EA043224B1 - COMPOSITIONS CONTAINING RECOMBINANT ACID ALPHA-GLUCOSIDASE - Google Patents

Description

Technical field

The principles and embodiments of the present invention generally relate to formulations containing recombinant acid α-glucosidase, and in particular to liquid formulations.

Prerequisites for the invention

Pompe disease, also known as acid maltase deficiency or type II glycogen storage disease, is one of several lysosomal storage disorders. Lysosomal storage disorders are a group of autosomal recessive genetic disorders characterized by the accumulation of cellular glycosphingolipids, glycogen, or mucopolysaccharides in intracellular compartments called lysosomes. Individuals with these diseases carry mutant genes encoding enzymes that are defective in catalyzing the hydrolysis of one or more of these compounds, which then accumulate in lysosomes. Other examples of lysosomal disorders include Gaucher's disease, GM1 gangliosidosis, fucosidosis, mucopolysaccharidoses, Hurler-Scheie disease, Niemann-Pick types A and B, and Fabry disease. Pompe disease is also classified as a neuromuscular disease or metabolic myopathy.

Pompe disease is estimated to occur in approximately 1 in 40,000 newborns and is caused by a mutation in the GAA gene, which codes for 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 main form of glucose storage in animals, by catalyzing its hydrolysis to glucose in lysosomes. Because individuals with Pompe disease produce a mutant, defective acid α-glucosidase that is inactive or under-active, glycogen catabolism is slower or non-existent, with glycogen accumulating in the lysosomes of various tissues, particularly striated muscle, leading to a wide range of clinical manifestations, including progressive muscle weakness and respiratory failure. In particular, tissues such as cardiac and skeletal muscles are affected.

Pompe disease can vary widely in 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. This disease was classified into main types: early onset or infantile form and late onset. Earlier onset of the disease and lower enzymatic activity are usually associated with a more severe course of the disease. Pompe disease of the infantile type is the most severe, resulting in a complete or near complete deficiency of acid α-glucosidase, and presents with symptoms that include severe lack of muscle tone, weakness, enlarged liver and heart, and cardiomyopathy. The tongue may become enlarged and protrude, and swallowing may become difficult. The most severely affected children die of respiratory and cardiac complications before the age of two. Late onset Pompe disease can present at any age over 12 months and is characterized by no cardiac involvement and a better short-term prognosis. Symptoms are associated with progressive skeletal muscle dysfunction and include generalized muscle weakness and atrophy of the respiratory muscles of the trunk, proximal lower extremities, and diaphragm. Some adult patients do not have major symptoms or movement restrictions. The prognosis usually depends on the degree of damage to the respiratory muscles. Most subjects with Pompe disease eventually become physically debilitated, requiring the use of a wheelchair and assisted ventilation, with frequent premature death due to respiratory failure.

Current treatment options for Pompe disease include enzyme replacement therapy (ERT) using recombinant human acid α-glucosidase (rhGAA). Conventional rhGAA based products are known as alglucosidase-alpha, Myozyme® or Lumizyme® manufactured by Genzyme, Inc. ERT is a long-term treatment required throughout the life of the patient and involves the administration of a replacement enzyme by intravenous infusion. The replacement enzyme is then transported into the bloodstream and enters the lysosomes within the cells, where its action is to break down stored glycogen, which compensates for the deficiency in the activity of the endogenous defective mutant enzyme and thus reduces the severity of disease symptoms.

The manner in which replacement enzymes such as rhGAA are prepared, stored, transported and administered to patients is complex. The enzymes used for ERT are generally relatively complex and require careful handling, making the choice of concomitant buffers, excipients, etc. difficult. key. If the enzyme is not stored properly, large amounts may be required, making treatment costly and ineffective.

Some traditional rhGAA-based products are provided to patients as lyophilized (freeze-dried) preservative-free powder in disposable vials. The rhGAA is then reconstituted in vials, then diluted and administered intravenously. Although

- 1 043224 Lyophilization helps preserve the enzyme from the time it is made until the time it is ready for administration to a patient, this process itself can damage the enzyme. Thus, great care should be taken when selecting components for a rhGAA formulation to ensure that they help maintain protein concentration and activity.

In addition, recombinant enzymes are often structurally different from wild-type enzymes. Even if the amino acids in the recombinant enzyme are identical to their wild-type counterparts, there may be differences in the chemical structure of the carbohydrates. Thus, when new recombinant enzymes are discovered, formulations specific to the chemical structure of the newly discovered enzymes must be developed for these enzymes.

Accordingly, there is a continuing need for formulations for the storage and transport of recombinant enzymes, such as rhGAA, that maintain enzyme activity and concentration.

Short description

One aspect of the present invention relates to a pharmaceutical composition. In an embodiment, one composition contains:

(a) a recombinant acid α-glucosidase, wherein the recombinant acid α-glucosidase is derived from expression in Chinese Hamster Ovary (CHO) cells and is characterized by an increased content of N-glycan units bearing one or two mannose-6-phosphate residues compared to the content of N-glycan units bearing one or two mannose-6-phosphate residues in alglucosidase-alpha;

(b) at least one buffer 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, wherein the formulation has a pH of about 5.0 to about 7.0.

Embodiment two involves modifying the pharmaceutical formulation according to embodiment one, wherein the recombinant acid α-glucosidase is present at a concentration of about 5 to about 50 mg/mL.

Embodiment three involves modifying the pharmaceutical formulation according to embodiment one or two, wherein the recombinant acid α-glucosidase is present at a concentration of approximately 15 mg/mL.

Embodiment four involves modifying a pharmaceutical formulation according to any one of Embodiments 1-3, wherein the formulation has a pH of about 5.5 to about 7.0.

Embodiment five involves modifying the pharmaceutical formulation according to any of Embodiments 1-4, wherein the formulation has a pH of approximately 6.0.

Option six provides for the modification of the pharmaceutical composition according to any one of embodiments 1-5, where at least one buffer contains citrate.

Option seven provides for the modification of the pharmaceutical composition according to any of embodiments 1-6, where at least one buffer contains a potassium, sodium or ammonium salt.

Option eight provides for the modification of the pharmaceutical composition according to any one of embodiments 1-7, where at least one buffer contains sodium citrate.

Option nine provides for the modification of the pharmaceutical composition according to any of embodiments 1-8, where at least one buffer is present at a concentration of from about 10 to about 100 mm.

Embodiment ten provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-9, wherein at least one buffer is present at a concentration of approximately 25 mM.

Embodiment 11 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-10 where trehalose, sucrose, glycine, or combinations thereof are excluded.

Embodiment 12 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-11, wherein at least one excipient is mannitol present at a concentration of about 10 to about 50 mg/mL.

Embodiment 13 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-12, wherein at least one excipient is polysorbate 80 present at a concentration of about 0.2 to about 0.5 mg/mL.

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Embodiment 14 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-13 wherein the mannitol is present at a concentration of approximately 20 mg/mL and polysorbate 80 is present at a concentration of approximately 0.5 mg/mL.

Embodiment 15 provides for a modification of the pharmaceutical formulation according to any of Embodiments 1-14 and further comprises: (d) water.

Embodiment 16 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-15 and further comprises: (d) an alkalizing agent and/or (e) an acidifying agent, wherein the alkalizing agent and the acidifying agent are present in amounts sufficient to maintain the pH of the pharmaceutical formulation in the range from about 5.0 to about 6.0.

Embodiment 17 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-16, wherein at least 30% of the human recombinant acid α-glucosidase molecules contain one or more N-glycan units bearing one or two mannose-6-phosphate residues.

Embodiment 18 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-17, wherein the recombinant human acid α-glucosidase contains an average of 0.5 to 7.0 moles of N-glycan units bearing one or two mannose-6-phosphate residues. per mole of human recombinant acid α-glucosidase.

Embodiment 19 provides for the modification of the pharmaceutical composition according to any one of embodiments 1-18, wherein the human recombinant acid α-glucosidase contains an average of at least 3 moles of mannose-6-phosphate residues per mole of human recombinant acid α-glucosidase and at least 4 moles of residues sialic acid per mole of human recombinant acid α-glucosidase.

Embodiment 20 provides for a modification of the pharmaceutical composition according to any one of embodiments 1-19, wherein the recombinant human α-glucosidase contains seven potential N-glycosylation sites, at least 50% of the recombinant human α-glucosidase acid molecules contain an N-glycan unit bearing two mannose-6-phosphate residues, in the first site, at least 30% of the recombinant human α-glucosidase acid molecules contain an N-glycan unit bearing one mannose-6-phosphate residue, in the second site, at least 30% of the molecules of the recombinant human acid α-glucosidase contain an N-glycan unit bearing two mannose-6-phosphate residues in the fourth site, and at least 20% of recombinant human acid α-glucosidase molecules contain an N-glycan unit bearing one mannose-6-phosphate residue. phosphate, at the fourth site.

Embodiment 21 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-20, wherein the pharmaceutical formulation consists essentially of:

(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, wherein the composition has a pH of about 5.0 to about 6.0.

Embodiment 22 provides for a modification of the pharmaceutical formulation according to any one of Embodiments 1-21, wherein the pharmaceutical formulation consists essentially of:

(a) recombinant acid α-glucosidase present at a concentration of approximately 15 mg/ml;

(b) sodium citrate buffer present at a concentration of approximately 25 mM;

(c1 ) mannitol present at a concentration of approximately 20 mg/ml;

(c2) polysorbate 80 present at a concentration of approximately 0.5 mg/ml; and (d) water;

(e) optionally an acidifying agent; and (f) optionally an alkalizing agent, wherein the composition has a pH of about 5.0 to about 6.0.

Another aspect of the present invention relates to a lyophilized pharmaceutical composition. Accordingly, Embodiment 23 refers to a pharmaceutical composition containing the composition according to any one of Embodiments 1-22 subjected to lyophilization. Embodiment 24 relates to a pharmaceutical composition containing a lyophilized mixture,

- 3 043224 containing:

(a) a recombinant acid α-glucosidase, wherein the recombinant acid α-glucosidase is derived from expression in Chinese Hamster Ovary (CHO) cells and is characterized by an increased content of N-glycan units bearing one or two mannose-6-phosphate residues compared to the content of N-glycan units bearing one or two mannose-6-phosphate residues in 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.

Another aspect of the present invention relates to a method for treating Pompe disease. Accordingly, Embodiment 25 provides for administering to a patient in need thereof the pharmaceutical composition according to Embodiments 1-22. Embodiment 26 provides for a modification of the method of Embodiment 25 further comprising diluting the pharmaceutical formulation prior to administration to a patient. Embodiment 27 relates to a method for 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.

Another aspect of the present invention relates to a process for the preparation of the pharmaceutical compositions described above. Accordingly, Embodiment 28 refers to a method for preparing a pharmaceutical composition according to any one of Embodiments 1 to 22, the method comprising: adding at least one buffer, at least one excipient, and recombinant acid α-glucosidase to water to form a solution; optionally adjusting the pH of the solution; and optionally adding an additional volume of water to the solution. Embodiment 29 includes a modification of the method of Embodiment 28 further including filtering the solution. Embodiment 30 includes a modification of the method according to embodiment 27 or 28, further providing for the storage of the solution.

Brief description of graphic materials

Additional features of the present invention will become apparent from the following written description and the accompanying figures, which show the following.

In FIG. 1A shows non-phosphorylated high mannose glycan, mono-M6P glycan and bisM6P glycan.

In FIG. 1B shows the chemical structure of the M6P group.

In FIG. 2A depicts the efficient targeting of rhGAA to target tissues (eg, muscle tissues of a subject with Pompe disease) via M6P-bearing glycans.

In FIG. 2B depicts non-productive drug clearance from non-target tissues (eg, liver and spleen) or binding of M6P-free glycans to non-target tissues.

