variant acid α-glucosidase amino acid sequences may contain deletions, substitutions and / or insertions relative to SEQ ID NO: 1, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more deletions, substitutions and / or insertions relative to the amino acid sequence set forth in SEQ ID NO: 1. The skilled artisan may select alternative vectors suitable for transformation of CHO cells for the production of such DNA constructs.

[0082] A variety of alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used, for example, with default settings. For example, polypeptides having at least 90%, 95% or 99% identity to a particular polypeptide described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When BLASTP is used, the percentage similarity is based on the BLASTP positive score, and the percentage sequence identity is based on the BLASTP identity score. BLASTP "identity" refers to the number and percentage of total residues in a high-scoring sequence pair that are identical; and BLASTP "positive" refers to the number and percentage of residues that have a positive alignment score and are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequence of a similar polypeptide can be deduced using the genetic code and obtained by conventional means, in particular by back-translating the amino acid sequence using the genetic code.

[0083] In some embodiments, it has been found that recombinant human acid α-glucosidase with superior ability to target cation-independent mannose-6-phosphate receptors (CIMPR) and cellular lysosomes, as well as glycosylation patterns that reduce non-productive clearance in vivo, can be produced using Chinese hamster ovary (CHO) cells. These cells can be induced to express recombinant human acid α-glucosidase with significantly higher levels of N-glycan units with one or more mannose-6-phosphate residues than conventional recombinant human acid α-glucosidase products, such as alglucosidase alpha. The recombinant human acid α-glucosidase produced by these cells has significantly more muscle cell-targeting mannose-6-phosphate (M6P) and bis-mannose-6-phosphate N-glycan residues than conventional acid α-glucosidases, such as Lumizyme®, as exemplified by ATB200, for example. Without being bound by theory, it is believed that this extensive glycosylation allows the ATB200 enzyme to be more effectively taken up by target cells and therefore more efficiently removed from the circulation than other recombinant human acid α-glucosidases, such as alglucosidase alpha, which has a much lower M6P and bis-M6P content. ATB200 has been shown to bind efficiently to CIMPR, be efficiently taken up by skeletal and cardiac muscles, and have a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces non-productive clearance in vivo.

[0084] It is also contemplated that the extensive glycosylation of ATB200 may contribute to the reduced immunogenicity of ATB200, for example, compared to alglucosidase alpha. As will be understood by those skilled in the art, glycosylation of proteins with conserved mammalian sugars generally increases the solubility of the product and reduces the aggregation and immunogenicity of the product. Glycosylation indirectly alters protein immunogenicity by minimizing protein aggregation and shielding protein immunogenic epitopes from the immune system (Guidance for Industry-Immunogenicity Assessment for Therapeutic Protein Products, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, August 2014). Thus, in at least one embodiment, administration of recombinant human acid alpha-glucosidase does not induce anti-drug antibodies. In at least one embodiment, administration of recombinant human acid alpha-glucosidase reduces the incidence of anti-drug antibodies in a subject below the level of anti-drug antibodies induced by administration of alglucosidase alpha.

[0085] As described in co-pending International Patent Application PCT / US2015 / 053252, cells such as CHO cells can be used to produce the rhGAA described therein, which can be used in the present invention. Examples of such CHO cell lines are GA-ATB200 or ATB200-001-X5-14, or subcultures thereof that produce rhGAA compositions as described therein. Such CHO cell lines can contain multiple copies of genes, such as 5, 10, 15, 20 or more copies per polynucleotide encoding GAA.

[0086] High M6P and bis-M6P rhGAA, such as ATB200 rhGAA, are prepared by transforming CHO cells with a DNA construct encoding GAA. Although CHO cells have been used previously to generate rhGAA, it was not observed that transformed CHO cells could be cultured and selected to produce rhGAA with a high content of M6P and bis-M6P glycans that target CIMPR.

[0087] Surprisingly, it has been found that it is possible to transform CHO cell lines, select transformants that produce rhGAA containing a high content of glycans with M6P or bis-M6P targeting CIMPR, and stably express this high M6P rhGAA.Thus, the method for producing these CHO cell lines is also described in co-pending International Patent Application PCT / US2015 / 053252.The method includes transforming CHO cells with DNA encoding GAA or GAA variants, stably integrating DNA encoding GAA into its chromosome, selecting CHO cells that stably express GAA, and selecting CHO cells that express GAA with a high content of glycans with M6P or bis-M6P, and optionally selecting CHO cells that have N-glycans with high sialic acid content and / or low non-phosphorylated high mannose content. These CHO cell lines can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell lines and recovering the compositions from the culture of the CHO cells.

[0088] In one or more embodiments, ATB200 is present in an amount ranging from about 5 to about 50 mg / mL. In further embodiments, ATB200 is present in an amount ranging from about 5, 8, 10 or 12 to about 18, 20, 25, 30, 35, 40, 45 or 50 mg / mL. In some embodiments, ATB200 is present in an amount of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mg / mL. In further embodiments, ATB200 is present in an amount of about 15 mg / mL.

[0089] pH and Buffers The pH of the formulation may range from about 5.0 to about 7.0 or from about 5.0 to about 6.0. In one or more embodiments, the pH of the formulation ranges from about 5.5 to about 6.0. In some embodiments, the formulation has a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5. In general, the amounts of the listed ingredients will result in a pH range of about 5.0 to about 6.0. However, the pH can be adjusted to a target pH by using pH adjusters (i.e., alkalizing and acidifying agents), such as sodium hydroxide and / or hydrochloric acid.

[0090] The formulation also includes a buffer selected from the group consisting of citrate, phosphate, and combinations thereof. As used herein, "buffer" refers to a buffer containing a weak acid and its conjugate base that helps prevent changes in pH. The citrate and / or phosphate may be sodium citrate or sodium phosphate. Other salts include potassium and ammonium salts. In one or more embodiments, the buffer includes citrate. In further embodiments, the buffer includes sodium citrate (e.g., a mixture of sodium citrate dihydrate and citric acid monohydrate). In one or more embodiments, the buffer including citrate may include sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffers are present.

[0091] Excipients The formulation also includes at least one excipient. Some embodiments of the present invention include an excipient that assists the isotonicity agent, acts as a bulking agent, or acts as a stabilizer. In one or more embodiments, the at least one excipient is selected from the group consisting of mannitol, polysorbate, and combinations thereof.

[0092] In one or more embodiments, the excipient comprises a component that can act as a tonicity agent and / or bulking agent, particularly mannitol. A tonicity agent is a component that helps ensure that the formulation has an osmotic pressure similar or equivalent to that of human blood. A bulking agent is a component that adds mass to the formulation (e.g., lyophilized) and provides the cake with the appropriate structure.

[0093] In one or more embodiments, the total amount of tonicity agent and / or bulking agent ranges from about 10 to about 50 mg / mL. In further embodiments, the total amount of tonicity agent and / or bulking agent ranges from about 10, 11, 12, 13, 14, or 15 to about 16, 20, 25, 30, 35, 40, 45, or 50 mg / mL. In some embodiments, the tonicity agent and / or bulking agent comprises mannitol. In one or more embodiments, the mannitol is present in an amount of about 10 to about 50 mg / mL. In further embodiments, the mannitol is present in an amount of about 10, 11, 12, 13, 14, or 15 to about 16, 20, 25, 30, 35, 40, 45, or 50 mg / mL. In still further embodiments, mannitol is present in an amount of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL. In some embodiments, mannitol is the only tonicity agent and / or bulking agent. Examples of other tonicity agents and / or bulking agents include sodium chloride, sucrose, and trehalose.

[0094] In some embodiments, the excipient includes a component capable of acting as a stabilizer, such as polysorbate 80. A stabilizer is a compound capable of preventing or minimizing aggregate formation at a hydrophobic air-water interface. In some embodiments, the stabilizer is a surfactant. In one or more embodiments, the total amount of stabilizer is in the range of about 0.1 to about 1.0 mg / mL. In further embodiments, the total amount of stabilizer is in the range of about 0.1, 0.2, 0.3, 0.4, or 0.5 to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In still further embodiments, the total amount of stabilizer is in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In some embodiments, polysorbate 80 is the only stabilizer. Thus, in one or more embodiments, polysorbate 80 is in the range of about 0.1 to about 1.0 mg / mL. In further embodiments, the total amount of polysorbate 80 ranges from about 0.1, 0.2, 0.3, 0.4, or 0.5 to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In still further embodiments, the total amount of polysorbate 80 is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL.

[0095] In one or more embodiments, it may be desirable to exclude certain compounds from the formulation. For example, when preparing a formulation for the treatment of a given disease, it may be desirable to exclude certain compounds that exacerbate the underlying disease. As mentioned above, individuals with Pompe disease have a reduced or absent ability to break down glycogen. Some commonly used excipients, such as sucrose, trehalose and glycine, can be converted to glucose in the body, which exacerbates Pompe disease. Thus, in some embodiments, sucrose, trehalose and / or glycine are excluded from the formulation. Similarly, certain ingredients may be excluded that are not the most suitable for the formulation in a given particular situation. For example, in some embodiments, poloxamer may be excluded.

[0096] Exemplary embodiments In one or more embodiments, the formulation comprises: (a) ATB200 (e.g., a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and containing an increased content of N-glycan units with one or two mannose-6-phosphate residues compared to the content of N-glycan units with one or two mannose-6-phosphate residues of alglucosidase alpha); (b) at least one buffering agent selected from the group consisting of citrate, phosphate, and combinations thereof; (c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof. and the pH of the formulation is from about 5.0 to about 7.0.

[0097] In some embodiments, the formulation comprises: (a) ATB200 (e.g., a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and containing an increased content of N-glycan units with one or two mannose-6-phosphate residues compared to the content of N-glycan units with one or two mannose-6-phosphate residues of alglucosidase alpha); (b1) sodium citrate; (b2) citric acid monohydrate; (c1) Mannitol; (c2) polysorbate 80; (d) water; (e) optionally, an acidifying agent; and (f) optionally an alkalizing agent; and the pH of the formulation is from about 5.0 to about 7.0. In a further embodiment, the pH of the formulation ranges from about 5.5 to about 6.0. In yet a further embodiment, the pH of the formulation is about 6.0.

[0098] In certain embodiments, the formulation comprises: (a) ATB200 (e.g., a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and containing an increased content of N-glycan units having one or two mannose-6-phosphate residues compared to the content of N-glycan units having one or two mannose-6-phosphate residues of alglucosidase alpha), present at a concentration of about 5-30 mg / mL or about 15 mg / mL; (b) a sodium citrate buffer, present at a concentration of about 10-100 mM or about 25 mM; (c1) mannitol, present in a concentration of about 10-50 mg / mL or about 20 mg / mL; (c2) polysorbate 80, present at a concentration of about 0.1 to 1 mg / mL or about 0.5 mg / mL; and (d) water; (e) optionally, an acidifying agent; and (f) optionally an alkalizing agent; and the pH of the formulation is from about 5.0 to about 7.0. In a further embodiment, the pH of the formulation ranges from about 5.5 to about 6.0. In yet a further embodiment, the pH of the formulation is about 6.0.

[0099] Preparation of formulations Another aspect of the present invention relates to a method for preparing the formulation described herein. In one or more embodiments, the formulation can be prepared from an enzyme solution. Using methods known in the art, this solution can be concentrated and buffer exchanged to a target concentration and buffer, if necessary. Additional ingredients (e.g., excipients and pH adjusters) can then be added. The formulation can then be filtered and stored in a storage container.

[0100] In one or more embodiments, the method for preparing any pharmaceutical formulation described herein comprises adding at least one buffer, at least one excipient, and recombinant acid α-glucosidase to water to provide a solution; optionally adjusting the pH of the solution; and optionally adding additional water to the solution.In a further embodiment, the method further comprises filtering the solution.In yet a further embodiment, the method further comprises storing the solution.

[0101] An exemplary, non-limiting process for the manufacture of the formulations described herein is as follows: 1. In a suitable manufacturing vessel, add Water for Injection approximately 85-90% of the batch volume. 2. Add and dissolve the desired excipients and buffers (e.g., sodium citrate dihydrate, citric acid monohydrate, polysorbate 80, mannitol) and mix until dissolved. 3. Add ATB200 drug substance and mix. 4. Adjust the pH to the desired pH (e.g., 6±0.1) using an adjuster (e.g., hydrochloric acid or sodium hydroxide solution) as necessary. 5. Sufficient water for injection is added to final volume and mixed. 6. Filter the solution through a sterile filter into a sterile receiver. 7. Aseptically fill drug solutions into vials and insert stoppers. 8. Cap all vials and store at 2-8°C.

[0102] In one or more embodiments, the formulation as prepared is in liquid form. That is, the formulation includes water. This liquid formulation may undergo a lyophilization (freeze-drying) process to provide a cake or powder. Thus, another aspect of the present invention relates to a pharmaceutical composition comprising any of the above formulations after lyophilization. The lyophilized mixture may include a buffer selected from the group consisting of ATB200, citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of trehalose, mannitol, polysorbate 80, and combinations thereof. In some embodiments, other components (e.g., other excipients) may be added to the lyophilized mixture. The pharmaceutical composition comprising the lyophilized formulation may be provided in a vial and then stored, shipped, reconstituted, and / or administered to a patient.

[0103] Patient Treatment and Administration Methods Another aspect of the invention relates to a method of treating Pompe disease and / or the use of the formulations described herein for the treatment of Pompe disease. The formulations can be administered as prepared or after lyophilization and reconstitution. Thus, in one or more embodiments, the method comprises administering any of the above pharmaceutical formulations to a patient in need thereof. In other embodiments, the method comprises reconstituting a lyophilized pharmaceutical composition and administering the reconstituted pharmaceutical composition to a patient in need thereof. In some embodiments, the reconstituted pharmaceutical composition has a similar or identical composition to the pharmaceutical formulation before lyophilization and / or as prepared. In either case, the pharmaceutical formulation or reconstituted pharmaceutical composition may be diluted before administration to a patient. In a further embodiment, the pharmaceutical formulation or reconstituted pharmaceutical composition is administered intravenously.

[0104] In one or more embodiments, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, the components are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active substance. If the composition is administered by injection, it can be dispensed with an infusion bottle containing pharmaceutical grade sterile water, saline or dextrose / water. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0105] In other embodiments, the pharmaceutical formulation or reconstituted pharmaceutical composition is administered by direct administration to a target tissue, such as the heart or skeletal muscle (e.g., intramuscularly) or the nervous system (e.g., direct injection into the brain; intracerebroventricularly; intrathecally). If desired, multiple routes can be used simultaneously.

[0106] The pharmaceutical formulation or reconstituted composition is administered in a therapeutically effective amount (e.g., a dosage sufficient to treat the disease, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease, when administered periodically). The therapeutically effective amount in treating a disease depends on the nature and extent of the effect of the disease and can be determined by standard clinical techniques. Additionally, in vitro or in vivo assays can be optionally used to help identify optimal dosage ranges. In at least one embodiment, the recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 1 mg / kg to about 100 mg / kg, for example about 5 mg / kg to about 30 mg / kg, typically about 5 mg / kg to about 20 mg / kg. In at least one embodiment, the recombinant human acid alpha-glucosidase is administered by intravenous infusion at a dose of about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 50 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In at least one embodiment, the recombinant human acid alpha-glucosidase is administered by intravenous infusion at a dose of about 20 mg / kg. In at least one embodiment, the recombinant human acid alpha-glucosidase is administered by intravenous infusion at a dose of about 20 mg / kg. The effective dose for a particular individual can be changed (e.g., increased or decreased) over time, depending on the needs of the individual. For example, the amount can be increased during times of physical illness or stress, or if anti-acid alpha-glucosidase antibodies become present or increase, or if symptoms of the disease worsen.

[0107] The therapeutically effective amount of recombinant human acid alpha-glucosidase (or a composition or medicament containing recombinant human acid alpha-glucosidase) is administered periodically and continuously depending on the nature and extent of the effect of the disease. As used herein, "regular" administration indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time administration). The interval can be determined by standard clinical techniques. In a preferred embodiment, recombinant human acid alpha-glucosidase is administered monthly, bimonthly; weekly; twice weekly; or daily. The administration interval for a single individual does not need to be a fixed interval and can be changed over time depending on the needs of the individual. For example, the interval of administration can be decreased when a person has a physical illness or stress, when anti-recombinant human acid alpha-glucosidase antibodies become present or increase, or when the symptoms of the disease worsen.

[0108] In one or more embodiments, the pharmaceutical formulation or reconstituted composition is co-administered with a pharmacological chaperone, such as oral administration of the chaperone and intravenous administration of the pharmaceutical formulation or reconstituted composition. In various embodiments, the pharmacological chaperone is miglustat. In at least one embodiment, miglustat is administered at an oral dose of about 200 to about 400 mg, or at an oral dose of about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg. In at least one embodiment, miglustat is administered at an oral dose of about 233 mg to about 400 mg. In at least one embodiment, miglustat is administered at an oral dose of about 250 to about 270 mg, or at an oral dose of about 250 mg, about 255 mg, about 260 mg, about 265 mg, or about 270 mg. In at least one embodiment, miglustat is administered as an oral dose of about 260 mg.

[0109] In at least one embodiment, miglustat and recombinant human acid alpha-glucosidase are administered simultaneously. In at least one embodiment, miglustat and recombinant human acid alpha-glucosidase are administered sequentially. In at least one embodiment, miglustat is administered prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered within 3 hours prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered approximately 2 hours prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered within 2 hours prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered approximately 1.5 hours prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered approximately 1 hour prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered about 50 to about 70 minutes prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered about 55 to about 65 minutes prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered about 25 to about 35 minutes prior to administration of recombinant human acid alpha-glucosidase. In at least one embodiment, miglustat is administered about 27 to about 33 minutes prior to administration of recombinant human acid alpha-glucosidase.

[0110] kit Another aspect of the invention relates to kits comprising the pharmaceutical formulations (including after lyophilization) described herein. In one or more embodiments, the kits include a container (e.g., a vial, tube, bag, etc.) that contains the pharmaceutical formulation (before or after lyophilization) and instructions for reconstitution, dilution, and administration. EXAMPLES

[0111] Enzyme Example 1: Limitations of existing Myozyme® and Lumizyme® rhGAA products To evaluate the ability of rhGAA in Myozyme® and Lumizyme®, the only currently approved treatments for Pompe disease, these rhGAA preparations were injected onto a CIMPR column (which binds rhGAA with M6P groups) and subsequently eluted with a free M6 gradient. Fractions were collected in a 96-well plate and GAA activity was assayed with 4MU-α-glucose substrate. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a percentage of total enzyme.

[0112] Figures 3A-B show the problems associated with conventional ERT (Myozyme® and Lumizyme®): 73% of Myozyme® rhGAA (Figure 3B) and 78% of Lumizyme® rhGAA (Figure 3A) did not bind to CIMPR (see the left-most peak in each figure). Only 27% of Myozyme® rhGAA and 22% of Lumizyme® rhGAA contained M6P that could be targeted to CIMPR on muscle cells.

[0113] The effective dose of Myozyme® and Lumizyme® corresponds to the amount of rhGAA containing M6P that targets CIMPR on muscle cells. However, most of the rhGAA in these two conventional products does not target the CIMPR receptor on target muscle cells. The administration of conventional rhGAA, in which most of the rhGAA is not targeted to muscle cells, increases the risk of allergic reaction or immune induction against non-targeted rhGAA.

[0114] Enzyme Example 2: Preparation of CHO cells producing ATB200 rhGAA with high content of mono- or bis-M6P-containing N-glycans CHO cells were transfected with DNA expressing rhGAA, and then transformants producing rhGAA were selected. A DNA construct for transforming CHO cells with DNA encoding rhGAA is shown in Figure 4. CHO cells were transfected with DNA expressing rhGAA, and then transformants producing rhGAA were selected.

[0115] After transfection, DG44 CHO (DHFR-) cells containing a stably integrated GAA gene were selected in hypoxanthine / thymidine-deficient (-HT) medium.

[0116] Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Cell pools expressing large amounts of GAA were identified by GAA enzyme activity assay and used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid media plates, picked by ClonePix system, and transferred to 24 deep-well plates. Individual clones were assayed for GAA enzyme activity to identify clones expressing high levels of GAA. Conditioned medium to determine GAA activity used 4-MU-α-glucosidase substrate. Clones producing higher levels of GAA, as measured by GAA enzyme assay, were further evaluated for viability, growth ability, GAA productivity, N-glycan structure, and stable protein expression. CHO cell lines, including the CHO cell line GA-ATB-200, expressing rhGAA with enhanced mono-M6P or bis-M6P N-glycans, were isolated using this procedure.