In FIG. 3A and 3B are respectively graphs showing the results of CIMPR binding affinity chromatography to Lumizyme® and Myozyme®. The dotted lines refer to the elution gradient with M6P. M6P elution displaces GAA molecules bound to CIMPR via an M6P-containing glycan. As shown in FIG. 2A, 78% of the active GAA form in Lumizyme® was eluted prior to the addition of M6P. In FIG. 2B shows that 73% of the active GAA form in Myozyme® was eluted prior to the addition of M6P. Only 22% or 27% rhGAA respectively in Lumizyme® or Myozyme® was eluted with M6P. These figures show that most of the rhGAA in these two conventional rhGAA products lack the M6P glycans that are required to target CIMPR in target muscle tissues.

In FIG. 4 shows a DNA construct for transforming CHO cells with DNA encoding rhGAA. CHO cells were transformed with a DNA construct encoding rhGAA.

Fig. 5A and 5B respectively are graphs showing the results of CIMPR binding affinity chromatography for rhGAA as Myozyme® and ATB200. As shown in FIG. 5B, approximately 70% of the rhGAA molecules in rhGAA ATB200 contain M6P.

Fig. 6 is a graph showing the results of chromatography to determine the binding affinity of CIMPR to ATB200 rhGAA with and without capture on an anion exchange column (AEX).

Fig. 7 is a graph showing Polywax elution profiles for rhGAA Lumizyme® and ATB200.

Fig. 8 is a table summarizing the Lumizyme® N-glycan structures compared to three different rhGAA ATB200 formulations, designated BP-rhGAA, ATB200-1 and ATB200-2.

In FIG. 9A-9H show the results of the site-specific N-glycosylation analysis of rhGAA ATB200.

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Fig. 10A is a graph comparing CIMPR binding affinity for rhGAA ATB200 (left curve) and for Lumizyme® (right curve).

Fig. 10B is a table showing the content of Bis-M6P in rhGAA Lumizyme® and ATB200.

Fig. 11A is a graph comparing rhGAA ATB200 activity (left curve) with Lumizyme® rhGAA activity (right curve) in normal fibroblasts at various GAA concentrations.

Fig. 11A is a table comparing rhGAA ATB200 activity (left curve) with Lumizyme® rhGAA activity (right curve) in fibroblasts from a Pompe disease subject at various GAA concentrations.

Fig. 11C is a table comparing the K _uptake of fibroblasts obtained from healthy subjects and subjects with Pompe disease.

Fig. 12A is a graph showing the amount of glycogen versus dose of human recombinant acid α-glucosidase in mouse heart muscle after contact with an inert medium (negative control), with 20 mg/ml alglucosidase-alpha (Lumizyme®) or with 5, 10 or 20 mg/kg ATB200.

Fig. 12B is a graph showing the amount of glycogen versus dose of human recombinant acid α-glucosidase in the mouse quadriceps after contact with an inert medium (negative control), with 20 mg/ml alglucosidase-alpha (Lumizyme®) or with 5, 10, or 20 mg/kg ATB200.

Fig. 12C is a graph showing the amount of glycogen versus dose of human recombinant acid α-glucosidase in mouse triceps after contact with an inert medium (negative control), with 20 mg/ml alglucosidase-alpha (Lumizyme®) or with 5, 10, or 20 mg/kg ATB200.

Fig. 13 is a table showing that the combination of rhGAA ATB-200 and the miglustat chaperone provides significantly better glycogen clearance in Gaa knockout mice compared to treatments with either rhGAA Lumizyme® or ATB200 without the miglustat chaperone.

Fig. 14 is a series of electron micrographs of cardiac muscle, diaphragm, and soleus muscle of wild-type and Gaa knockout mice treated with inert medium, alglucosidase-alpha, and ATB200 in the presence and absence of miglustat, showing the levels of membrane protein associated with lysosomes (LAMP-1).

Fig. 15 is a series of electron micrographs of cardiac muscle, diaphragm, and soleus muscle of wild-type and Gaa knockout mice treated with inert medium, alglucosidase-alpha, and ATB200 in the presence and absence of miglustat, showing glycogen levels stained with Schiff's reagent. -periodic acid (PAS).

Fig. 16 is a series of electron micrographs (1000x) of quadriceps from wild type and Gaa knockout mice treated with inert medium, alglucosidase alfa and ATB200 in the presence and absence of miglustat, stained with methylene blue to show vacuoles (indicated by arrows).

Fig. 17 is a series of electron micrographs (40x) of quadriceps of wild type and Gaa knockout mice treated with inert medium, alglucosidase-alpha, and ATB200 in the presence and absence of miglustat, showing levels of microtubule-associated light chain 3 autophagy markers. protein 1A/1B conjugated with phosphatidylethanolamine (LC3A II), and p62, insulin-dependent glucose transporter GLUT4 and insulin-independent glucose transporter GLUT1.

Fig. 18A and 18B are graphs showing wire hang and muscle grip strength data in wild-type and Gaa knockout mice treated with inert medium, alglucosidase-alpha, and ATB200 in the presence of miglustat.

Fig. 19A-19G are graphs showing glycogen levels in quadriceps, triceps, and cardiac muscle cells of wild type and Gaa knockout mice treated with inert medium, alglucosidase-alpha, and ATB200 in the presence and absence of miglustat.

Fig. 20 is a series of photomicrographs (100x and 200x) of vastus lateralis (VL) muscle fibers of wild-type and Gaa knockout mice treated with inert medium, alglucosidase-alpha and ATB200 in the presence and absence of miglustat, showing dystrophin signals.

In FIG. Figure 21 shows the design of a phase 1/2, open-label, fixed-sequence, escalating-dose study, the first to be conducted in humans, to evaluate the safety, tolerability, PK, PD, and efficacy of intravenous infusions of ATB200 given in combination with oral miglustat. , in adults with Pompe disease.

- 5 043224

Fig. 22A-22B are graphs showing total GAA protein concentration versus time profiles in plasma in human subjects following a dose 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.

Fig. 22C is a graph showing the plasma AUC for total GAA protein in human subjects following a dose of 20 mg/kg ATB200, 20 mg/kg ATB200 and 130 mg miglustat, or 20 mg/kg ATB200 and 260 mg miglustat.

Fig. 22D is a graph showing plasma concentration versus time profiles of total GAA protein in two separate human subjects following a dose of 20 mg/kg of ATB200 and 260 mg of miglustat.

Fig. 23 is a graph showing the plasma concentration-time profiles of miglustat in human subjects after administration of a dose of 130 mg or 260 mg of miglustat.

Fig. 24A-24D are graphs showing changes in levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), and hexose tetrasaccharide (Hex4) in human patients after administration of increasing doses of ATB200 (5, 10, and 20 mg/kg) with subsequent co-administration of ATB200 (20 mg/kg) and miglustat (130 and 260 mg).

Detailed description

Before describing several illustrative embodiments of the present invention, it should be understood that the present invention is not limited to the construction details or method steps set forth in the following description. The present invention is capable of other embodiments and may be practiced or carried out in various ways. It will be understood that the following embodiments may be combined not only as indicated below, but also in other suitable combinations in accordance with the scope of the present invention.

Surprisingly, it has been found that by careful selection of buffers and excipients, a formulation for the recombinant GAA ATB200 protein can be provided that has excellent stability and can be subjected to formulation, storage, transport, reconstitution, and administration processes while maintaining enzyme activity and efficacy at minimizing enzyme precipitation. Accordingly, one aspect of the present invention relates to a formulation containing rhGAA, a buffer and at least one excipient. In one or more embodiments, rhGAA contains ATB200. In some embodiments, the formulation is a liquid formulation. Details and various embodiments regarding the various ingredients of the formulation are provided below.

Definitions.

The terms used in this description, as a rule, have their usual meanings in the art, in the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to the practitioner in describing the compositions and methods of the present invention and their preparation and use.

In the present description, except when the context otherwise requires due to language or necessary implied meaning, the word contain or variants such as contains or containing are used in the inclusive sense, i.e. to indicate the presence of the recited features, but without excluding the presence or addition of additional features in various embodiments of the present invention.

As used herein, the term Pompe disease, also referred to as acid maltase deficiency, glycogen storage disease type II (GSDII), and glycogenosis type II, is intended to refer to a genetic lysosomal storage disorder characterized by mutations in the GAA gene, which codes for the enzyme human acid α-glucosidase. . The term includes, without limitation, early and late onset forms of the disease, including, but not limited to, onset Pompe disease in infancy, adolescence, and adulthood.

As used herein, the term acidic α-glucosidase is intended to mean a lysosomal enzyme that hydrolyzes the α-1,4 bonds between the D-glucose units of glycogen, maltose and isomaltose. Alternative names include, without limitation, lysosomal α-glucosidase (EC: 3,2.1,20); glucoamylase; 1,4-α-D-glucanglucohydrolase; amyloglucosidase; gammaamylase and exo-1,4-a-glucosidase. Human acid α-glucosidase is encoded by the GAA gene (Gene ID 2548 at the National Center for Biotechnology Information (NCBI)), which has been mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). The complete amino acid sequence of wild-type GAA is shown under SEQ ID NO: 1 as described in US Pat. No. 8,592,362 and GenBank accession number AHE24104.1 (GI: 568760974).

To date, more than 500 mutations have been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations resulting in incorrect folding or incorrect processing of the acid α-glucosidase enzyme include T1064C (Leu355Pro) and

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C2104T (Arg702Cys). In addition, GAA mutations that affect enzyme maturation and processing include Leu405Pro and Met519Thr. The activity of the acid α-glucosidase protein requires the presence of the conserved WIDMNE hexapeptide at amino acid residues 516-521. As used herein, the abbreviation GAA is meant to mean the enzyme acid α-glucosidase, while the italicized abbreviation GAA is meant to mean the human gene encoding human acid α-glucosidase. Thus, the abbreviation rhGAA is meant to mean the enzyme recombinant human acid α-glucosidase.

As used herein, the term alglucosidase-alpha is meant to mean recombinant human acid α-glucosidase identified as [199-arginine,223histidine]prepro-a-glucosidase (human); registration number 420794-05-0 according to the Chemical Abstract Service. Alglucosidase-alpha has been approved for marketing in the US since January 2016 under the names Lumizyme® and Myozyme® by Genzyme.

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

As used herein, the term glycan is intended to mean a polysaccharide chain covalently linked to an amino acid residue in a protein or polypeptide. As used herein, the term N-glycan or N-linked glycan is meant to mean a polysaccharide chain attached to an amino acid residue in a protein or polypeptide by forming a covalent bond with the nitrogen atom of the amino acid residue. For example, an N-glycan may be covalently attached to the side chain nitrogen of an aspartic residue. Glycans may contain one or more monosaccharide units, and the monosaccharide units may be covalently linked to form a straight chain or a branched chain. In at least one embodiment, the N-glycan units attached to ATB200 may contain one or more monosaccharide units, each independently selected from N-acetylglucosamine, mannose, galactose, or sialic acid. N-glycan units in a protein can be determined using any suitable analytical technique such as mass spectrometry. In some embodiments, N-glycan units can be determined by tandem mass spectrometry liquid chromatography (LC-MS/MS) using instruments such as the Orbitrap Velos Pro™ mass spectrometer from Thermo Scientific, the Orbitrap Fusion Lumos Tribid™ mass spectrometer from Thermo Scientific or Waters Xevo® G2-XSQTof mass spectrometer.

As used herein, the term high mannose N-glycan is intended to mean an N-glycan having one to six or more mannose units. In at least one embodiment, the high mannose N-glycan unit may comprise a 6uc(Nacetylglucosamine) chain linked to an aspartic residue and further linked to a branched polymannose chain. As used herein, the interchangeable terms M6P or mannose-6-phosphate are intended to mean a mannose unit phosphorylated at position 6; those. having a phosphate group linked to a hydroxyl group at position 6. In at least one embodiment, one or more mannose units of one or more N-glycan units are phosphorylated at position 6 to form mannose-6 phosphate units.

As used herein, the term complex N-glycan is meant to mean an N-glycan containing one or more galactose and/or sialic acid units. In at least one embodiment, the N-glycan complex can be a high mannose N-glycan wherein one or more mannose units are further linked to one or more monosaccharide units each independently selected from N-acetylglucosamine, galactose, and sialic acid.

As used herein, a therapeutically effective dose and an effective amount are intended to mean an amount of acid α-glucosidase that is sufficient to elicit a therapeutic response in a subject. A therapeutic response may be any response that the user (eg, consultant physician) recognizes as an effective response to therapy, encompassing any surrogate clinical markers or symptoms described herein and known in the art. Thus, in at least one embodiment, the therapeutic response may be a reduction in intensity or inhibition of one or more symptoms or markers of Pompe disease known in the art. Symptoms or markers of Pompe disease include, without limitation, reduced tissue acid α-glucosidase activity; cardiomyopathy; cardiomegaly; progressive muscle weakness, especially in the trunk or lower extremities; deep hypotension; macroglossia (and in some cases protrusion of the tongue); difficulty swallowing, sucking and/or eating; respiratory failure

- 7 043224 accuracy; hepatomegaly (moderate); lethargy of the muscles of the face; areflexia; intolerance to physical activity; shortness of breath on exertion; orthopnea; sleep apnea; morning headaches; drowsiness; lordosis and/or scoliosis; decreased deep tendon reflexes; low back pain and inconsistency with key stages of motor development.

As used herein, the term enzyme replacement therapy or ERT is intended to mean the administration of a non-native purified enzyme to an individual deficient in that enzyme. The protein to be administered can be obtained from natural sources or through recombinant expression. The term also refers to the administration of a purified enzyme to an individual otherwise requiring or benefiting from the administration of a purified enzyme. In at least one embodiment, such an individual suffers from an enzyme deficiency. The enzyme administered may be a purified recombinant enzyme obtained in vitro, or a protein purified from isolated tissue or fluid, such as, for example, placenta or animal milk, or from plants.

As used herein, the term "pharmaceutically acceptable" is meant to mean molecular entities and compositions that are physiologically tolerable and do not typically cause adverse reactions when administered to a human. As used herein, the term "pharmaceutically acceptable" preferably means approved by a regulatory agency of the federal or state government or referred to in the USP or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans.

As used herein, the term excipient refers to a substance other than those included in the active ingredients, and is usually an inactive material. The adjuvant may assist in transporting the active drug to the site where the drug is to be effective, control the release of the active drug, or promote solubilization, or may perform a variety of other functions. Examples of excipients include, without limitation, buffering agents, surfactants, antimicrobials, antioxidants, excipients, stabilizer, tonicity regulators, and the like.

As used herein, the term buffer is intended to mean a solution containing both a weak acid and its conjugate weak base, the pH of which changes only slightly when an alkali or acid is added. As will be explained further below, in some embodiments, the buffer used in the pharmaceutical formulation is a nitrate and/or phosphate buffer.

As used herein, the terms subject or patient are intended to mean 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.

As used herein, the terms approximately and approximately are intended to mean an acceptable degree of error for the quantity being measured, taking into account the nature or accuracy of the measurements. For example, the degree of error may be indicated by the number of significant numbers provided for the measurement, as understood in the art, and includes, without limitation, a ±1 change in the most accurate significant number provided for the measurement. Typical illustrative degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of the specified value or range of values. Alternatively, and in particular in biological systems, the terms about and approximately can mean values that are within an order of magnitude, preferably within a 5-fold and more preferably within a 2-fold change in said value. Numerical values given in this document are approximate, unless otherwise indicated, which means that the term approximately or approximately may be indicative unless specifically stated.

Reference in this specification to one embodiment, certain embodiments, different embodiments, one or more embodiments, or an embodiment means that a particular feature, structure, material, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, phrases such as in one or more embodiments, in certain embodiments, in different embodiments, in the same embodiment, or in an embodiment appearing in different places in this specification do not necessarily refer to the same embodiment of this specification. inventions. In addition, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

rhGAAATB200.

The formulation contains ATB200, which is a recombinant GAA (rhGAA) protein suitable for use in enzyme replacement therapy. Details regarding the structure and

- 8 043224 ATB200, as well as options, are presented below.

In at least one embodiment, ATB200 is expressed in Chinese hamster ovary (CHO) cells and is characterized by an increased content of N-glycan units bearing one or more mannose-6-phosphate residues compared to N-glycan units bearing one or more residues of mannose-6-phosphate, in alglucosidase-alpha.

There are seven possible N-linked glycosylation sites in rhGAA. Because each glycosylation site is heterogeneous in the type of N-linked oligosaccharides (N-glycans) present, rhGAA is composed of a complex mixture of proteins with N-glycans having varying binding affinities for the M6P receptor and other carbohydrate receptors. RhGAA, which contains high-mannose N-glycans having one M6P group (mono-M6P), binds to CIMPR at low (~6000 nM) affinity, while rhGAA, which contains two M6P groups in the same N-glycan (6c-M6P ), binds with high (~2 nM) affinity. Representative constructs for non-phosphorylated mono-M6P and bis-M6P glycans are shown in FIG. 1A. The mannose-6-P group is shown in FIG. 1b. Once inside the lysosomes, rhGAA can enzymatically break down stored glycogen. However, traditional rhGAA have low overall levels of M6P and bis-M6P-bearing glycans and thus do not provide adequate targeting to muscle cells resulting in poor delivery of rhGAA to lysosomes. Efficient targeting of rhGAA drug is shown in FIG. 2A. Most of the rhGAA molecules in these conventional products do not have phosphorylated N-glycans, resulting in a lack of affinity for CIMPR. The mannose receptor can also be cleared of high mannose non-phosphorylated glycans resulting in unproductive ERT clearance (FIG. 2B).

Also present in rhGAA are other types of N-glycans, complex carbohydrates that contain galactose and sialic acids. Because complex N-glycans are non-phosphorylated, they lack affinity for CIMPR. However, complex-type N-glycans with accessible galactose residues have a moderate to high affinity for the hepatic hepatocyte asialoglycoprotein receptor, resulting in rapid unproductive clearance of rhGAA (FIG. 2B).

In at least one embodiment, the acid α-glucosidase is recombinant human acid α-glucosidase, referred to herein as ATB200, as described in co-pending International Patent Application PCT/US2015/053252. ATB200 has been shown to bind to cation-independent mannose-6phosphate receptors (CIMPR) with high affinity (K _D ~2-4 nM) and is efficiently internalized by Pompe disease fibroblasts and skeletal muscle myoblasts (K _uptake n _U i ~7-14 nM). ATB200 has been characterized in vivo and shown to have a shorter apparent plasma half-life (t _1/2 ~45 min) than alglucosidase-alpha (t _1/2 ~60 min).

In one or more embodiments, the human recombinant acid α-glucosidase has a wild-type GAA amino acid sequence listed under SEQ ID NO: 2 that corresponds to amino acid residues 57-952 and is identical to human wild-type (WT) GAA after its natural intracellular proteolytic processing. , which removes the first 56 residues containing the signal peptide and the precursor peptide. The amino acid sequence of ATB200 was confirmed by trypsin digestion followed by liquid chromatography/mass spectroscopy, as well as by protein sequencing. In contrast, the current standard of enzyme replacement therapy (ERT) using rhGAA (commercially available products containing alglucosidase-alpha in most countries is Myozyme® and in the US Lumizyme®, Genzym, Sanofi) differs from WT GAA and contains substitutions of 3 amino acid residues: at position 199, histidine is replaced by arginine, at position 223, arginine is replaced by histidine, and at position 780, valine is replaced by isoleucine.

In at least one embodiment, the recombinant human acid α-glucosidase undergoes post-translational and/or chemical modifications at one or more amino acid residues of the protein.

For example, methionine and tryptophan residues can be oxidized. As another example, aspartic residues can be deamidated to aspartic acid. As another example, aspartic acid may be isomerized to isoaspartic acid. Accordingly, in some embodiments, the enzyme is initially expressed as having the amino acid sequence listed under 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 invention.

In at least one embodiment, the recombinant human acid α-glucosidase has the wild type GAA amino acid sequence set forth under SEQ ID NO: 1 as described in US Patent No. 8,592,362 and has GenBank Accession Number AHE24104. . By

- 9 043224 in at least one embodiment, the recombinant human acid α-glucosidase is glucosidase alpha, a human acid α-glucosidase enzyme encoded by the most predominant of the nine observed GAA gene haplotypes.

In at least one embodiment, the recombinant human acid α-glucosidase is initially expressed as having the full-length 952 amino acid wild-type GAA sequence set forth under SEQ ID NO: 1, and the recombinant human acid α-glucosidase is intracellularly processed to remove part of the amino acids, for example, the first 56 amino acids. Accordingly, the recombinant human acid α-glucosidase secreted by the host cell may have a shorter amino acid sequence than the recombinant human acid α-glucosidase that is originally expressed in the cell. In at least one embodiment, the shorter protein may have an amino acid sequence as set forth under SEQ ID NO: 2, which differs from SEQ ID NO: 1 only in that the first 56 amino acids containing the signal peptide and the precursor peptide have been deleted from thereby resulting in a protein having 896 amino acids. Other differences 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 at compared to the amino acid sequence described under SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the rhGAA-based product contains a mixture of human recombinant acid α-glucosidase molecules having different amino acid lengths.

In at least one embodiment, the recombinant human acid α-glucosidase undergoes post-translational and/or chemical modifications at one or more amino acid residues of the protein. For example, methionine and tryptophan residues can be oxidized. As another example, N-terminal glutamine can form pyroglutamate. As another example, aspartic residues can be deamidated to aspartic acid. As another example, aspartic acid residues can be isomerized to isoaspartic acid. As another example, unpaired cysteine residues in a protein can form disulfide bonds with free glutathione and/or cysteine. Accordingly, in some embodiments, the enzyme is initially expressed as having the amino acid sequence listed under 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 invention.

Preferably, no more than 70%, 65%, 60%, 55%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total number of human recombinant acid α-glucosidase molecules lack the N-glycan unit bearing one or more mannose-6-phosphate residues, or no 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 human recombinant acid α-glucosidase molecules contain at least one N -glycan link bearing one or more residues of mannose-6-phosphate, or have the ability to bind to CIMPR.

Human recombinant acid α-glucosidase molecules can have 1, 2, 3 or 4 mannose-6-phosphate (M6P) groups in their glycans. For example, only one N-glycan in a molecule of human recombinant acidic α-glucosidase can carry M6P (monophosphorylated), a single N-glycan can carry two M6P groups (bisphosphorylated), or each of two different N-glycans in the same molecule of recombinant acidic Human α-glucosidase can carry distinct M6P groups. Human recombinant acid α-glucosidase molecules can also have N-glycans that do not carry M6P groups. In another embodiment, the N-glycans average more than 3 mol/mol M6P and more than 4 mol/mol sialic acid, whereby the recombinant human acid α-glucosidase contains an average of at least 3 mol of mannose-6-phosphate residues per mol of recombinant human acid α-glucosidase; and at least 4 mol of sialic acid per mol 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 human recombinant acid α-glucosidase may be in the mono-M6P-glycan form, e.g., about 6.25% of the total glycans can carry one M6P group, and on average at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans in human recombinant acid α-glucosidase are in the form of a bis-M6P-glycan, and on average less than 25% of the total number of human recombinant acid α-glucosidase molecules do not contain a phosphorylated glycan that binds to CIMPR.

The human recombinant acid α-glucosidase can have an average content of M6P-bearing N-glycans in the range of 0.5 to 7.0 mol/mol of human recombinant acid α-glucosidase, or any intermediate value in the 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 mol/mol recombinant human acid α-glucosidase. Recombinant human acid α-glucosidase can be fractionated to obtain preparations based on recombinant human acid α-glucosidase with different average number of glycans bearing

- 10 043224

M6P or carrying bis-M6P, thus allowing for further individual tailoring of targeted delivery of recombinant human α-acid α-glucosidase to lysosomes in target tissues by selecting a particular fraction or by selectively pooling different fractions.

In some embodiments, the recombinant human acid α-glucosidase will carry an average of 2.0 to 8.0 moles of M6P 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 mol M6P/mol recombinant human acid α-glucosidase.