[0117] Enzyme Example 3: Capture and purification of ATB200 rhGAA Multiple batches of rhGAA according to the present invention were produced in shake flasks and perfusion bioreactors using the CHO cell line GA-ATB-200, and CIMPR binding was measured. For purified ATB200 rhGAA from different production batches, similar CIMPR receptor binding (about 70%) as shown in Figure 5B and Figure 6 was observed, indicating that ATB200 rhGAA can be consistently produced. As shown in Figures 3A-B and 5A-B, Myozyme® and Lumizyme® rhGAA showed significantly lower CIMPR binding than ATB200 rhGAA.

[0118] Enzyme Example 4: Analytical Comparison of ATB200 to Lumizyme® ATB200 rhGAA was fractionated by terminal phosphate using weak anion exchange ("WAX") liquid chromatography. Elution profiles were generated by eluting ERT with increasing amounts of salt. Profiles were monitored by UV (A280 nm). ATB200 rhGAA was obtained and purified from CHO cells. Lumizyme® was obtained from a commercial source. Lumizyme® showed a high peak on the left side of its elution profile. ATB200 rhGAA showed four prominent peaks eluting to the right of Lumizyme® (Figure 7). This confirms that ATB200 rhGAA was significantly more phosphorylated than Lumizyme® since this assessment is due to terminal charge and not CIMPR affinity.

[0119] Enzyme Example 5: Oligosaccharide characteristics of ATB200 rhGAA Purified ATB200 rhGAA and Lumizyme® glycans were evaluated by MALDI-TOF to determine the individual glycan structures found on each ERT (Figure 8). The ATB200 sample was found to contain less non-phosphorylated high mannose type N-glycans than Lumizyme®. The higher content of M6P glycans in ATB200 than Lumizyme® indicates that ATB200 rhGAA targets muscle cells more effectively. The higher percentage of mono- and bis-phosphorylated structures determined by MALDI is consistent with the CIMPR profile, which showed significantly greater binding of ATB200 to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contains at least one natural bis-M6P N-glycan structure. The higher content of this bis-M6P N-glycan on ATB200 rhGAA directly correlated with high affinity binding to CIMPR in the M6P receptor plate binding assay (KD approx. 2-4 nM), FIG. 10A.

[0120] ATB200 rhGAA was also analyzed for site-specific N-glycan profiles using two different LC-MS / MS analytical techniques. In the first analysis, the protein was denatured, reduced, alkylated, and digested prior to LC-MS / MS analysis. During protein denaturation and reduction, 200 μg protein sample, 5 μl 1 mol / L Tris-HCl (final concentration 50 mM), 75 μL 8 mol / L guanidine HCl (final concentration 6 M), 1 μL 0.5 mol / L EDTA (final concentration 5 mM), 2 μL 1 mol / L DTT (final concentration 20 mM) and Milli-Q® water were added to a 1.5 mL tube to provide a total volume of 100 μL. The sample was mixed and incubated at 56° C. for 30 minutes in a dry bath. During alkylation, the denatured and reduced protein samples were mixed with 5 μL of 1 mol / L iodoacetamide (IAM, final concentration 50 mM) and then incubated in the dark for 30 min. After alkylation, 400 μL of pre-chilled acetone was added to the sample and the mixture was frozen at -80°C for 4 h. The sample was then centrifuged at 13000 rpm at 4°C for 5 min and the supernatant was removed. 400 μl of pre-chilled acetone was added to the pellet, which was then centrifuged at 13000 rpm at 4°C for 5 min and the supernatant was removed. The sample was air-dried on ice in the dark to remove acetone residues. 40 μL of 8M urea and 160 μL of 100 μM NH4HCO3 were added to the sample to dissolve the protein. During trypsin digestion, 50 μg of protein was added with trypsin digestion buffer to a final volume of 100 μl, and 5 μL of 0.5 μg / mL trypsin (protein to enzyme ratio 20 / 1 w / w) was added. The solution was mixed well and incubated overnight (16 ± 2 hours) at 37°C. The reaction was stopped by adding 2.5 μl of 20% TFA (final concentration 0.5%). Samples were then analyzed using a Thermo Scientific Orbitrap Velos Pro™ mass spectrometer.

[0121] In a second LC-MS / MS analysis, ATB200 samples were prepared following similar denaturation, reduction, alkylation and digestion procedures, except that iodoacetic acid (IAA) was used as the alkylating reagent instead of IAM, and analyzed using a Thermo Scientific Orbitrap Fusion Lumos Tribid™ mass spectrometer.

[0122] The results of the first and second analyses are shown in Figures 9A-9H. In Figures 9A-9H, the results of the first analysis are represented by the left bar (dark gray), and the results of the second analysis are represented by the right bar (light gray). In Figures 9B-9G, the symbolic nomenclature of glycans follows Varki, A., Cummings, RD, Esko JD, et al., Essentials of Glycobiology, 2nd edition (2009).

[0123] As can be seen from Figures 9A-9G, the two analyses showed similar results, although there were some differences between the results. This variation could be due to many factors, including the equipment used and the completeness of the N-glycan analysis. For example, if some species of phosphorylated glycans are not identified and / or quantified, the total number of phosphorylated glycans could be underestimated and the percentage of rhGAA with phosphorylated glycans at that site could be underestimated. As another example, if some species of non-phosphorylated glycans are not identified and / or quantified, the total number of non-phosphorylated glycans could be overestimated and the percentage of rhGAA with phosphorylated glycans at that site could be overestimated. Figure 9A shows the N-glycosylation site occupancy of ATB200. As can be seen from Figure 9A, the first, second, third, fourth, fifth and sixth N-glycosylation sites are mostly occupied by both assays, with more than 90% and up to about 100% of the ATB200 enzymes having glycans detected at each potential site. However, the seventh potential N-glycosylation site is glycosylated about half the time.

[0124] Figure 9B shows the N-glycosylation profile of the first site, N84. As can be seen from Figure 9B, the major glycan species is bis-M6P glycan. In both the first and second analyses, it was detected that more than 75% of ATB200 had bis-M6P glycan at the first site.

[0125] Figure 9C shows the N-glycosylation profile of the second site, N177. As can be seen from Figure 9C, the major glycan species are mono-M6P glycan and non-phosphorylated high mannose glycan. In both the first and second analyses, it was detected that more than 40% of ATB200 had mono-M6P glycan at the second site.

[0126] Figure 9D shows the N-glycosylation profile of the third site, N334. As can be seen from Figure 9D, the major glycan species are non-phosphorylated high mannose glycans, di-, tri-, and tetra-antennary complex glycans, and hybrid glycans. In both the first and second analyses, it was detected that more than 20% of ATB200 had sialic acid residues at the third site.

[0127] Figure 9E shows the N-glycosylation profile of the fourth site, N414. As can be seen from Figure 9E, the major glycan species are bis-M6P and mono-MGP glycans. In both the first and second analyses, it was detected that more than 40% of ATB200 had bis-M6P glycan at the fourth site. In both the first and second analyses, it was also detected that more than 25% of ATB200 had mono-M6P glycan at the fourth site.

[0128] Figure 9F shows the N-glycosylation profile of the fifth site, N596. As can be seen from Figure 9F, the major glycan species is a fucosylated di-antennary complex glycan. In both the first and second analyses, it was detected that more than 70% of ATB200 had a sialic acid residue at the fifth site.

[0129] Figure 9G shows the N-glycosylation profile of the sixth site, N826. As can be seen from Figure 9F, the major glycan species are di-, tri-, and tetra-antennary complex glycans. In both the first and second analyses, it was detected that more than 80% of ATB200 had sialic acid residues at the sixth site.

[0130] Analysis of glycosylation at the seventh site, N869, showed approximately 40% glycosylation, with the most common glycans being A4S3S3GF (12%), A5S3G2F (10%), A4S2G2F (8%) and A6S3G3F (8%).

[0131] Figure 9H shows a summary of phosphorylation at each of the seven potential N-glycosylation sites. As can be seen from Figure 9G, both the first and second analyses detected high phosphorylation levels at the first, second and fourth sites. Both analyses detected that 80% or more of ATB200 was mono- or di-phosphorylated at the first site, 40% or more of ATB200 was mono-phosphorylated at the second site, and 80% or more of ATB200 was mono- or di-phosphorylated at the fourth site.

[0132] Another glycan analysis of ATB200 was performed according to hydrophilic interaction liquid chromatography-fluorescence detection-mass spectrometry (HILIC-FLD-MS) method.

[0133] The results of the HILIC-FLD-MS analysis are shown in Table A below. In Table A, the first number of the three-digit number indicates the number of branches of the glycan, the second number indicates the number of core fucose units, and the third number indicates the number of terminal sialic acid units. Using this nomenclature, "303" represents a triantennary glycan (first 3) with 0 core fucose (second 0) and 3 terminal sialic acids (last 3), "212" represents a biantennary glycan with 1 core fucose and 2 terminal sialic acids, "404" represents a tetraantennary glycan containing 0 core fucose and 4 terminal sialic acids, and so on.

[0134] JPEG2025060645000002.jpg224164

[0135] JPEG2025060645000003.jpg224164

[0136] JPEG2025060645000004.jpg84164

[0137] Based on this HILIC-FLD-MS analysis, the ATB200 tested is expected to have an average fucose content of 2-3 moles per mole ATB200, a GlcNAc content of 20-25 moles per mole ATB200, a galactose content of 8-12 moles per mole ATB200, a mannose content of 22-27 moles per mole ATB200, an M6P content of 3-5 moles per mole ATB200, and a sialic acid content of 4-7 moles per mole ATB200.

[0138] Enzyme Example 6: CIMPR affinity characteristics of ATB200 In addition to the higher percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of the interaction. Lumizyme® and ATB200 rhGAA receptor binding was measured using CIMPR plate binding assay. Briefly, GAA was captured using a CIMPR-coated plate. Various concentrations of rhGAA were applied to the immobilized receptor, and unbound rhGAA was washed away. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 10A, ATB200 rhGAA bound to CIMPR significantly better than Lumizyme®.

[0139] 10B shows the relative content of bis-M6P glycans in Lumizyme®, conventional rhGAA, and ATB200 according to the present invention. In Lumizyme®, on average, only 10% of the molecules have bis-phosphorylated glycans. This is in contrast to ATB200, where on average, all rhGAA molecules have at least one bis-phosphorylated glycan.