Up to 60% of the N-glycans in human recombinant 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 to 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 acid α-glucosidase carry sialic acid and a terminal galactose (Gal) residue. This range includes all intermediate values and sub-ranges, for example, from 7 to 30% of the total number of N-glycans in human recombinant acid α-glucosidase can carry sialic acid and terminal galactose. In several other embodiments, no more than 5%, 10%, 15%, 16%, 17%, 18%, 19%, or 20% of the N-glycans in human recombinant acid α-glucosidase have only terminal galactose and do not contain sialic acid. This range includes all intermediate values and sub-ranges, for example, from 8 to 19% of the total number of N-glycans in the recombinant human α-glucosidase in the composition may have only terminal galactose and do not contain sialic acid.

In other embodiments, 40%, 45%, 50%, 55-60% of the total N-glycans in human recombinant acid α-glucosidase are complex-type N-glycans; or not more than 1, 2, 3, 4, 5, 6, 7% of the total number of N-glycans in human recombinant acid α-glucosidase are N-glycans of the hybrid type; not more than 5%, 10%, or 15% of the high-mannose-type N-glycans in human recombinant acid α-glucosidase are unphosphorylated; at least 5% or 10% of the high-mannose type N-glycans in human recombinant acid α-glucosidase are mono-M6P-phosphorylated; and/or at least 1 or 2% of the high-mannose type N-glycans in human recombinant acid α-glucosidase are bis-M6P-phosphorylated. These values include all intermediate values and sub-ranges. Recombinant human acid α-glucosidase may fall within one or more of the content ranges described above.

In some embodiments, the recombinant human acid α-glucosidase will carry 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 mol residues/mol recombinant human acid α-glucosidase. Without wishing to be bound by theory, it is believed that the presence of N-glycan units bearing sialic acid residues can prevent unproductive clearance of human recombinant acid α-glucosidase by asialoglycoprotein receptors.

In one or more embodiments, rhGAA has M6P and/or sialic acid units at specific N-glycosylation sites on a recombinant human lysosomal protein. For example, there are seven potential N-linked glycosylation sites in rhGAA. These potential glycosylation sites are at the following positions of SEQ ID NO: 2: N84, N177, N334, N414, N596, N826 and N869. Similarly, for the full length amino acid sequence of SEQ ID NO: 1, these potential glycosylation sites are at the following positions: N140, N233, N390, N470, N652, N882 and N925. Other rhGAA variants may have similar glycosylation sites depending on the location of the aspartic residues. Generally, the sequences ASN-XSER or ASN-X-THR in the amino acid sequence of a protein indicate potential glycosylation sites, except that X cannot be HIS or PRO.

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 (eg, N84 in SEQ ID NO: 2 and N140 in 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 may be phosphorylated at the first N-glycosylation site. This phosphorylation may result from the presence of mono-M6P and/or 6c-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 rhGAA carry a mono-M6P moiety in 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 rhGAA carry the 6c-M6P moiety in the first N-glycosylation site.

In one or more embodiments, at least 20% of rhGAA are phospho

- 11 043224 rylated at the second N-glycosylation site (eg N177 in SEQ ID NO: 2 and N223 in 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 may be phosphorylated at the second N-glycosylation site. This phosphorylation may result from the presence of mono-M6P and/or 6c-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 rhGAA carry a mono-M6P moiety in second N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 6θ, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA carry a 6c-M6P moiety in second N-glycosylation site. In one or more embodiments, at least 5% of rhGAA is phosphorylated at the third N-glycosylation site (eg, N334 in SEQ ID NO: 2 and N390 in SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA are phosphorylated at the third N-glycosylation site. For example, the third N-glycosylation site may have a combination of non-phosphorylated high mannose glycans, two-, three-, and four-antennary complex glycans, and hybrid glycans as major forms. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of rhGAA are sialylated at the third N-glycosylation site.

In one or more embodiments, at least 20% of rhGAA is phosphorylated at the fourth N-glycosylation site (eg, N414 in SEQ ID NO: 2 and N470 in 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 may be phosphorylated at the fourth N-glycosylation site. This phosphorylation may result from the presence of mono-M6P and/or 6c-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 rhGAA carry a mono-M6P moiety in fourth site of N-glycosylation. 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 rhGAA carry the 6c-M6P moiety in fourth site of N-glycosylation. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of rhGAA are sialylated at the fourth N-glycosylation site.

In one or more embodiments, at least 5% of rhGAA is phosphorylated at the fifth N-glycosylation site (eg, N596 in SEQ ID NO: 2 and N692 in SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA are phosphorylated at the fifth N-glycosylation site. For example, the fifth N-glycosylation site may have fucosylated two-antennary complex glycans as the main forms. In some embodiments, at least 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 6θ, 65, 70, 75, 80, 85, 90, or 95% rhGAA are sialylated at the fifth N-glycosylation site.

In one or more embodiments, at least 5% of rhGAA is phosphorylated at the sixth N-glycosylation site (eg, N826 in SEQ ID NO: 2 and N882 in SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA are phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site may have a combination of two-, three-, and four-antennary complex glycans as major forms. In some embodiments, at least 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% rhGAA are sialylated at the sixth N-glycosylation site.

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

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

Recombinant human α-glucosidase is preferably produced using Chinese hamster ovary (CHO) cells such as CHO cell lines GA-ATB200 or ATB200-001-X5-14, or a subculture or derivative of such a CHO cell culture. DNA constructs that express

- 12 043224 allele variants of acid α-glucosidase or amino acid sequences of other variants of acid α-glucosidase, such as, for example, at least 90, 95 or 99% identical to SEQ ID NO: 1, can be constructed and expressed in CHO cells . These amino acid sequences of acid α-glucosidase variants may contain deletions, substitutions and/or insertions compared 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 compared to the amino acid sequence described under SEQ ID NO: 1. Alternative vectors suitable for transformation of CHO cells can be selected by those skilled in the art to obtain such DNA constructs.

To calculate the identity of two sequences, various alignment algorithms and/or programs can be used, including FASTA or BLAST, which are available as part of the GCG sequence analysis software package (University of Wisconsin, Madison, Wisconsin) and can be used, for example, with default settings. For example, polypeptides are provided that are at least 90%, 95%, or 99% identical to the specific polypeptides described herein, and preferably exhibit substantially the same functions, as well as polynucleotides encoding such polypeptides. Unless otherwise stated, the calculation of the similarity score will be based on the application of BLOSUM62. When using BLASTP, percent similarity is based on a positive BLASTP score and percent sequence identity is based on a BLASTP identity score. Identical in BLASTP shows the number of residues in pairs of high-scoring sequences that are identical and their proportion of the total number of residues; and the Positive score in BLASTP shows the number and proportion of residues for which the alignment scores are positive and are similar to each other. The present invention contemplates and embraces amino acid sequences having these degrees of identity or similarity, or any intermediate degree of identity or similarity, to the amino acid sequences disclosed herein. Polynucleotide sequences of similar polypeptides are derived from the genetic code and can be obtained using conventional methods, in particular by restoring their amino acid sequences using the genetic code.

In some embodiments, a human recombinant acid α-glucosidase having superior ability to target cation-independent mannose-6-phosphate receptors (CIMPR) and cellular lysosomes, as well as glycosylation patterns that reduce unproductive clearance from it in vivo, can be obtained by using Chinese hamster ovary (CHO) cells. These cells can be induced to express human recombinant acid α-glucosidase with significantly higher levels of N-glycan units bearing one or more mannose-6-phosphate residues than traditional human recombinant acid α-glucosidase products such as alglucosidase-alpha. . The recombinant human α-glucosidase produced by these cells, exemplified by ATB200, has a significantly higher content of N-glycan residues mannose-6-phosphate (M6P) and bis-mannose-6-phosphate, which provide a targeted effect on muscle cells than the traditional one. acid α-glucosidase such as Lumizyme®. Without wishing to be bound by any theory, it is believed that this extensive glycosylation allows the ATB200 enzyme to be more efficiently taken up by target cells, and therefore undergo more efficient clearance from the circulation, than other recombinant human acid α-glucosidases, such as, for example, alglucosidase-alpha, which has a significantly lower content of M6P and 6c-M6P. ATB200 has been shown to potently bind to CIMPR and is efficiently taken up by skeletal muscle and cardiac muscle, and has a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces waste in vivo.

It is also contemplated that extensive glycosylation of ATB200 may contribute to a reduction in the immunogenicity of ATB200 compared to, for example, alglucosidase-alpha. As will be recognized by those skilled in the art, glycosylation of proteins with conservative mammalian sugars generally improves the solubility of the product and reduces the aggregation and immunogenicity of the product. Glycosylation indirectly alters the immunogenicity of a protein by minimizing protein aggregation as well as by shielding immunogenic epitopes of the protein from the immune system (Guidance for Industry - Immunogenicity Assessment for Therapeutic Protein Products, U.S. Department of Health and Human Services, Food and Drug Administration , Center for Drug Evaluation and Research, Center for Biological Product Evaluation and Research, August 2014). Therefore, in at least one embodiment, the administration of recombinant human α-acid α-glucosidase does not induce the production of antibodies to the drug. In at least one embodiment, administration of human recombinant acid α-glucosidase induces the production of anti-drug antibodies in the subject to a score lower than the level of anti-drug antibodies,

- 13 043224 the production of which is induced by the introduction of alglucosidase-alpha.

As described in co-pending International Patent Application PCT/US2015/053252, cells such as CHO cells can be used to produce the rhGAA described herein, and this rhGAA can be used according to the present invention. Examples of such a CHO cell line are GA-ATB200 or ATB200-001-X5-14 or a subculture thereof which produces the rhGAA-based composition described herein. Such CHO cell lines may contain multiple copies of a gene, such as 5, 10, 15, or 20 or more copies of a GAA-encoding polynucleotide.

rhGAA rich in M6P and 6c-M6P, such as rhGAA ATB200, can be obtained by transforming CHO cells with a DNA construct that encodes GAA. Although CHO cells have previously been used to produce rhGAA, it has not been recognized that transformed CHO cells can be cultured and selected to produce rhGAA having a high content of M6P and bis-M6P glycans that provide a targeted effect on CIMPR.

Surprisingly, it has been found that it is possible to transform CHO cell lines, select transformants that produce glycan-rich rhGAA bearing M6P or 6c-M6P that target CIMPR, and stably express the M6P-rich rhGAA. Thus, methods for obtaining these CHO cell lines are also described in the international patent application PCT/US2015/053252, which is simultaneously pending. The method involves transforming a CHO cell with DNA encoding GAA or a GAA variant, selecting a CHO cell whose chromosome(s) stably integrates DNA encoding GAA and which stably expresses GAA, and selecting a CHO cell that expresses GAA having a high content of glycans bearing M6P or 6c-M6P; and optionally selecting a CHO cell having N-glycans high in sialic acid and/or having a low content of non-phosphorylated high-mannose N-glycans. These CHO cell lines can be used to produce rhGAA and rhGAA based compositions by culturing the CHO cell lines and extracting said composition from the CHO cell culture.

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 additional embodiments, ATB200 is present in an amount of about 15 mg/mL.

pH value and buffer.

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 is in the range of 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. Typically, when the specified amounts of components are observed, the pH will be in the range of from about 5.0 to about 6.0. However, the pH can be adjusted to achieve the target pH by using pH adjusters (ie alkalizing agents and acidifying agents) such as sodium hydroxide and/or hydrochloric acid.

The formulation also contains a buffer that is selected from the group consisting of a citrate, phosphate, and combinations thereof. As used herein, a buffer means a buffer solution containing a weak acid and an acid conjugate base that helps prevent changes in pH. The citrate and/or phosphate may be sodium citrate or sodium phosphate. Other salts include potassium or ammonium salts. In one or more embodiments, the buffer contains citrate. In additional embodiments, the buffer contains sodium citrate (eg, a mixture of sodium citrate dihydrate and citric acid monohydrate). In one or more embodiments, citrate-containing buffer solutions may contain sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffer are present.