[0140] Enzyme Example 7: ATB200 rhGAA was internalized more efficiently by fibroblasts than Lumizyme® The relative cellular uptake of ATB200 and Lumizyme® rhGAA was compared using normal and Pompe fibroblast cell lines. Comparisons included 5-100 nM ATB200 rhGAA according to the invention versus 10-500 nM conventional rhGAA Lumizyme®. After 16 hours of incubation, external rhGAA was inactivated with TRIS base and cells were washed three times with PBS before harvesting. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and graphed against total cellular protein, the results of which are presented in Figure 11A-B.

[0141] ATB200 rhGAA was also shown to be efficiently internalized into cells (FIGS. 11A and 11B), respectively, demonstrating that ATB200 rhGAA was internalized into both normal and Pompe fibroblasts and was more highly internalized than conventional Lumizyme® rhGAA. ATB200 rhGAA saturates cell receptors at approximately 20 nM, whereas Lumizyme® requires approximately 250 nM. The uptake efficiency constant (K 取り込み ) is 2-3 nm for ATB200 and 56 nM for Lumizyme®, as shown in Figure 11C. These results suggest that ATB200 rhGAA is a well-targeted treatment for Pompe disease.

[0142] Enzyme example 8: Glycogen reduction in Gaa knockout mice 12A-12C show the effect of administration of alglucosidase alfa (Lumizyme®) and ATB200 in Gaa knockout mice. Animals received two IV bolus doses (every other week). Tissues were collected two weeks after the final dose and analyzed for acid α-glucosidase activity and glycogen content.

[0143] As can be seen from Figures 12A-12C, ATB200 was found to deplete tissue glycogen in acid alpha-glucosidase (Gaa) knockout mice in a dose-dependent manner. A 20 mg / kg dose of ATB200 consistently cleared glycogen stores at a greater rate than the 5 mg / kg and 10 mg / kg dose levels in Gaa knockout mice. However, as seen in Figures 12A-12C, ATB200 administered at 5 mg / kg showed a similar reduction in glycogen in mouse heart and skeletal muscle (quadriceps and triceps) as Lumizyme® administered at 20 mg / kg, while ATB200 administered at 10 and 20 mg / kg showed a significantly better reduction in glycogen levels in skeletal muscle than Lumizyme®.

[0144] FIG. 15 shows the effect of administration of alglucosidase alfa (Lumizyme®) and ATB200 in Gaa knockout mice. 12 week old GAA KO mice were injected with Lumizyme® or ATB200, 20 mg / kg IV every 4 weeks. Tissues were collected 14 days after the last enzyme dose for glycogen measurements. FIG. 13 shows the relative reduction of glycogen in quadriceps and triceps skeletal muscles, with ATB200 producing a greater reduction of glycogen than Lumizyme®.

[0145] Enzyme Example 9: Muscle Physiology and Morphology in Gaa Knockout Mice Gaa knockout mice were given two IV boluses of 20 mg / kg recombinant human acid α-glucosidase (alglucosidase alfa or ATB200) every other week. Control mice were treated with vehicle only. Soleus, quadriceps and septum tissues were collected 2 weeks after the last dose of recombinant human acid α-glucosidase. Soleus and septum tissues were analyzed by staining with periodic acid-Schiff reagent (PAS) for glycogen levels and by measuring the levels of the lysosomal-associated membrane protein upregulated in Pompe disease (LAMP1) marker for lysosomal proliferation. Semi-thin sections of quadriceps embedded in epoxy resin (Epon) were stained with methylene blue and viewed under an electron microscope (1000×) to determine the extent of the presence of vacuoles. Quadriceps muscle samples were analyzed immunohistochemically to determine the levels of the autophagy markers microtubule-associated protein type 1A / 1B light chain 3 phosphatidylethanolamine conjugate (LC3A II) and p62, the insulin-dependent glucose transporter GLUT4, and the insulin-independent glucose transporter GLUT1.

[0146] In a similar experiment, Gaa knockout mice were given four IV boluses of 20 mg / kg recombinant human acid α-glucosidase (alglucosidase alfa or ATB200) every other week. Control mice were treated with vehicle only. Cardiac muscle tissue was harvested 2 weeks after the last dose of recombinant human acid α-glucosidase and analyzed for glycogen levels by staining with periodic acid-Schiff reagent (PAS) and for lysosomal proliferation by measuring the levels of LAMP1.

[0147] As seen in Figure 14, administration of ATB200 showed a reduction in lysosomal proliferation in heart and skeletal muscle (soleus) tissues compared to conventional treatment with alglucosidase alpha. Furthermore, as seen in Figure 15, administration of ATB200 showed a reduction in punctate glycogen levels in heart and skeletal muscle (soleus) tissues compared to conventional treatment with alglucosidase alpha.

[0148] Similarly, as seen in FIG. 16, ATB200 significantly reduced the number of vacuoles in muscle fibers in the quadriceps of Gaa knockout mice compared to untreated mice and mice treated with alglucosidase alpha. As seen in FIG. 17, the levels of both LC3II and p62 are increased in Gaa knockout mice compared to wild-type mice. Furthermore, the levels of insulin-dependent glucose transporter GLUT4 and insulin-independent glucose transporter GLUT1 are increased in Gaa knockout mice compared to wild-type mice. The elevated GLUT4 and GLUT1 levels associated with acid alpha-glucosidase deficiency may contribute to increased glucose uptake into muscle fibers, resulting in increased glycogen synthesis both basally and after food intake.

[0149] Enzyme Example 10: Muscle function in Gaa knockout mice In a long-term study of 12 biweekly doses, 20 mg / kg ATB200 + 10 mg / kg miglustat progressively increased functional muscle strength in GaaKO mice from baseline as measured by both grip strength and wire hang test (Figures 18A-18B). Alglucosidase alfa (Lumizyme®)-treated mice receiving the same ERT dose (20 mg / kg) were observed to decline under identical conditions throughout the majority of the study (Figures 18A-18B). Similar to the short-term study, ATB200 / miglustat had substantially better glycogen clearance after 3 months (Figures 19A-19C) and 6 months (Figures 19D-19G) of treatment than alglucosidase alfa. ATB200 / miglustat also reduced autophagy and intracellular accumulation of LAMP1 and dysferlin after 3 months of treatment compared to alglucosidase alfa (Figure 20). In Figure 18A, * indicates statistical significance compared to Lumizyme alone (p<0.05, two-tailed t-test). In Figures 19A-19G, * indicates statistical significance compared to Lumizyme® alone (p<0.05, one-way ANOVA analysis using Dunnett's multiple comparisons method).

[0150] Taken together, these data indicate that ATB200 / miglustat efficiently targeted muscle, reversed cell dysfunction, and improved muscle function. Importantly, the obvious improvement in muscle structure and the reduction in autophagy and intracellular accumulation of LAMP1 and dysferlin may be a good surrogate for improved muscle physiology that correlates with improved functional muscle strength. These results suggest that monitoring autophagy and these important muscle proteins is a rational and practical method to evaluate the effect of therapeutic treatments for Pompe disease in GaaKO mice, which may prove to be useful biomarkers from muscle biopsies in clinical trials.

[0151] Figure 20 shows that 6 months of ATB200 administration with or without miglustat reduces intracellular accumulation of dystrophin in GaaKO mice. ATB200 ± miglustat reduced dystrophin accumulation to a greater extent than Lumizyme®.

[0152] Formulation Example 1: pH and Buffers Analytical methods for formulation examples Analyses of the examples described herein for appearance, pH, protein concentration, etc. were performed according to the following methods unless otherwise stated.

[0153] exterior The appearance of the samples, including transparency, color and visible particles, was examined under a black and white background using a YB-2 light box.

[0154] pH The pH of the samples was measured with a SevenMulti™ pH meter.

[0155] Protein concentration Protein concentrations were determined by UV280 readings using a NanoDrop™ 2000 spectrophotometer. All measurements were repeated twice with 2.5 μL samples each time and averaged.

[0156] Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) For examples of pH and buffers, freeze-thaw and excipients, separation of protein monomers and their high molecular weight species and fragments was performed on an Agilent 1260 HPLC system using a TSKgel® G3000 SWXL column (Tosoh Bioscience, 7.8×300 mm, 5 μm, 25° C.). The mobile phase consisted of 50 mM sodium phosphate, 100 mM sodium chloride and 20 mM sodium citrate (pH 6.0±0.2). The chromatographic system used a flow rate of 1.0 mL / min, an injection volume of 50 μL (typically 1 mg / mL) and a run time of 20 min with an isocratic gradient. Signal was detected by a UV detector at 280 nm (reference: 360 nm).

[0157] In the PS80 example, separation of protein monomers and their high molecular weight species and fragments was performed on an Agilent 1260 HPLC system using a BioSepTM-SEC-s3000 column (Pennomenex, 7.8 x 300 mm, 5 μm, 25 °C). The mobile phase consisted of 50 mM sodium phosphate and 100 mM sodium chloride (pH 6.2 ± 0.2). The chromatographic system used a flow rate of 1.15 mL / min, an injection volume of 50 μL (typically 1 mg / mL), and a run time of 25 min with an isocratic gradient. Signal was detected at 280 nm by a UV detector.

[0158] SDS-PAGE (non-reducing) Reportable values ​​are the purity and molecular weight of proteins in non-reducing SDS-PAGE. Samples were denatured under non-reducing conditions in the presence of excess SDS to obtain a uniform negative charge. In an applied electric field (165 V), these SDS-coated species were separated on the basis of apparent molecular weight through a polyacrylamide gel. Separated bands were detected by Coomassie blue staining.

[0159] 4-MUG enzyme activity 10 μL of the sample was diluted and hydrolyzed (GAA for 60 min at 37° C.) to produce the fluorescent product 4-MU. The reaction was stopped by adding 125 μL of 1 M glycine or 0.1 M NaOH.

[0160] A series of 4-MU standards were analyzed with the samples, and a standard calibration curve was generated based on the fluorescence signal. Conversion of RFU to 4-MU amounts was achieved by software-mediated comparison with a standard curve regressed according to a four-parameter logistic regression model. GAA enzyme activity (nmol 4-MU released / hr / ml GAA) in the samples was then calculated based on the 4-MU standard curve.

[0161] 4-MUG enzyme concentration 10 μL of sample and GAA reference standard were diluted and hydrolyzed to generate the fluorescent product 4-MU (GAA for 60 min at 37° C.) The reaction was stopped by adding 125 μL of 1 M NaOH.