Excipients.

The composition also contains at least one excipient. Some embodiments of the present invention contain adjuvants that aid in the regulation of tonicity, acting as fillers or as stabilizers. In one or more embodiments, at least one excipient is selected from the group consisting of mannitol, polysorbate, and combinations thereof.

In one or more embodiments, the excipient contains an ingredient that can act as a tonicity regulator and/or excipient, in particular mannitol. Tonicity agents are components that contribute to the

- 14 043224 to the requirement that the composition has an osmotic pressure the same as the osmotic pressure of human blood or similar to it. Fillers are ingredients that give additional volume to the composition (for example, lyophilized) and provide the necessary structure of the lyophilisate.

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

In some embodiments, the adjuvant contains an ingredient that can act as a stabilizer, such as polysorbate 80. Stabilizers are compounds that can prevent or minimize aggregation of the formulation at hydrophobic air/water interfaces. In some embodiments, the implementation of 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 additional embodiments, the total amount of stabilizer is in the range 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 other embodiments, the total amount of stabilizer 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 In some embodiments, polysorbate 80 is the sole stabilizer. Thus, in one or more embodiments, the content of polysorbate 80 is in the range of about 0.1 to about 1.0 mg/mL. In additional embodiments, the content of polysorbate 80 is in the range 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 yet other embodiments, the implementation of the content of polysorbate 80 is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 0.9 or 1.0 mg / ml.

In one or more embodiments, it may be desirable to exclude certain compounds from the formulation. For example, in the course of formulating a formulation for the treatment of a given disease, it would be desirable to exclude certain compounds that may exacerbate the underlying disease. As noted above, individuals with Pompe disease have reduced or no capacity for glycogen catabolism. Several commonly used excipients, such as sucrose, trehalose, and glycine, can be converted to glucose in the body, which in turn can exacerbate Pompe disease. Thus, in some embodiments, sucrose, trehalose and/or glycine are excluded from the formulation. Similarly, it is possible to exclude certain components that in certain contexts are not suitable for the formulation. For example, in some embodiments, poloxamers can be excluded.

Illustrative Embodiments.

In one or more embodiments, the implementation of the composition contains:

(a) ATB200 (e.g., recombinant acid α-glucosidase, wherein the recombinant acid α-glucosidase is derived from expression in Chinese Hamster Ovary (CHO) cells and is characterized by an increased content of N-glycan units bearing one or two mannose-6-phosphate residues compared to the content of N-glycan units bearing one or two mannose-6phosphate residues in alglucosidase-alpha);

(b) at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof;

(c) at least one excipient selected from the group consisting of or consisting of mannitol, polysorbate 80, and combinations thereof, and wherein the formulation has a pH of from about 5.0 to about 6.0 or 7 ,0.

In some embodiments, the implementation of the composition contains:

(a) ATB200 (e.g., recombinant acid α-glucosidase, wherein the recombinant acid α-glucosidase is derived from expression in Chinese Hamster Ovary (CHO) cells and is characterized by an increased content of N-glycan units bearing one or two mannose-6 residues

- 15 043224 phosphate, in comparison with the content of N-glycan units bearing one or two mannose-6-phosphate residues in alglucosidase-alpha);

(b1) sodium citrate;

(b2) citric acid monohydrate;

(c1) mannitol;

(c2) polysorbate 80;

(d) water;

(e) optionally an acidifying agent; and (f) optionally, or consists of an alkalizing agent, and wherein the composition has a pH of from about 5.0 to about 6.0 or 7.0. In further embodiments, the pH is in the range of about 5.5 to about 6.0. In still other embodiments, the pH is about 6.0.

In a specific embodiment, the composition contains:

(a) ATB200 (e.g., recombinant acid α-glucosidase, wherein the recombinant acid α-glucosidase is derived from expression in Chinese Hamster Ovary (CHO) cells and is characterized by an increased content of N-glycan units bearing one or two mannose-6-phosphate residues compared to the content of N-glycan units bearing one or two mannose-6phosphate residues in alglucosidase-alpha), present at a concentration of about 5-30 mg/ml or about 15 mg/ml;

(b) sodium citrate buffer present at a concentration of about 10100 mM or about 25 mM;

(c1 ) mannitol present at a concentration of about 10-50 mg/ml or about 20 mg/ml;

(c2) polysorbate 80 present at a concentration of about 0.1-1 mg/ml or about 0.5 mg/ml; and (d) water;

(e) optionally an acidifying agent; and (f) optionally an alkalizing agent, and wherein the composition has a pH of about 5.0 to about 6.0 or 7.0. In further embodiments, the pH is in the range of about 5.5 to about 6.0. In still other embodiments, the pH is about 6.0.

Getting composition.

Another aspect of the present invention relates to methods for obtaining the compositions described in this document. In one or more embodiments, the implementation of the composition can be obtained from the solution of the enzyme. Concentration of this solution and buffer exchange to obtain the target concentration and required buffers can be carried out using methods known in the art. Further components (eg excipients and pH adjusters) can then be added. The composition can then be filtered, placed in a storage container and stored.

In one or more embodiments, the method for preparing any of the pharmaceutical compositions described herein comprises: adding at least one buffer, at least one excipient, and recombinant acid α-glucosidase to water to form a solution; optional adjustment of the pH of the solution; and optionally adding an additional volume of water to the solution. In additional embodiments, the method further comprises filtering the solution. In still other embodiments, the method further comprises storing the solution.

An exemplary non-limiting method for preparing the formulation described herein is provided below.

1. Approximately 85-90% of the total water for injection is added to a suitable production tank.

2. Add and dissolve the required excipients and buffers (eg 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 target pH (eg 6±0.1), if necessary, using regulators (eg hydrochloric acid or sodium hydroxide solution).

5. Add enough water for injection to reach the final volume and mix.

6. Filter the solution through a sterile filter into a sterile receiver.

7. Under aseptic conditions, the drug solution is poured into vials and sealed with stoppers.

- 16 043224

8. Cap all vials and store at 2 to 8°C.

In one or more embodiments, the fresh formulation will be in liquid form. It is understood that the composition contains water. This liquid formulation can be subjected to a lyophilization (freeze-drying) process to obtain a lyophilisate or powder. Accordingly, another aspect of the present invention relates to a pharmaceutical composition containing any of the formulations described above, subjected to lyophilization. The lyophilized mixture may contain: ATB200; a buffer selected from the group consisting of 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 ingredients (eg, other excipients) may be added to the lyophilized mixture. A pharmaceutical composition comprising a lyophilized preparation may be provided in a vial, after which it may be stored, transported, reconstituted and/or administered to a patient.

A method of treatment and administration to a patient.

Another aspect of the present invention relates to a method for the treatment of Pompe disease and/or the use of the compositions described herein for the treatment of Pompe disease. The formulation can be administered immediately after preparation or after lyophilization and reconstitution. Thus, in one or more embodiments, the method comprises administering to a patient in need thereof any of the pharmaceutical formulations described above. In other embodiments, the method includes reconstituting the 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 the same component composition as the pharmaceutical composition prior to lyophilization and/or freshly prepared pharmaceutical composition. In any case, the pharmaceutical composition or reconstituted pharmaceutical composition may be diluted prior to administration to a patient. In additional embodiments, the implementation of the pharmaceutical composition or reconstituted pharmaceutical composition is administered intravenously.

In one or more embodiments, the intravenous composition is a solution in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic to reduce pain at the injection site. Typically, the ingredients are supplied either singly or mixed together in a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet, indicating the amount of active agent. If the composition is to be administered by infusion, it may be dispensed into an infusion bottle containing sterile pharmaceutical grade water, saline, or a dextrose/water mixture. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

In other embodiments, the pharmaceutical composition or reconstituted pharmaceutical composition is administered by direct injection into a target tissue such as cardiac or skeletal muscle (e.g., intramuscularly) or into the nervous system (e.g., by direct injection into the brain; intraventricular; intrathecal). More than one path can be used at the same time if necessary.

The pharmaceutical composition or reconstituted composition is administered in a therapeutically effective amount (e.g., at a dosage level that, when administered at regular intervals, is sufficient to treat the disease, in particular to reduce the severity of symptoms associated with the disease, prevent or delay the onset of the disease, and/or to reducing the severity or frequency of symptoms of the disease). The amount that will be therapeutically effective in the treatment of a disease will depend on the nature and extent of the effects of the disease and can be determined using standard clinical techniques. Additionally, in vitro or in vivo assays may optionally be used to assist in selecting optimal dose ranges. In at least one embodiment, human recombinant acid α-glucosidase is administered by intravenous infusion at a dose of from about 1 mg/kg to about 100 mg/kg, such as from about 5 mg/kg to about 30 mg/kg , typically from about 5 mg/kg to about 20 mg/kg. In at least one embodiment, human recombinant acid α-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 , approximately 30 mg/kg, approximately 35 mg/kg, approximately 40 mg/kg, approximately 50 mg/kg, approximately 50 mg/kg, approximately 60 mg/kg, approximately 70 mg/kg, approximately 80 mg/kg, approximately 90 mg/kg or approximately 100 mg/kg. In at least one embodiment, recombinant human α-glucosidase is administered by intravenous infusion at a dose of about 20 mg/kg. The effective dose for a particular individual may vary (eg, increase or decrease) over time.

- 17 043224 depending on the needs of the individual. For example, the amount can be increased during times of medical illness or stress, or when anti-acid α-glucosidase antibodies appear or increase, or when the symptoms of the disease worsen.

A therapeutically effective amount of recombinant human recombinant acid α-glucosidase (or composition or drug product containing recombinant human recombinant acid α-glucosidase) is administered at regular intervals depending on the nature and severity of the effects of the disease and on an ongoing basis. Administration at regular intervals, as used herein, indicates that a therapeutically effective amount is administered intermittently (as opposed to a single dose). The interval can be determined using standard clinical techniques. In preferred embodiments, human recombinant acid α-glucosidase is administered monthly, once or twice a month; once a week; twice a week or daily. The administration interval for the needs of an individual does not have to be a fixed interval and may vary over time depending on the needs of the individual. For example, the interval between doses can be reduced during times of medical illness or stress, or when antibodies to recombinant human recombinant acid α-glucosidase develop or increase, or when symptoms of the disease worsen.

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 mg to about 400 mg, or 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 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 approximately 260 mg.

In at least one embodiment, miglustat and recombinant human α-glucosidase are administered simultaneously. In at least one embodiment, miglustat and recombinant human acid α-glucosidase are administered sequentially. In at least one embodiment, the miglustat is administered prior to the administration of the recombinant human acid α-glucosidase. In at least one embodiment, the miglustat is administered less than three hours prior to administration of the human recombinant acid α-glucosidase. In at least one embodiment, the miglustat is administered approximately two hours prior to administration of the recombinant human α-acid α-glucosidase. In at least one embodiment, the miglustat is administered less than two hours prior to administration of the human recombinant acid α-glucosidase. In at least one embodiment, miglustat is administered approximately 1.5 hours prior to administration of the recombinant human α-acid α-glucosidase. In at least one embodiment, the miglustat is administered approximately one hour prior to administration of the human recombinant acid α-glucosidase. In at least one embodiment, the miglustat is administered from about 50 minutes to about 70 minutes prior to administration of the human recombinant acid α-glucosidase. In at least one embodiment, miglustat is administered from about 55 minutes to about 65 minutes prior to administration of the human recombinant acid α-glucosidase. In at least one embodiment, the miglustat is administered about 30 minutes prior to administration of the recombinant human α-acid α-glucosidase. In at least one embodiment, miglustat is administered from about 25 minutes to about 35 minutes prior to administration of the human recombinant acid α-glucosidase. In at least one embodiment, the miglustat is administered from about 27 minutes to about 33 minutes prior to administration of the human recombinant acid α-glucosidase.

Kit.

Another aspect of the present invention relates to kits containing the pharmaceutical compositions described herein (including subjected to lyophilization). In one or more embodiments, the kit contains a container (eg, vial, vial, pouch, etc.) containing pharmaceutical formulations (lyophilized or not) and instructions for reconstitution, reconstitution, and administration.