[0162] A series of GAA reference standards were analyzed with the samples, and a standard calibration curve was generated based on the fluorescence signal. Conversion of RFU to 4-MU amounts was achieved by software-mediated comparison with a standard curve that was regressed according to a four-parameter logistic regression model. The GAA enzyme concentration (nanomoles 4-MU released / hr / ml GAA) in the samples was then calculated based on the GAA standard curve.

[0163] Dynamic Light Scattering (DLS) Using a micropipette, 40 μL aliquots of undiluted sample were transferred into 40 μL disposable cuvettes. Triplicate measurements were performed for each sample.

[0164] Particle:HIAC 200 μL of each sample was diluted to 2000 μL with filtered reference buffer. Samples were tested in triplicate, with 450 μL used for each test. The average number of particles of 1 μm, 3 μm, 5 μm, 10 μm and 25 μm size per mL was reported.

[0165] MicroCal Differential Scanning Calorimetry (DSC) Capillary cell differential scanning calorimetry (DSC) is used to measure the thermal stability of proteins by detecting the difference in the amount of heat required to raise the temperature of a sample and the baseline as a function of temperature. Specifically, it is used to measure the thermal transition midpoint (Tm), which is an indicator of the relative stability of a protein in solution.

[0166] Samples were diluted to approximately 1 mg / mL in reference buffer. An aliquot of 400 μL of reference buffer was added to each odd-numbered well of a 96-well plate, and an aliquot of 400 μL of each sample was added to the corresponding even-numbered well. Scanning temperatures ranged from 10 °C to 110 °C.

[0167] Modulated Differential Scanning Calorimetry (mDSC) The glass transition temperature (Tg') and eutectic temperature (Te) were tested using a Netzsch differential scanning calorimeter (DSC204 F1). 15 μL of sample was loaded into a loading disk for testing. First, the temperature was lowered from 20°C to -60°C at a rate of 10°C / min, and the Te value was obtained from the cooling curve in this step. Second, the temperature was increased from -60°C to 40°C at a rate of 10°C / min, and the Tg' value was analyzed from the heating curve.

[0168] M6P M6P was liberated from the samples by hydrolysis (4M TFA, 100°C, 4 h) and dried in a centrifugal vacuum evaporator. Dried M6P and reference standards were suspended in purified water before analysis. A CarboPac PA10 BioLCTM analytical column (4mm x 250mm, 3.5μm, 100Å, 30°C) and a CarboPac PA10 BioLCGuard column (4mm x 50mm, 3.5μm, 100Å, 30°C) were used. The mobile phase consisted of phase A (100mM NaOH) and phase B (1M NaOAc, 100mM NaOH). The chromatographic system used a flow rate of 1mL / min, an injection volume of 25μL, and a run time of 30 minutes with a gradient. The signal was detected by pulsed amperometric detection. The M6P content in the samples was calculated based on the standard curve.

[0169] Sialic acid Sialic acid was released from the drug molecule by hydrolysis (2M HAc, 80°C, 2 h), then all samples and mixed standard solutions were labeled with DMB (50°C, 17±0.5 h in the dark) and separated on an Agilent 1260 HPLC system using a Zorbax Eclipse Plus C18 column (Agilent, 4.6 mm×100 mm, 3.5 μm, 45°C). The mobile phase consisted of phase A (9% ACN, 7% MeOH) and phase B (100% ACN). The chromatographic system used a flow rate of 0.5 mL / min, an injection volume of 20 μL, and a run time of 20 min with a gradient. The signal was detected by a fluorescence detector (λex=373 nm, λem=448 nm). The Neu5Gc and Neu5Ac contents in the samples were calculated based on the standard curve.

[0170] Ten buffer formulations were prepared with a pH ranging from 4.0 to 8.0, containing 25 mM sodium phosphate or 25 mM sodium citrate with or without 50 mM NaCl, as shown in Table 1. The ATB200 4-MUG enzyme concentration was 1 mg / mL.

[0171] JPEG2025060645000005.jpg83164

[0172] Sample preparation The materials used in the pH and buffer evaluation experiments are as listed in Table 2 below.

[0173] JPEG2025060645000006.jpg49164

[0174] 25 mM sodium phosphate buffers of pH 4.0 (P40), 5.0 (P50), 6.0 (P60), 7.0 (P70) and 8.0 (P80), 25 mM sodium phosphate buffer of pH 6.0 (P60 without NaCl), 25 mM sodium citrate buffer with 50 mM NaCl of pH 5.0 (C50), 5.5 (C55), 6.0 (C60) and 6.5 (C65) were prepared. For pH 4.0, the pH in the sodium phosphate buffer (pH 4.0) was adjusted using HCl.

[0175] The ATB200 enzyme solution was first concentrated using an ultrafiltration centrifuge at 15°C and 3500 rpm for 40 min. The concentrated enzyme solution was then buffer-exchanged into the three different buffers mentioned above by two rounds of ultrafiltration at 15°C and 3500 rpm for 50 and 55 min. The buffer-exchanged enzyme solution was then analyzed for protein concentration and 4-MUG enzyme concentration.

[0176] Finally, an appropriate volume of each buffer was added to adjust the final ATB200 enzyme concentration to 1.0 mg / mL, which was confirmed by both UV_A280 absorbance and 4-MUG enzyme concentration.

[0177] The solution was sterile filtered through a 0.22 μm polyethersulfone (PES) filter.

[0178] Each formulation was aseptically filled into 2 mL glass vials in a biosafety hood with fill volumes ranging from 500 μL to 1000 μL according to the sample volume requirements of the analytical study. The vials were stoppered and immediately crimp-oversealed after filling.

[0179] Sample Testing Vials of each formulation were stored at 5° C. and 40° C. for up to 8 weeks and agitated on a rotary shaker at 100 rpm at 25° C. for up to 5 days (see Table 3). Formulations were sampled initially (T0), at 5 days (5D), 2 weeks (2W), 4 weeks (4W) and 8 weeks (8W) for tests of appearance, pH, UV concentration, 4-MUG enzyme concentration, SEC, HIAC, DLS, 4-MUG enzyme activity and DSC as described in Table 3.

[0180] JPEG2025060645000007.jpg60164

[0181] result Thermal Stability Results – MicroCal DSC The results of the thermal stability measurements are shown in Table 4 below.

[0182] JPEG2025060645000008.jpg88164

[0183] Higher Tm 発症 indicates better thermal stability of the protein in a particular formulation. Thus, the formulations showing the best thermal stability were P50, C55, C50, P60 without NaCl, P60 and C60. These results indicate that the ATB200 enzyme has better thermal stability in weak acidic buffers than in basic conditions.

[0184] Appearance - Stirring The visual results of the stirring experiment are shown in Table 5 below:

[0185] JPEG2025060645000009.jpg121164

[0186] As can be seen, after 5 days of stirring, P60, C50, C55, C60, and C65 without NaCl remained colorless, clear, and free of visible particles, while visible particles were observed in P40, P50, P60, P70, and P80. This data indicates that sodium citrate buffer stabilizes the formulation better than sodium phosphate buffer after stirring.

[0187] pH The results of the pH measurements are shown in Table 6 below:

[0188] JPEG2025060645000010.jpg104164

[0189] As can be seen from the data, both phosphate and citrate buffers with 50 mM NaCl in the pH range of 5.0-6.5 were able to maintain the desired pH throughout. There was no significant change in the pH of the samples during buffer exchange, concentration adjustment, storage for 8 weeks at 5°C and 40°C, and agitation.

[0190] In contrast, pH decreased in P60 without NaCl after 8 weeks of storage at both 5°C and 40°C, and pH increased in P40 after buffer exchange and 8 weeks of storage at both temperatures. However, agitation did not result in a change in pH in both P40 and P60 without NaCl.

[0191] Protein concentration The results of the protein concentration determination are shown in Table 7 below:

[0192] JPEG2025060645000011.jpg104164

[0193] Storage at 5° C. for 8 weeks and stirring for 5 days did not affect the protein concentration. No significant changes were observed between all formulations.

[0194] In contrast, during storage at 40°C, protein concentrations increased slightly for P50, P60, P60 without NaCl, C60, and C65, decreased slightly for C50, and were maintained for P40, P70, P80, and C55. Visible particles formed in the 40°C samples, so the 40°C / 8 week samples were retested after centrifugation at 12000 rpm for 1 min. The results showed a decrease in protein concentration after centrifugation in samples containing particles, suggesting that particles present in the samples have an effect on absorbance at 280 nm.

[0195] 4-MUG enzyme concentration The results of the 4-MUG enzyme concentration measurements are shown in Table 8 below.

[0196] JPEG2025060645000012.jpg104164

[0197] After buffer exchange, the 4-MUG enzyme concentration of P80 immediately decreased to 0.27 mg / mL, indicating that pH 8.0 significantly affected the enzyme activity. After 2 weeks of storage at both 5°C and 40°C, the enzyme concentration decreased to zero. After 8 weeks of storage at 5°C, the enzyme concentrations of most formulations were stable, except for P80, which decreased slightly in P40.

[0198] In contrast, storage at 40 °C clearly affected the enzyme concentration. After 2 weeks, the enzyme concentration disappeared in P70, dropped dramatically in P40 and C65, and was obviously decreased in P50. After 4 weeks, the enzyme concentration continued to decrease. Finally, after 8 weeks, the enzyme concentration was close to 0 in P40 and C65, decreased to 0.33 mg / mL in pH 5.0 buffers (P50 and C50), and decreased to 0.5-0.7 mg / mL in pH 5.5-6.0 buffers (P60, P60 without NaCl, C55, and C60). Among them, P60 without NaCl finally had the highest enzyme concentration (0.73 mg / mL). The results indicate that the enzyme concentration was best preserved in terms of 4-MUG enzyme concentration in the pH 5.5-6.0 range, but there was no significant difference between sodium phosphate buffer and sodium citrate buffer.

[0199] Except for P80, enzyme concentrations did not change significantly during the agitation experiments.

[0200] 4-MUG enzyme activity The results of the 4-MUG enzyme activity measurements are shown in Table 9 below:

[0201] JPEG2025060645000013.jpg97164

[0202] The change trend of 4-MUG enzyme activity corresponded to the change trend of 4-MUG enzyme concentration.

[0203] P80 showed the worst stability in 4-MUG enzyme activity under the test conditions. The enzyme activity of P80 showed about 70% decrease after buffer exchange. Then, the enzyme activity was almost completely lost after 2 weeks storage at 5°C and 40°C. The basal conditions significantly affected the enzyme activity.