- 18 043224

Examples

Enzyme Example 1 Restrictions on currently available MYOZYME® and LUMIZYME® rhGAA based products.

To assess the properties of rhGAA in Myozyme® and Lumizyme®, the only currently approved treatments for Pompe disease, these rhGAA preparations were injected into a CIMPR column (which binds to rhGAA having M6P groups) and then eluted with a gradient free M6. Fractions were collected in a 96-well plate, and GAA activity was analyzed using a substrate representing 4MU-α-glucose. Relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a percentage of total enzyme.

In FIG. 3A-B show problems with traditional ERT agents (Myozyme® and Lumizyme®): 73% rhGAA in Myozyme® (Fig. 3B) and 78% rhGAA in Lumizyme® (Fig. 3A) did not bind to CIMPR, see the leftmost peaks on each figure. Only 27% of rhGAA in Myozyme® and 22% of rhGAA in Lumizyme® contained M6P, which can efficiently target it to CIMPR in muscle cells.

An effective dose of Myozyme® and Lumizyme® corresponds to the amount of rhGAA containing M6P that targets CIMPR in muscle cells. However, most of the rhGAA in these two conventional products does not target the CIMPR receptor in the target muscle cells. Administration of conventional rhGAA, where most of the rhGAA does not target muscle cells, increases the risk of an allergic reaction or induction of an immune response to the untargeted rhGAA.

Enzyme Example 2 Preparation of cho cells producing rhgaa atb200 having a high content of n-glycans bearing mono- or bis-m6p.

CHO cells were transfected with DNA that expresses rhGAA, followed by selection of transformants that produce rhGAA. A DNA construct for transforming CHO cells with DNA encoding rhGAA is shown in FIG. 4. CHO cells were transfected with DNA that expresses rhGAA, followed by selection of rhGAA producing transformants.

After transfection, CHO-DG44 (DHFR-) cells containing a stably integrated GAA gene were selected on hypoxanthine/thymidine (-HT)-free medium. Upregulation of GAA expression in these cells was induced by treatment with methotrexate (MTX 500 nM). Cell populations that expressed GAA at high levels were identified by GAA enzyme activity assays and used to create single clones producing rhGAA. Individual clones were obtained in culture dishes with semi-liquid medium, collected using the ClonePix system and transferred to plates with 24 deep wells. Individual clones were analyzed for GAA enzyme activity to identify clones expressing GAA at a high level. Used conditioned media for determining the activity of GAA was a substrate - 4-MU-a-glucosidase. Clones producing GAA at higher levels, as measured by GAA enzyme assays, were further evaluated for viability, growth ability, GAA production productivity, N-glycan structure, and stable protein expression. CHO cell lines, including the CHO cell line GA-ATB-200 expressing N-glycan-enhanced rhGAA with mono-M6P or 6c-M6P, were isolated using this procedure.

Enzyme example 3. Capture and purification of rhGAA ATB200.

Several batches of rhGAA according to the present invention were prepared in shake flasks and perfusion bioreactors using the CHO GA-ATB-200 cell line and binding to CIMPR was measured. Purified rhGAA ATB200 from various production lots showed binding to the CIMPR receptor similar to that shown in FIG. 5B and FIG. 6 (~70%) indicating that rhGAA ATB200 can be stably produced. As shown in FIG. 3A-B and 5A-B, Myozyme® and Lumizyme® showed significantly less binding to CIMPR than rhGAA ATB200.

Enzyme Example 4 Analytical Comparison of ATB200 and LUMIZYME®.

Weak anion exchange liquid chromatography (WAX) was used to fractionate rhGAA ATB200 at terminal phosphate. Elution profiles were obtained by eluting the ERT agent with increasing amounts of salt. The profiles were controlled using UV radiation (A280 nm). rhGAA ATB200 was obtained from CHO cells and purified. Lumizyme® was obtained from a commercial source. Lumizyme® showed a high peak on the left side of its elution profile. rhGAA ATB200 showed four distinct elution peaks to the right of Lumizyme® (FIG. 7). This confirms that rhGAA ATB200 was phosphorylated to a greater extent than Lumizyme®, since this assessment was done by terminal charge and not by CIMPR affinity.

Enzyme Example 5 Characterization of oligosaccharides B rhGAA ATB200.

Purified glycans rhGAA ATB200 and Lumizyme® were evaluated using MALDI-TOF to determine the structures of individual glycans detected in each tool for ERT (Fig. 8). ATB200 samples were found to contain lower amounts of non-phosphorylated high-mannose type N-glycans than Lumizyme®. Higher content of M6P-glycans in ATB200 than in Lumizyme®,

- 19 043224 allows rhGAA ATB200 to target muscle cells with greater efficiency. The high percentage of monophosphorylated and bisphosphorylated structures as determined by MALDI is consistent with the CIMPR binding profiles, which illustrate a significantly greater degree of ATB200 binding to the CIMPR receptor. Analysis of N-glycans by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contains at least one natural bis-M6P-N-glycan structure. This higher content of 6uc-M6P-Nglycans in rhGAA ATB200 directly correlates with high affinity CIMPR binding in plate M6P receptor binding assays (KD was approximately 2-4 nM), FIG. 10A.

rhGAA ATB200 was also analyzed for site-specific N-glycan profiles using two different LC-MS/MS based analytical techniques. In the first assay, the protein was denatured, reduced, alkylated, and cleaved prior to LC-MS/MS analysis. During protein denaturation and reduction 200 µg protein sample, 5 µl 1 mol/l TrisHCl (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 of 1 mol/l DTT (final concentration 20 mM) and Milli-Q® water were added to a 1.5 ml tube to give a total volume of 100 μl. The sample was mixed and incubated at 56°C for 30 min in a dry heat bath. During alkylation, the denatured and reduced protein sample was mixed with 5 µl of 1 mol/l iodoacetamide (IAM, final concentration 50 mM), then incubated at 10-30°C in the dark for 30 min. After alkylation, 400 µl of pre-chilled acetone was added to the sample, and the mixture was subjected to freeze-refrigeration at -80°C for 4 hours. Then, the sample was centrifuged for 5 min at 13,000 rpm and 4°C, and the supernatant was removed. 400 µl of pre-chilled acetone was added to the pellet, which was then centrifuged for 5 min at 13,000 rpm and 4° C., and the supernatant was removed. The sample was then air dried on ice in the dark to remove residual acetone. To dissolve the protein, 40 μl of 8M urea and 160 μl of 100 mM NH ₄ HCO ₃ were added to the sample. During trypsin digestion, trypsin digestion buffer was then added to 50 μg of protein to a final volume of 100 μl and 5 μl of 0.5 mg/ml trypsin was added (protein to enzyme ratio 20/1 w/w). The solution was thoroughly mixed and incubated overnight (16±2 h) at 37°C. 2.5 μl of 20% TFA was added to quench the reaction (0.5% final concentration). The sample was then analyzed using an Orbitrap Velos Pro™ mass spectrometer from Thermo Scientific.

In a second LC-MS/MS analysis, an ATB200 sample was prepared following a similar denaturation, reduction, alkylation, and cleavage procedure, except that iodoacetic acid (IAA) was used as the alkylating reagent instead of IAM, and then analyzed by mass Thermo Scientific Orbitrap Fusion Lumos Tribid™ spectrometer.

The results of the first and second analyzes are shown in FIG. 9A-9H. In FIG. 9A-9H, the results of the first analysis are represented by the bars on the left (dark gray), and the results of the second analysis are represented by the bars on the right (light gray). In FIG. 9B-9G, the symbol nomenclature for representing glycans is that of Varki, A., Cummings, R.D., Esko J.D., et al., Essentials of Glycobiology, 2nd edition (2009).

In FIG. 9A-9G, it can be seen that the two assays gave similar results, although there was some difference between the results. This difference may be due to a number of factors, including the instrument used and the completeness of the N-glycan analysis. For example, if some forms of phosphorylated glycans have not been identified and/or quantified, then the total number of phosphorylated glycans may be underrepresented, and the percentage of rhGAA bearing phosphorylated glycans at a given site may be underrepresented. As another example, if some forms of non-phosphorylated glycans have not been identified and/or quantified, then the total number of non-phosphorylated glycans may be underrepresented, and the percentage of rhGAA bearing phosphorylated glycans at a given site may be overrepresented. quantity. In FIG. 9A shows N-glycosylation site occupancy in ATB200. As can be seen in FIG. 9A, the first, second, third, fourth, fifth, and sixth N-glycosylation sites were predominantly occupied, with both analyzes revealing that at each potential site, more than 90% to about 100% of the ATB200 enzyme molecules had a glycan. However, the seventh potential N-glycosylation site was glycosylated in approximately half of the cases.

In FIG. 9B shows the N-glycosylation profile for the first site, N84. In FIG. 9B, it can be seen that the major form of the glycans were bis-M6P glycans. Both the first and second analyzes revealed that more than 75% of ATB200 had a bis-M6P glycan at the first site.

In FIG. 9C shows the N-glycosylation profile for the second site, N177. As can be seen in FIG. 9C, the major forms of glycans were mono-M6P glycans and non-phosphorylated high mannose glycans. Both the first and second analyzes revealed that more than 40% of ATB200 had a monoM6P glycan at the second site.

In FIG. 9D shows the N-glycosylation profile for the third site, N334. In FIG. 9D you can see

- 20 043224 that the main forms of glycans were non-phosphorylated high-mannose glycans, two-, three- and four-antennary complex glycans and hybrid glycans. Both the first and second analyzes revealed that more than 20% of ATB200 had a sialic acid residue at the third site.

In FIG. 9E shows the N-glycosylation profile for the fourth site -N414. In FIG. 9E, it can be seen that the main forms of glycans were 6c-M6P and mono-MGP glycans. Both the first and second analyzes revealed that more than 40% of ATB200 had a bis-M6P-glycan at the fourth site. Both the first and second analyzes also revealed that more than 25% of ATB200 had a mono-M6P-glycan at the fourth site.

In FIG. 9F shows the N-glycosylation profile for the fifth site, N596. In FIG. 9F, it can be seen that the main forms of glycans were fucosylated two-antennary complex glycans. Both the first and second analyzes revealed that more than 70% of ATB200 had a sialic acid residue at the fifth site.

In FIG. 9G shows the N-glycosylation profile for the sixth site, N826. As can be seen in FIG. 9G, the main forms of glycans were two-, three-, and four-antennary complex glycans. Both the first and second analyzes revealed that more than 80% of ATB200 had a sialic acid residue at the sixth site.

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

In FIG. 9H shows a summary of phosphorylation information for each of the seven potential N-glycosylation sites. In FIG. 9G, it can be seen that both the first and second assays showed high levels of phosphorylation at the first, second, and fourth sites. In both assays, more than 80% of ATB200 was mono- or diphosphorylated at the first site, more than 40% of ATB200 was monophosphorylated at the second site, and more than 80% of ATB200 was mono- or diphosphorylated at the fourth site.

Another analysis of ATB200 glycans was performed according to the hydrophilic interaction liquid chromatography-fluorescence detection mass spectrometry (HILIC-FLDMS) method.

The results of the analysis of HILIC-FLD-MS are presented in table. A below. In table. A The first digit in the three-digit digit indicates the number of branches in the glycan, the second digit indicates the number of basic fucose units, and the third digit indicates the number of terminal sialic acid units. Using this nomenclature, 303 is a three-antennary glycan (first 3) with 0 basic fucose (2nd 0) and 3 terminal sialic acids (last 3), 212 is a two-antennary glycan with 1 basic fucose and 2 terminal sialic acids, 404 represents is a four-antennary glycan with 0 basic fucose and 4 terminal sialic acids, etc.