[0204] After 8 weeks of storage at 5°C, the enzyme activity of PS80 was almost completely lost, a 20% decrease was observed in P40, and no significant changes were observed in the other formulations.

[0205] In contrast, storage at 40°C caused a clear decrease in enzyme activity in all formulations. After 2 weeks, enzyme activity was almost completely lost in P70, dramatically decreased in P40 and C65, and clearly decreased in P50. Enzyme activity continued to decrease to different degrees from 2 to 4 weeks. At the end of the experiment, enzyme activity was almost completely lost in C65, dropped to 211.5 U / L in P40, about 1550 U / L in P50 and C50, and 2500-3000 U / L in C60, P60, and C55. Of the formulations, P60 without NaCl maintained the highest activity of 3549.7 U / L.

[0206] Based on the results of the 4-MUG enzyme activity test, the enzyme was most stable at pH in the range of 5.5 to 6.0, with no distinction being observed between sodium phosphate and sodium citrate buffers.

[0207] Purity: SEC-HPLC The results of the SEC measurements are shown in Table 10 below.

[0208] JPEG2025060645000014.jpg196164

[0209] After buffer exchange, the SEC purity of several formulations was significantly decreased compared to the starting material (SEC monomer: 98.9%). For P80, the SEC purity dropped to 44.9% with 54.5% aggregates formed; for P40, the monomer% decreased by 4.9% compared to the pre-exchange DS and more LMW fragments were formed than HMW molecules (4.7% vs. 1.3%); for P70, a slight decrease in monomer% (1.2%) was found, corresponding to an increase in aggregates; for P60 without NaCl, the monomer% decreased by 2.3%.

[0210] After 8 weeks of storage at 5 °C, the SEC purity of formulations P60, P60 without NaCl, and C65 was well maintained, whereas the SEC purity of the other formulations was significantly decreased compared to T0. For P40, the SEC purity dramatically decreased to 74.3% (T0: 94.0%) with the formation of 23.4% LMW fragments; for P50 and C50, there was a slight decrease in monomer (5-7%) and an increase in LMW fragments (5-7%). For P80, a decrease of 18.2% was observed, which was mainly shifted towards aggregates (14.8%). Similarly for P70, there was a decrease of 10.7% in monomer and an increase of 9.2% in HMW fragments. For C55, the proportions of monomer, HMW and LMW were slightly changed.

[0211] Storage at 40°C resulted in dramatic changes in monomer% in all formulations. For P70 and P80, SEC monomer dropped to 0-0.5% after 2 weeks, while for C65 22.4% remained, mostly in aggregates after 8 weeks. For P40, P50 and C50, there was a significant drop in monomer (50-90%), mainly due to the formation of LMW fragments. For P60, P60 without NaCl, and C60, SEC monomer% decreased to 80.8%, 83.6% and 77.5%, respectively, after 8 weeks.

[0212] The SEC results showed that pH 6.0 worked best for the stability of ATB200, and no significant difference was found between sodium phosphate and sodium citrate buffers. Furthermore, the absence of sodium chloride in the formulation did not affect the stability of ATB200.

[0213] During the stirring experiments, the SEC purity slightly decreased in P60 and P60 without NaCl. The monomer % decreased obviously in P40, P50, P70, P80, C50, C60 and C65.

[0214] Polydispersity: DLS The results of the DLS measurements are shown in Table 11 below.

[0215] JPEG2025060645000015.jpg132164

[0216] JPEG2025060645000016.jpg163164

[0217] JPEG2025060645000017.jpg164164

[0218] The DLS data reflected the hydrodynamic radius of the protein molecules and the polydispersity of the particles. Since a milky color was observed in some samples, DLS was used to analyze the subvisible particles. The DLS results were generally consistent with what the appearance suggested.

[0219] During 8 weeks of storage at 5°C, the hydrodynamic radius and polydispersity index (PDI) of the protein molecules were stable and comparable for P60, P70, P80, P60 without NaCl, and C60. The hydrodynamic radius varied from approximately 10 nm to several hundred nm for all other five formulations, especially P40 after 8 weeks.

[0220] During 8 weeks of storage at 40°C, there were dramatic changes in hydrodynamic radius and PDI in all formulations, however, it is difficult to compare the formulations on the basis of results due to the complex profiles of aggregation.

[0221] In the stirring experiments, the hydrodynamic radius and PDI of P80 increased dramatically; for P40, P50, and C50, the hydrodynamic radius and PDI increased slightly; and no significant changes were observed for P60, P70, P60 without NaCl, C55, C60, and C65.

[0222] According to the above DLS data, P60, P70, P60 without NaCl, C55, C60, and C65 were better than P40, P50, P80, and C50.

[0223] Particle:HIAC The results of the HIAC measurements of the stirring experiments are shown in Table 12 below:

[0224] JPEG2025060645000018.jpg188164

[0225] During the stirring experiment, P70 showed a significant increase in particle count after stirring for 5 days at 25°C, while all other formulations had comparable particle counts.

[0226] overview Overall, P60 and C60 were superior as the most stable formulations compared to the other formulations. Therefore, it was concluded that ATB200 was the most stable in the range of 5.0-6.0. However, no distinction could be made between phosphate and citrate buffers. Furthermore, the absence of sodium chloride in the formulation showed no significant effect on the stability of ATB200.

[0227] Formulation example 2: Freezing and thawing Three formulations were evaluated for stability during the freeze-thaw process. The three formulations are summarized below in Table 13. The target concentration of ATB200 enzyme was 5 mg / mL.

[0228] JPEG2025060645000019.jpg55164

[0229] Sample preparation ATB200 DS was buffer exchanged into three formulation buffers using dialysis cassettes (20000 MWCO). Dialysis was performed at 5°C with gentle agitation, once every 6-10 hours, with three buffer exchanges each.

[0230] After each dialysis, an appropriate volume of formulation buffer was added to adjust the final UV concentration to 5 mg / mL. The solutions were then sterile filtered through 0.22 μm PES filters. Each formulation was then aseptically filled into 2 mL glass vials in a biosafety hood with fill volumes ranging from 500 μL to 1000 μL according to the sample volume requirements of the analytical study. The vials were stoppered and immediately crimp-over-sealed after filling.

[0231] Prior to freeze-thaw, a vial was taken for each formulation and the remaining sample vials were subjected to a freeze-thaw cycle. Post-freeze-thaw sample vials were taken at pre-determined sampling points.

[0232] Sample Testing Three freeze-thaw processes were tested as follows:

[0233] Process 1: Uncontrolled freezing and thawing. Samples were frozen in a -80°C freezer and thawed in a 25°C chamber.

[0234] Process 2: Controlled freezing and uncontrolled thawing. The samples were placed in a Frosty container and frozen in a -80°C freezer. The Frosty container used isopropanol to achieve a controlled freezing rate of 1°C / min. The frozen container was placed in a 25°C chamber to thaw the samples. The temperature increase rate was approximately 1°C / min.

[0235] Process 3: Controlled freezing and thawing. The samples were frozen and thawed using a freeze dryer. The minimum sample temperature achieved during freezing was -47°C. The sample temperature was increased to 25°C. The temperature ramp rate for both freezing and thawing was controlled at approximately 0.5°C / min. The experiment was repeated to confirm the results.

[0236] Five or three freeze-thaw cycles were performed with each process. The following tests were performed on pre- and post-freeze-thaw samples: appearance, concentration (UV and enzyme) and SEC-HPLC. A summary of the test parameters is provided in Table 14 below.

[0237] JPEG2025060645000020.jpg70164

[0238] result Appearance - Round 1 The results of the first round of appearance measurements are shown below in Tables 15A and 15B:

[0239] JPEG2025060645000021.jpg73164

[0240] JPEG2025060645000022.jpg72164

[0241] At TO (before freeze-thaw), all formulations appeared colorless, slightly opalescent, and free of visible particles relative to the corresponding formulation buffer.

[0242] After five rapid freeze-thaw cycles (Process 1), all formulations appeared to contain more visible particles compared to their TO. CP60 contained the fewest particles of the three after 5FT.

[0243] After five cycles of slow freeze-thaw (Process 2), all formulations appeared to contain more visible particles than T0, but fewer than those processed with Process 1. Fewer particles were observed in the CP60 sample after 5 FT cycles than the P60 and C60 samples after 1 FT cycle.

[0244] After 3 cycles of slow freeze-thaw (Process 3), all formulations appeared to have more visible particles than TO. There were no differences among the three formulations.

[0245] Appearance - Round 2 The results of the second round of appearance measurements are shown in Table 23 below:

[0246] JPEG2025060645000023.jpg113164

[0247] In the second round of slow freeze-thaw testing (Process 3), more FT cycles were performed than in the first round. A large number of visible particles appeared in P60 after 5FT compared to T0, whereas a small number of particles were observed in both C60 and CP60.

[0248] 4-MUG enzyme concentration and activity The results of the 4-MUG enzyme concentration and activity measurements are shown below in Table 17 (Processes 1-2 and Process 3, first round) and Table 18 (Process 3, second round).

[0249] JPEG2025060645000024.jpg59164

[0250] JPEG2025060645000025.jpg75164

[0251] After five cycles of rapid freeze-thaw (Process 1), the enzyme concentration of 4-MUG was slightly decreased in P60 compared to the T0 sample, but no significant difference was observed in C60 and CP60.

[0252] After five cycles of slow freeze-thaw using Frosty containers (Process 2), no significant differences were observed in any of the three formulations compared to the TO samples.

[0253] After three cycles of slow freeze-thaw using a freeze dryer (Process 3), there was a clear decrease in enzyme concentration in both P60 and CP60 compared to T0, but no significant change in C60. The freeze-thaw process 3 was repeated for five cycles, and the same trend was observed. The enzyme concentration of the P60 sample decreased by 18.8% after one cycle of F / T, and further deteriorated after three cycles (53.1% decrease) and five cycles (68.9% decrease). For CP60, the enzyme concentration began to decrease after three cycles, eventually decreasing to the same level as P60 after five cycles. For C60, there was little change after five cycles of slow freeze-thaw.

[0254] The results of 4-MUG enzyme concentration showed that the freezing / heating rate, number of F / T cycles and buffer type could affect the stability of ATB200. The citrate buffer system (C60) provided a good stabilizing effect regardless of the freeze-thaw process used.

[0255] The change trend of 4-MUG enzyme activity was the same as that of 4-MUG enzyme concentration.

[0256] During the rapid freeze-thaw test (Process 1), a slight decrease in enzyme activity was observed in P60 after five cycles, whereas no significant changes were observed in C60 and CP60.