_________________________Table A_________________________

Peak FLD Peak MS RT (min) Glycan structure % area peak 1 1 BisP Map 8 2.83% 2 2 3 13.41 BisP Map 7 17.58% 14.30 BisP Map 6 1.02% 3 4 5 6 20.89 Mopor Map 6 2.34% 4 21.65 Mopor Map 5 1.16% 5 23.51 Mopor Map 8 1.28%

- 21 043224

6 7 24.33 MonoP Man 7 4.35% 7 8 25.61 MonoP Man 7 (+)GlcNAc 0.50% 8 9 28.76 MonoP hMan6 101 0.48% 9 10 30.54 MonoP Man 6 (+)GlcNAc 0.68% 10 eleven 33.50 Man 6 3.97% 12 33.65 303 0.74% eleven 13 34.97 Man 7 0.20% 12 14 35.64 403 0.39% 13 15 36.61 302 0.36% 14 16 38.07 302 0.61% 15 17 38.53 man 5 1.85% 16 18 39.57 302 0.48% 19 39.78 hMan5 101 0.42% 20 40.05 hMan5 100 (-)Gal 0.30% 17 21 40.77 301 (-)Gal 0.52% 22 40.58 301 0.50% 18 23 41.47 300_(-)Gal 0.80% 19 24 42.17 301 (-)Gal 0.11% 25 42.13 301 0.58% 20 26 42.89 301 (-)Gal 0.07% 27 42.79 301 0.80% 21 28 43.41 300 0.85% 29 43.28 101 0.39% 22 thirty 43.94 202 0.63% 23 31 44.45 401 0.39% 24 32 45.04 MonoP_hMan6_l 11 0.36% 25 33 45.69 MonoP hMan6 111 1.45% 34 45.90 100 0.23% 35 45.90 400 0.19% 26 36 46.87 201 0.49% 37 47.15 202 0.34% 27 38 48.19 414 0.37% 28 39 48.94 202 1.97% 29 40 50.79 MonoP Man 6 110 (-)Gal 1.31% 41 51.37 414 0.62% thirty 42 52.22 313 0.74% 43 52.42 201 (-)Gal 0.46% 44 52.42 201 1.18% 45 53.11 hMan6 111 0.20% 31 46 53.83 200 (-)Gal 0.80% 47 54.23 201 1.27% 48 54.75 413 0.30%

- 22 043224

32 49 55.47 200 1.30% 33 50 57,45,58,34 414 (+)GlcNAcGal 0.14% 51 56,62,56,91,57, 99 413 0.94% 52 56,11,57,26,57, 99 312 0.98% 34 53 60.19 413 0.33% 54 59.39 413 (+)GlcNAcGal 0.42% 55 59.80 312 0.52% 56 59.49 412 0.18% 35 57 60.75 413 0.78% 58 60.89 413_(+)GlcNAcGal 0.07% 36 59 61.79 413 0.20% 60 61.75 312 0.16% 61 62.12 412 0.64% 37 62 63.87 311 0.73% 63 63,18,64,32 412 0.29% 64 63.84 413 (+)GlcNAcGal 0.45% 65 63.5, 64.36 311 (-)Gal 0.42% 38 66 65.73, 66.20 311 0.68% 67 65.85, 66.49 412 0.72% 68 65.91 310 (-)Gal 0.28% 39 69 67.37 212 1.42% 70 67.57 310 0.34% 40 71 68.67 412 (+)GlcNAcGal 0.24% 72 68.36 412 0.53% 41 73 68.36 412 (+)GlcNAcGal 0.17% 74 69.03 412 0.35% 75 69.30 413_(+)2(GlcNAcGal) 0.16% 42 76 70.66 412 (+)GlcNAcGal 0.73% 43 77 71.74 211 1.09% 78 71.23 211 (-)Gal 0.19% 44 79 72.46 212 3.66% 45 80 74.82 221 (-)Gal(+)GalNAc 0.38% 81 74,43,74,96 41 l_(+)GlcNAcGal 0.66% 46 82 75.92 410 0.42% 47 83 76,73,77,87 211 (-)Gal 1.24% 84 77.23 211 3.64% 48 85 79.05 211 1.52% 86 79.38 210_(-)2Gal 0.45% 49 87 80.11 210(-)Gal 1.58% 50 88 81.15 210 2.41% 51 89 84.22-87.15 311 1.26% 52 90 95.35 Mono Acetyl NANA 212 0.99% 53 91 96.23 Mono Acetyl NANA 211 0.76% 54 92 97.37 Bis Acetyl NANA 212 0.42%

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

Enzyme Example 6 Affinity Characterization for ATB200 for CIMPR.

In addition to the higher percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of this interaction. Binding of Lumizyme® and rhGAA ATB200 to receptors was determined using a CIMPR binding assay on plates. Briefly, CIMPR coated plates were used to capture GAA. Various concentrations of rhGAA were applied to the immobilized receptor, and unbound rhGAA was washed out. The amount of rhGAA remaining was determined by GAA activity. In FIG. 10A shows that rhGAA ATB200 bound CIMPR significantly better than Lumizyme®.

In FIG. 10B shows the relative content of bis-M6P-glycans in Lumizyme®, a traditional

- 23 043224 rhGAA and ATB200 in accordance with the present invention. In the case of Lumizyme®, on average only 10% of the molecules have a bisphosphorylated glycan. In contrast, in the case of ATB200, on average, each rhGAA molecule has at least one bisphosphorylated glycan.

Enzyme Example 7 rhGAA ATB200 was more efficiently internalized by fibroblasts than LUMIZYME®.

The relative uptake of rhGAA ATB200 and Lumizyme® cells was compared using a normal fibroblast cell line and a Pompe disease fibroblast cell line. Comparisons included 5-100 nM rhGAA ATB200 according to the present invention and 10-500 nM conventional rhGAA Lumizyme®. After a 16 hour incubation, extrinsic rhGAA was inactivated with TRIS base and cells were washed 3 times with PBS before being harvested. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and the data plotted against total cellular protein level and the results are shown in FIG. 11A-B.

It was also shown that rhGAA ATB200 was efficiently internalized into cells (FIGS. 11A and 11B, which respectively show that rhGAA ATB200 is internalized into both normal and Pompe disease fibroblast cells, and that it is internalized to a greater extent). degree than traditional rhGAA Lumizyme®). rhGAA ATB200 saturates cellular receptors at approximately 20 nM, while Lumizyme® requires approximately 250 nM. The absorption efficiency constant ( _Knabsorbed n _UR ) extrapolated from these results is 2-3 nM for ATB200 and 56 nM for Lumizyme® as shown in FIG. 11c. These results suggest that rhGAA ATB200 is a well-targeted treatment for Pompe disease.

Enzyme Example 8 Decreased glycogen levels in GAA knockout mice.

In FIG. 12A-12C show the effects of administration of alglucosidase-alpha (Lumizyme®) and ATB200 on glycogen clearance in Gaa knockout mice. Animals received two IV bolus injections (once every two weeks); tissues were collected two weeks after the last dose and analyzed for acid α-glucosidase activity and glycogen content.

As seen in FIG. 12A-12C, ATB200 was found to deplete tissue glycogen stores in a dose-dependent manner in acid α-glucosidase (Gaa) gene knockout mice. The 20 mg/kg dose of ATB200 consistently removed more stored glycogen in Gaa knockout mice than the 5 and 10 mg/kg doses. However, as seen in FIG. 12A-12C, ATB200 administered at a dose of 5 mg/kg showed a decrease in glycogen levels in mouse cardiac and skeletal muscles (quadriceps and triceps) similar to that of Lumizyme® administered at a dose of 20 mg/kg, while ATB200 administered at 10 and 20 mg/kg, showed a significantly better reduction in skeletal muscle glycogen levels than Lumizyme®.

In FIG. 15 shows the effects of administration of alglucosidase-alpha (Lumizyme®) and ATB200 on glycogen clearance in Gaa knockout mice. Twelve week old GAA KO mice were treated with Lumizyme® or ATB200, 20 mg/kg IV once every other week for 4 injections. Tissues were collected 14 days after the last enzyme dose to measure glycogen levels. In FIG. 13 shows the relative reduction in skeletal muscle glycogen levels in the quadriceps and triceps, with ATB200 providing a greater reduction in glycogen levels than Lumizyme®.

Enzyme Example 9 Muscle Physiology and Morphology in GAA Knockout Mice.

Gaa knockout mice received two IV boluses of human recombinant acid α-glucosidase (alglucosidase-alpha or ATB200) at 20 mg/kg once every two weeks. Control mice were treated with vehicle medium only. Soleus, quadriceps, and diaphragm tissue were harvested two weeks after the last dose of human recombinant acid α-glucosidase. Soleus and diaphragm tissue were analyzed for glycogen levels by Periodic Acid Schiff (PAS) staining and for lysosome proliferation by measuring levels of a lysosome-associated membrane marker protein (LAMP1), whose expression is upregulated in Pompe disease. Semithin sections of quadriceps embedded in epoxy resin (Epon) were stained with methylene blue and examined by electron microscopy (1000x) to determine the degree of presence of vacuoles. Quadriceps samples were analyzed by immunohistochemistry to determine the levels of phosphatidylethanolamine-conjugated microtubule-associated protein 1A/1B light chain 3 autophagy markers (LC3A II) and p62, insulin-dependent glucose transporter GLUT4, and insulin-independent glucose transporter GLUT1.

In a similar study, Gaa knockout mice received four IV boluses of human recombinant acid α-glucosidase (alglucosidase-alpha or ATB200) at 20 mg/kg once every two weeks. Control mice were treated with vehicle medium only. Cardiac muscle tissue was sampled two weeks after the last dose of recombinant acid α

- 24 043224 human glucosidase and analyzed for glycogen levels by Periodic Acid Schiff (PAS) staining and for lysosome proliferation by measuring LAMP1 levels.

In FIG. 14 shows that ATB200 administration showed a reduction in lysosome proliferation in cardiac muscle tissue, diaphragm tissue and skeletal muscle tissue (soleus muscle tissue) compared to conventional treatment with alglucosidase-alpha. In addition, in FIG. 15 shows that with the introduction of ATB200 showed a decrease in glycogen levels in the form of pinpoint deposits in cardiac and skeletal muscle tissues (soleus tissue) in comparison with conventional treatment with alglucosidase-alpha.

Also in FIG. 16 shows that ATB200 significantly reduced the number of vacuoles in the quadriceps muscle fiber in Gaa knockout mice compared to untreated mice and mice treated with alglucosidase-alpha. In FIG. 17 shows that both LC3 II and p62 levels were elevated in Gaa knockout mice compared to wild-type mice. In addition, levels of the insulin-dependent glucose transporter GLUT4 and the non-insulin-dependent glucose transporter GLUT1 were increased in Gaa knockout mice compared to wild-type mice. Elevated levels of GLUT4 and GLUT1, associated with acid α-glucosidase deficiency, may increase glucose uptake by muscle fibers and increase glycogen synthesis both in the basal state and after a meal.

Enzyme Example 10 Muscle function in GAA knockout mice.

In longer studies with 12 biweekly doses, the combination of 20 mg/kg ATB200 with 10 mg/kg miglustat gradually increased functional muscle strength in Gaa KO mice from baseline as measured by both grip strength tests and wire hang tests (FIGS. 18A-18B). Alglucosidase-alpha (Lumizyme®) treated mice that received a similar dose of ERT agent (20 mg/kg) experienced a reduction in performance under identical conditions for most of the study (FIGS. 18A-18B). As with the shorter term study, the ATB200/miglustat combination had significantly better glycogen clearance after 3 months (FIGS. 19A-19C) and 6 months (FIGS. 19D-19G) treatment than alglucosidase-alpha. The ATB200/miglustat combination also reduced autophagy and intracellular accumulation of LAMP1 and dysferlin after 3 months of treatment (Figure 20) compared to alglucosidase-alpha. In FIG. 18A * indicates a statistically significant result compared to Lumizyme® alone (p<0.05, 2-tailed t-test). In FIG. 19A-19G * indicates a statistically significant result compared to Lumizyme® alone (p<0.05, Dunnett's multiple comparison, one-way ANOVA).