[0257] During slow freeze-thaw testing using a temperature change of 1° C. / min (Process 2), no obvious changes were observed in any of the samples.

[0258] In the slow freeze-thaw test using a freeze dryer (Process 3), the enzyme activity of P60 obviously decreased, and that of CP60 decreased slightly. However, there was almost no change in C60. In the second round of the freeze-thaw experiment in Process 3, the enzyme activity of P60 decreased by 19.1% after one cycle, 54.1% after three cycles, and 71.2% after five cycles. In CP60, the enzyme activity began to decrease after three cycles, and decreased to a level similar to that of P60 after five cycles. The change in C60 after five cycles was negligible.

[0259] Purity: SEC-HPLC The results of the purity measurements are shown below in Table 20 (Processes 1-2 and Process 3, first round) and Table 21 (Process 3, second round).

[0260] JPEG2025060645000026.jpg86164

[0261] JPEG2025060645000027.jpg72164

[0262] No change in SEC % monomer was detected in any sample after 5 cycles of fast F / T (Process 1) or slow F / T (Process 2).

[0263] In the first slow F / T test (Process 3), the P60 sample showed a clear SEC monomer % decrease (mainly due to the formation of HMW species) after 3 cycles, while no clear change occurred for C60 and CP60. In the second slow F / T test (0.5 °C / min) with Process 3, the monomer in the P60 sample started to decrease after 1 cycle and finally decreased by 68.1% after 5 cycles. And for CP60, the monomer % started to decrease after 3 cycles, but to a much lesser extent than P60, but reached the same level as P60 after 5 cycles. There was no change in the monomer percentage in C60 after up to 5 cycles.

[0264] The SEC purity results were consistent with the performance in enzyme concentration and activity, confirming that the freezing / heating rate, number of F / T cycles and buffer type can affect ATB200 stability during the freeze-thaw process. Buffers containing sodium phosphate also did not protect ATB200 during freeze-thaw cycles based on the SEC results.

[0265] overview ATB200 formulated in citrate buffer (C60) survived multiple freeze-thaw cycles better than the other two buffers (P60 and CP60). Regardless of the freeze-thaw process, ATB200 remained stable in citrate buffer.

[0266] Formulation Example 3: Excipients Eight formulations (E1-8) were prepared with various excipients. The ATB200 enzyme concentration in the excipient evaluation test was 5mg / mL. Three buffers, two stabilizers and one surfactant were selected to evaluate protein stability in the formulations as shown in Table 22 below.

[0267] JPEG2025060645000028.jpg67164

[0268] Sample preparation A 25 mM sodium phosphate buffer containing 50 mM NaCl at pH 6.0, a 25 mM sodium citrate buffer containing 50 mM NaCl at pH 6.0, and a 25 mM sodium phosphate-citrate combination buffer containing 50 mM NaCl at pH 6.0 were prepared separately. The ATB200 enzyme solution was exchanged into the three buffers using a dialysis cassette. Dialysis was performed at 5°C with gentle stirring, with three buffer exchanges each, one every 6 to 10 hours.

[0269] After dialysis, trehalose, mannitol or PS80 was added to the dialysate to prepare the formulations shown in Table 22. Finally, an appropriate amount of formulation buffer was added to adjust the final ATB200 concentration to 5 mg / mL. The solution was sterile filtered through a 0.22 μm PES filter.

[0270] Each formulation was aseptically filled into 2 mL glass vials in a biosafety hood with a fill volume of approximately 1 mL. The vials were stoppered and crimped immediately after filling.

[0271] Sample Testing Formulations E1-8 were tested under four different conditions. Vials of each formulation were stored at 5°C for 12 weeks (12W) and 40°C for 8 weeks (8W), subjected to 5 cycles of freeze-thaw (0.5°C / min), and agitated at 100 rpm for 5 days at 25°C. The sampling and testing plan is described in Table 23. Samples were tested at initial (T0), 2 weeks (2W), 4 weeks (4W), 8 weeks (8W) and 12 weeks (12W).

[0272] JPEG2025060645000029.jpg72164

[0273] result exterior The appearance results of the freeze-thaw experiment measurements are shown in Table 24 (using a cooler) and Table 25 (using a freeze dryer) below.

[0274] JPEG2025060645000030.jpg90164

[0275] JPEG2025060645000031.jpg99164

[0276] During the freeze-thaw study, visible particles increased slightly with increasing freeze-thaw cycles for F5 and F6 without PS80. No differences were observed for the other formulations.

[0277] overview Formulations without PS80 (F5 and F6) did not perform as well as formulations with PS80 in freeze-thaw experiments, as evidenced by the formation of visible particles after multiple freeze-thaw cycles.

[0278] Formulation Example 4: Hemolysis of human whole blood A series of dilutions (1:2, 1:3, 1:4, 1:5 and 1:10) of blood from human donors were prepared in saline. The dilutions used in the assay gave an OD of 0.8 to 1.2 at 540 nm when mixed with deionized water and were referred to as blood matrix. Four types of samples were tested: test article, placebo, positive control and negative control. The test article featured ATB200 rhGAA in a formulation containing 25 mM sodium citrate, 2% mannitol and 0.05% polysorbate 80 at pH 6.0. The placebo was the same as the test article except for the absence of ATB200 rhGAA. The positive control was sterile water for injection, pH 5. The negative control was saline (0.9NaCl), pH 5.

[0279] Test substance (ATB200) at 300, 600 and 1000 μg / ml with saline, placebo with saline, negative control (saline) and positive control (water) were mixed with blood matrix from human donors. Samples were incubated at 37°C for 1 hour without stirring. After incubation, the tubes were centrifuged at room temperature, approximately 100xg for 10 minutes. The amount of hemoglobin in the supernatant of each sample was analyzed spectrophotometrically at 540 nm.

[0280] The % hemolysis of a test substance was calculated using the formula: Determined by JPEG2025060645000032.jpg15154.

[0281] Hemolysis rate for water + blood is 100%. Saline was the negative control. Hemolysis below 10% was considered insignificant. The % hemolysis was calculated for each concentration of test article and placebo.

[0282] Table 26 below shows the results of the samples tested.

[0283] JPEG2025060645000033.jpg115164

[0284] Incubation of human blood matrix with three samples of placebo diluted with saline in the same proportions as the three test substance doses did not cause significant hemolysis of human blood. The % hemolysis of the samples diluted with placebo was calculated to be -1.3, -1.3, and 0.9%, respectively. Incubation of human blood matrix with 300, 600, and 1000 μg / ml ATB200 did not cause significant hemolysis of human blood. The % hemolysis of the test substance samples was calculated to be -0.1, -1.5%, and -1.5%, respectively.

[0285] In conclusion, the ATB200 formulation was compatible with human blood at all dilutions.

[0286] Formulation Example 5: Aggregation in human plasma and serum The test article dosing solution (3 concentrations) and placebo (3 concentrations) were mixed with equal volumes of human plasma and serum from donors. The test article featured ATB200 rhGAA in a formulation containing 25 mM sodium citrate, 2% mannitol, and 0.05% polysorbate 80 at pH 6.0. The placebo was the same as the test article, except for the absence of ATB200 rhGAA.

[0287] One ml of each dose of test substance or placebo was mixed with equal volumes of plasma, serum and saline. Samples were incubated at room temperature for 30 minutes. After incubation, tubes were examined macroscopically and microscopically for precipitation or clotting. An aliquot from each tube was centrifuged at 14,000 rpm in a microcentrifuge for 10 minutes. Each tube was examined for the presence or absence of a pellet. Precipitation / clotting and pellets were scored as follows: 0=negative 1 = Very little sediment or pellet 2 = Minimal sedimentation or pellet 3 = moderate sedimentation or pellet 4 = Significant sedimentation or pellet

[0288] Table 27 below shows the results of the samples tested.

[0289] JPEG2025060645000034.jpg115164

[0290] No macroscopic or microscopic precipitation was observed in human plasma or serum when mixed with each ATB200 concentration. No precipitation was observed in placebo samples. No pellets were observed when all placebo or ATB200 samples were centrifuged.

[0291] Based on the results of this study, the ATB200 formulation was found to be compatible with human plasma and serum at final concentrations up to and including 1000 μg / ml.

[0292] Example: Pharmacokinetic and safety data of recombinant acid alpha-glucosidase ATB200 co-administered with miglustat in ERT-experienced and ERT-naive patients with Pompe disease This study was designed to primarily evaluate the safety, tolerability, and pharmacokinetics (PK) of ATB200 administered in combination with miglustat. The PK / pharmacodynamics (PD) translational model in Gaa knockout mice predicts that a combination of 20 mg / kg ATB200 and a high dose (e.g., 260 mg) of miglustat in humans will provide optimal glycogen reduction.

[0293] In the following description, a "high dose" of miglustat refers to a dose of about 260 mg, and a "low dose" refers to a dose of about 130 mg.

[0294] The objective was to evaluate preliminary total GAA protein, ATB200 and miglustat PK data, and safety markers from 10 patients in this Phase 1 / 2 study.

[0295] This shows the experimental design of an open-label, fixed sequential, dose-escalating, first-in-human, Phase 1 / 2 study to evaluate the safety, tolerability, PK, PD, and efficacy of intravenous infusion of ATB200 co-administered with oral miglustat in adults with Pompe disease (Figure 21). Evaluation of mean total GAA protein and miglustat PK outcomes from the first 8 Cohort 1 patients through Visit 9 and the first 2 Cohort 3 patients was performed. a Safety data from the two sentinel patients in Cohort 1 were reviewed at each dose level prior to dosing in Cohorts 2 and 3. b In stages 2 and 3, miglustat was administered orally before the start of the ATB200 intravenous infusion. For all doses, ATB200 was infused intravenously for 4 hours. c The first two patients in cohorts 2 and 3 served as sentinel patients for each cohort.

[0296] Key entry criteria Men and women aged 18-65 years who have been diagnosed with Pompe disease based on a documented deficiency of GAA enzyme activity or by GAA genotyping. -Received ERT with alglucosidase alfa for 2-6 years (2 years or more for cohort 2) prior to study entry (cohort 1) Currently receiving alglucosidase alfa every other week and have completed the last two infusions without any drug-related adverse events leading to discontinuation (Cohorts 1 and 2) Must be able to walk 200-500 meters in the 6-minute walk test (Cohorts 1 and 3) · Forcible standing lung capacity must be between 30% and 80% of predicted normal (Cohorts 1 and 3) Must be wheelchair bound and unable to walk unassisted (Cohort 2) PK analysis: Blood samples were collected for plasma total GAA protein and activity concentrations. Stage 1: Before the start of ATB200 infusion and 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12 and 24 hours after infusion Stages 2 and 3: 1, 2, 3, 4, 4.5, 5, 6, 7, 9, 11, 13 and 25 hours after oral administration of miglustat Blood samples for plasma miglustat concentrations were taken immediately before oral administration of miglustat (time 0) and at 1, 1.5, 2, 2.5, 3, 4, 5, 6, 9, 11 and 25 hours after oral administration of miglustat. Plasma miglustat was determined by a validated LC-MS / MS assay. Total GAA protein concentrations in plasma for ATB200 at 5, 10 and 20 mg / kg were determined by validated LC-MS / MS quantification of rhGAA-specific "signature" peptide(s).

[0297] Preliminary analyses were completed for eight patients in cohort 1 who completed stages 1 and 2 and two patients in cohort 3 who initiated stage 3. First-time ERT-switched patients were representative of the Pompe disease population and had been on ERT for a mean of 5.02 years (Table 28).

[0298] JPEG2025060645000035.jpg58164

[0299] Total GAA Protein When administered alone, ATB200 increases slightly above dose proportionality (Table 29 and Figures 22A-22D). Variability appears to increase with miglustat dose (Figure 22C). Coadministration of 20 mg / kg ATB200 with a high dose of miglustat (260 mg) increased total GAA protein exposure (AUC) by approximately 25% compared to 20 mg / kg ATB200 alone. The distribution half-life (α phase) increased by 45%, suggesting that high doses of miglustat stabilize ATB200 in plasma. The increase in distribution half-life is accompanied by an increase in AUC from time to maximum plasma concentration, up to approximately 12 hours after administration. The increase in AUC and half-life can be observed on a logarithmic scale during the terminal elimination phase (Figure 22B). ATB200 showed a relatively high distribution volume. The plasma total GAA protein profile appears to be similar between ERT-naive (cohort 3) and ERT-experienced (cohort 1) patients (Figures 22A and 22D).

[0300] JPEG2025060645000036.jpg127164

[0301] Miglustat Penalty Miglustat demonstrated dose-proportional kinetics (Table 30 and Figure 23). Plasma miglustat appeared similar between single and multiple doses.

[0302] JPEG2025060645000037.jpg49164

[0303] Pharmacodynamics By Visit 11 in ERT-experienced patients from Cohort 1 (Figures 24A and 24B): Alanine aminotransferase (ALT) was decreased in 5 / 8 patients and normalized in 4 / 4 patients with elevated baseline levels Aspartate aminotransferase (AST) was reduced in 6 / 8 patients and normalized in 3 / 4 patients with elevated baseline levels Creatine phosphokinase (CPK) was decreased in 6 / 8 patients and normalized in 2 / 6 patients with elevated baseline levels Urinary glucose tetrasaccharide (HEX4) concentrations were decreased in 8 of 8 patients

[0304] By week 4, two patients in the treatment-naive cohort (cohort 3) had decreased levels of all four biomarkers (Figures 24C and 24D).

[0305] In Figures 24A-24D, data are expressed as mean ± standard error.

[0306] safety No serious adverse events (AEs) or infusion-related reactions were reported in any patient after a total of 155 or more infusions Treatment-emergent AEs, reported in 11 / 13 (84%) patients, were generally mild and transient. Treatment-related AEs reported in 7 / 13 (53%) patients: nausea (n=1), fatigue (n=1), headache (n=1), tremor (n=2), acne (n=1), tachycardia (n=1) and hypotension (n=1).

[0307] conclusion ATB200 alone and in combination with miglustat has been safe and well tolerated, with no infusion-related reactions to date. ATB200 alone showed a greater than dose-proportional increase in exposure that was further enhanced by miglustat, suggesting a stabilizing effect of the chaperone on ATB200. After switching from standard of care to ATB200 / miglustat, patients generally showed improvement in biomarkers of muscle damage, with many showing normalization by week 18. The first two treatment-naive patients treated with ATB200 / miglustat showed robust reductions in all biomarkers of muscle damage

[0308] array JPEG2025060645000038.jpg206164JPEG2025060645000039.jpg214164JPEG2025060645000040.jpg214164JPEG2025060645000041.jpg141164

Claims

1. 1. A pharmaceutical formulation comprising: (d) a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units having one or two mannose-6-phosphate residues compared to the content of N-glycan units having one or two mannose-6-phosphate residues of alglucosidase alpha; (e) at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof; and (f) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof. wherein the pH of the formulation is from about 5.0 to about 7.

0.

2. 2. The pharmaceutical formulation of claim 1, wherein the recombinant acid α-glucosidase is present at a concentration of about 5 mg / mL to about 50 mg / mL.

3. 3. The pharmaceutical formulation of claim 1 or 2, wherein the recombinant acid α-glucosidase is present at a concentration of about 15 mg / mL.

4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the pH of the formulation is from about 5.5 to about 7.

0.

5. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the pH of the formulation is about 6.

0.

6. The pharmaceutical formulation according to any one of claims 1 to 5, wherein the at least one buffering agent comprises citrate.

7. The pharmaceutical formulation according to any one of claims 1 to 6, wherein the at least one buffering agent comprises a potassium, sodium or ammonium salt.

8. The pharmaceutical formulation according to any one of claims 1 to 7, wherein the at least one buffering agent comprises sodium citrate.

9. The pharmaceutical formulation according to any one of claims 1 to 8, wherein the at least one buffering agent is present at a concentration of about 10 mM to about 100 mM.

10. The pharmaceutical formulation according to any one of claims 1 to 9, wherein the at least one buffering agent is present in a concentration of about 25 mM.

11. The pharmaceutical formulation of any one of claims 1 to 10, wherein trehalose, sucrose, glycine or combinations thereof are excluded.

12. 12. The pharmaceutical formulation of any one of claims 1 to 11, wherein the at least one excipient is mannitol present in a concentration of about 10 mg / mL to about 50 mg / mL.

13. 13. The pharmaceutical formulation of any one of claims 1 to 12, wherein the at least one excipient is polysorbate 80 present in a concentration of about 0.2 mg / mL to about 0.5 mg / mL.

14. 14. The pharmaceutical formulation of any one of claims 1 to 13, wherein the mannitol is present in a concentration of about 20 mg / mL and the polysorbate 80 is present in a concentration of about 0.5 mg / mL.

15. The pharmaceutical formulation of any one of claims 1 to 14, further comprising: (d) water.

16. A pharmaceutical formulation according to any one of claims 1 to 15, (e) an alkalizing agent; and / or (f) an acidifying agent; wherein said alkalizing agent and said acidifying agent are present in an amount to maintain a pH of said pharmaceutical formulation at about 5.0 to about 6.

0.

17. 17. The pharmaceutical preparation of any one of claims 1 to 16, wherein at least 30% of the molecules of recombinant human acid α-glucosidase contain one or more N-glycan units bearing one or two mannose-6-phosphate residues.

18. 18. The pharmaceutical formulation of any one of claims 1 to 17, wherein the recombinant human acid alpha-glucosidase comprises an average of 0.5 to 7.0 moles of N-glycan units having one or two mannose-6-phosphate residues per mole of recombinant human acid alpha-glucosidase.

19. 19. The pharmaceutical formulation of any one of claims 1 to 18, wherein the recombinant human acid alpha-glucosidase comprises, on average, at least 3 moles of mannose-6-phosphate residues per mole of recombinant human acid alpha-glucosidase and at least 4 moles of sialic acid residues per mole of recombinant human acid alpha-glucosidase.

20. 20. The pharmaceutical formulation of any one of claims 1 to 19, wherein the recombinant human acid α-glucosidase comprises seven potential N-glycosylation sites, at least 50% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having two mannose-6-phosphate residues at a first site, at least 30% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having one mannose-6-phosphate residue at a second site, at least 30% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having two mannose-6-phosphate residues at a fourth site, and at least 20% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having one mannose-6-phosphate residue at a fourth site.

21. 21. The pharmaceutical formulation according to any one of claims 1 to 20, comprising: (a) the recombinant acid α-glucosidase; (b1) sodium citrate; (b2) citric acid monohydrate; (c1) mannitol; (c2) Polysorbate 80; (d) water; (e) optionally, an acidifying agent; and (f) optionally an alkalizing agent; and wherein the pH of said formulation is from about 5.0 to about 6.

0.

22. A pharmaceutical formulation according to any one of claims 1 to 21, comprising: (a) the recombinant acid α-glucosidase is present at a concentration of about 15 mg / mL; (b) a sodium citrate buffer present at a concentration of about 25 mM; (c1) mannitol, present in a concentration of about 20 mg / mL; (c2) polysorbate 80, present at a concentration of about 0.5 mg / mL; and (d) water; (e) optionally, an acidifying agent; and (f) optionally, an alkalizing agent; and wherein the pH of said formulation is from about 5.0 to about 6.

0.

23. A pharmaceutical composition comprising the formulation according to any one of claims 1 to 22 after lyophilization.

24. 1. A pharmaceutical composition comprising: (a) a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units having one or two mannose-6-phosphate residues compared to the content of N-glycan units having one or two mannose-6-phosphate residues of alglucosidase alpha; (b) a buffer selected from the group consisting of citrate, phosphate, and combinations thereof; and (c) at least one excipient selected from the group consisting of trehalose, mannitol, polysorbate 80, and combinations thereof; 23. A pharmaceutical composition comprising a lyophilized mixture comprising:

25. Use of a pharmaceutical preparation according to any one of claims 1 to 22 for the treatment of Pompe disease.

26. 26. The use of claim 25, further comprising diluting the pharmaceutical formulation prior to administration to a patient.

27. 25. Use of the pharmaceutical composition of claim 23 or 24 for treating Pompe disease, wherein the pharmaceutical composition is reconstituted and the reconstituted pharmaceutical composition is administered to a patient in need thereof.

28. A method for preparing a pharmaceutical formulation according to any one of claims 1 to 22, said method comprising: adding said at least one buffering agent, at least one excipient, and recombinant acid α-glucosidase to water to provide a solution; Optionally, adjusting the pH of the solution; and Optionally, adding additional water to the solution; A method comprising:

29. 30. The method of claim 28, further comprising filtering the solution.

30. 30. The method of claim 28 or 29, further comprising storing the solution.

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