Taken together, these data indicate that the ATB200/miglustat combination was effective in targeting muscle, reversing cellular dysfunction and improving muscle function. Importantly, apparent improvements in muscle structure, as well as reduced autophagy and intracellular accumulation of LAMP1 and dysferlin, may be good surrogates for improved muscle physiology, which correlates with improvements in functional muscle strength. These results suggest that monitoring autophagy and the levels of these key muscle proteins, which may prove to be useful biomarkers in muscle biopsy specimens in clinical trials, may be a viable practical way to assess the effectiveness of therapeutic treatments for Pompe disease in Gaa KO mice.

In FIG. 20 shows that administration of ATB200 with or without miglustat for 6 months reduced intracellular accumulation of dystrophin in Gaa KO mice. A more significant reduction in dystrophin accumulation was observed with the ATB200 ± miglustat combination than with Lumizyme®.

Composition Example 1. pH and buffer.

Analytical Methods for Composition Examples.

The analyzes of the examples described herein for appearance, pH, protein concentration, etc. were performed according to the methods below, unless otherwise noted.

Appearance.

The appearance of the samples, including transparency, color, and particulate matter, was examined against black and white backgrounds using a YB-2 Illuminated Viewer.

pH value.

The pH of the sample was measured using a SevenMulti™ pH meter.

protein concentration.

Protein concentration was determined by reading at UV280 using a NanoDrop™ 2000 spectrophotometer. All measurements were repeated twice with 2.5 μl of sample each time and an average value was obtained.

Size Exclusion High Performance Liquid Chromatography (SEC-HPLC).

- 25 043224

For pH and buffer, freeze-thaw and excipient examples, separation of protein monomers and its macromolecular forms and fragments was performed using a TSKgel® G3000 SWXL column (Tosoh Bioscience, 7.8x300 mm, 5 µm, 25°C) on an Agilent 1260 system HPLC. 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 sample chromatography time of 20 minutes with an isocratic gradient. Signals were detected with a UV detector at 280 nm (reference: 360 nm).

In the PS80 example, separation of protein monomers and its high molecular weight forms and fragments was performed using a BioSep™-SEC-s3000 column (Phenomenex, 7.8x300 mm, 5 μm, 25°C) on an Agilent 1260 HPLC system. 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 chromatography time of 25 minutes with an isocratic gradient. Signals were detected with a UV detector at 280 nm.

SDS-PAGE (under non-reducing conditions).

Reportable values are the purity and molecular weight of the protein in SDS-PAGE under non-reducing conditions. The samples were denatured under non-reducing conditions in the presence of excess SDS to achieve a uniform negative charge. When exposed to an electric field (165 V), these SDS-coated forms were separated based on their identified molecular weight in a polyacrylamide gel. Separated bands were revealed by staining with Coomassie blue.

Enzymatic activity against 4-MUG.

µl of sample was diluted and digested (with GAA, 37° C. for 60 min) to give 4-MU fluorescent product. Added 125 μl of 1 M glycine or 0.1 M NaOH to stop the reaction.

A number of 4-MU standards were analyzed with samples to obtain a standard calibration curve based on the fluorescent signal. The conversion of RFU to 4-MU amount was obtained by software-mediated comparison with a standard curve that was regressed according to a 4-parameter logistic regression model. The enzymatic activity of GAA (nmol 4-MU released/h/ml GAA) in the sample was then calculated based on the standard curve for 4-MU.

Enzyme concentration as determined by 4-MUG.

μl of sample and GAA reference standard were diluted and digested (with GAA, 37° C. for 60 min) to give 4-MU fluorescent product. Added 125 μl of 1M NaOH to stop the reaction.

A number of GAA reference standards were analyzed with samples to obtain a standard calibration curve based on the fluorescent signal. The conversion of RFU to 4-MU amount was obtained by software-mediated comparison with a standard curve that was regressed according to a 4-parameter logistic regression model. Then the enzymatic concentration of GAA (nmol 4-MU released/h/ml GAA) in the sample was calculated based on the GAA standard curve.

Dynamic Light Scattering (DLS).

A micropipette was used to transfer a 40 µl aliquot of undiluted sample into a 40 µl disposable cuvette. For each sample, measurements were taken in triplicate.

Particles: HIAC.

200 µl of each sample was diluted in 2000 µl of filtered reference buffer. The sample was tested three times and 450 µl was used for each test. The average number of particles of each size was recorded: 1, 3, 5, 10 and 25 µm per ml.

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 a reference as a function of temperature. In particular, it is used to measure the thermal midpoint (Tm), which is an indication of the relative stability of a protein in solution.

Samples were diluted with reference buffer to approximately 1 mg/mL. A 400 μl aliquot of reference buffer was added to each odd-numbered well of a 96-well plate, while a 400 μl aliquot of each sample was added to the corresponding even-numbered well. The scanning temperature is in the range of 10 to 110°C.

Modulated differential scanning calorimetry (mDSC).

The glass transition temperature (Tg') and the eutectic point temperature (Te) were tested using a Netzsch differential scanning calorimeter (DSC 204 F1). For testing, 15 µl of sample was loaded onto a loading disk. First, the temperature was lowered from 20°C

- 26 043224 to -60°C at a rate of 10°C/min, and during this stage, the value of Te was obtained from the cooling curve. Then the temperature was raised from -60°C to 40°C at a rate of 10°C/min, and the Tg' value was analyzed from the heating curve.

M6P.

M6P was released from the sample by hydrolysis (4M TFA, 100°C, 4 h) and dried using a centrifugal vacuum evaporator. Dried M6P and reference standard were suspended in purified water prior to analysis. A CarboPac PA10 BioLC™ analytical column (4 mm x 250 mm, 3.5 µm, 100 A, 30°C) and a CarboPac PA10 BioLCGuard column (4 mm x 50 mm, 3.5 µm, 100 A, 30°C) were used. The mobile phase consisted of phase A (100 mM NaOH) and phase B (1 M NaOAc, 100 mM NaOH). The chromatographic system used a flow rate of 1 ml/min, an injection volume of 25 μl, and a chromatography time of 30 min with a gradient. Signals were detected by pulsed amperometric detection. The content of M6P in the sample was calculated based on the standard curve.

sialic acid.

Sialic acids were released from the drug molecules by hydrolysis (2M HAc, 80°C, 2 h) and then all samples were labeled and the standard solution was mixed with DMB (50°C, 17±0.5 h in the dark) before separation using Zorbax Eclipse Plus C18 columns (Agilent, 4.6 mm x 100 mm, 3.5 µm, 45°C) on an Agilent 1260 HPLC system. 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 chromatography time of 20 min with a gradient. Signals were detected using a fluorescent detector (λex=373 nm, λex=448 nm). The content of Neu5Gc and Neu5Ac in the sample was calculated from the standard curve.

Received ten formulations with a buffer, characterized by a pH in the range of 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 concentration of the ATB200 enzyme, determined using 4-MUG as a substrate, was 1 mg/ml.

Table 1

The composition of the compositions

Sample Buffer NaCl pH value P40 25 mM sodium phosphate 50 mM 4.0* P50 5.0 P60 6.0 R70 7.0 P80 8.0 P60 without NaCl 25 mM sodium phosphate Absent 6.0 C50 25 mM sodium citrate 50 mM 5.0 C55 5.5 C60 6.0 C65 6.5

* HCl was used to bring the pH to 4.0.

Sample preparation.

The material used in the pH and buffer evaluation study is as described in Table 1. 2 below.

table 2

Raw material information from pH and buffer evaluation study

Conc, mg/ml SEC, % Enzymatic activity, U/l SDS- PAGE, % Sialic acid, mol/mol protein М6Р, mol/mol protein sno, ppt UV 280 Conc, enzyme 3.6* * 1.72 98.9 9094.2 99.7 5.37 3.1 422

* The buffer for this ATB200 enzyme solution was 50 mM sodium phosphate (pH 6.2), 50 mM NaCl, 2% mannitol.

** The extinction coefficient was 1.51 units. opt. dens.*ml*mg ^-1 *cm ^-1 .

A 25 mM sodium phosphate buffer was prepared containing 50 mM NaCl at pH 4.0 (P40), 5.0 (P50), 6.0 (P60), 7.0 (P70), and 8.0 (P80), 25 mM sodium phosphate buffer at pH 6.0 (P60 without NaCl), and 25 mM sodium citrate buffer containing 50 mM NaCl at pH 5.0 (C50), 5.5 (C55), 6.0 (C60) and 6.5 (C65). For pH 4.0, HCl was used to adjust the pH in sodium phosphate buffer (pH 4.0).

The ATB200 enzymatic solution was first concentrated using a centrifuge ultrafiltration device at 15° C. and 3500 rpm for 40 minutes. The concentrated enzyme solution was then buffer exchanged with the three different buffers described above through two cycles of ultrafiltration at 15° C. and 3500 rpm for 50 minutes and 55 minutes. The buffer exchanged enzyme solutions were then analyzed for protein concentration and enzyme concentration using 4-MUG as a substrate.

Finally, an appropriate volume of each buffer was added to bring the final ATB200 enzyme concentration to 1.0 mg/mL. The final ATB200 concentration was confirmed by both UVA280 uptake and enzyme concentration determined using 4-MUG as a substrate.

The solutions were sterile filtered using a 0.22 μm polyethersulfone (PES) filter.

Each formulation was aseptically filled into 2 ml glass vials in a bioshielded laminar flow cabinet with a fill volume of 500 µl ~ 1000 µl according to the required amount of sample for analytical tests. The vials were stoppered and then rolled up with a crimp cap immediately after filling.

Sample testing.

Vials with each formulation were stored at 5°C and 40°C for up to 8 weeks, and shaken at 25°C at 100 rpm on an orbital shaker for up to 5 days (see Table 3). A sample of the composition was taken at the beginning (T0), after 5 days (5D), 2 weeks (2W), 4 weeks (4W) and 8 weeks (8W), as described in table. 3 for appearance analysis, pH determination, UV concentration, concentration, enzyme determined by 4-MUG, SEC, HIAC, DLS, 4-MUG enzyme activity and DSC.

Table 3

Parameters, Tests in pH Evaluation Study and Buffer ATB200

Storage conditions THAT 5D 2W 4W 8W 5°С X, y - X X Χ,Ζ 40°C - X X X Shaking, -100 rpm, 25°C X - - -

X: Appearance, pH value, UV concentration, 4MUG enzyme concentration, SEC, HIAC*, DLS, 4-MUG enzyme activity.

Y:DSC.

*: HIAC tested for T0 samples and shake samples only.

Results.

Thermal stability results - MicroCal DSC.

The results of measurements of thermal stability are shown below in table. 4.

Table 4

Sample Tm start (°C) TM1 (°C) P40 56.34 66.25 P50 63.49 73.42 P60 60.47 72.05 R70 47.55 64.09 P80 37.21 47.72 P60 without NaCl 61.22 72.25 C50 62.66 72.60 C55 62.89 73.39 C60 59.26 70.83 C65 53.45 67.24

A higher Tm _onset indicates better thermal stability of the protein in a particular composition. Accordingly, the formulations exhibiting the highest thermal stability were P50, C55, C50, P60 without NaCl, P60 and C60. These results indicate that the ATB200 enzyme is characterized by better thermal stability in a weak acidic buffer than in basic conditions.

Appearance - shaking.

The results of the study of appearance when shaking are shown below in table. 5.

- 28 043224

Table 5

Sample THAT Shaking, at 100 rpm, 25°C, 5 days P40 Colorless, transparent, free of visible particles Colorless, transparent, small visible particles P50 Colorless, transparent, very small visible particles P60 Colorless, transparent, very small visible particles R70 Colorless, transparent, small visible particles P80 Colorless, transparent, visible particles P60 without NaCl Colorless, transparent, free of visible particles C50 Colorless, transparent, free of visible particles C55 Colorless, transparent, free of visible particles C60 Colorless, transparent, free of visible particles C65 Colorless, transparent, free of visible particles

The table shows that after shaking for 5 days, P60 without NaCl, C50, C55, C60 and C65 remained colorless, transparent and did not contain visible particles, but visible particles were observed in P40, P50, P60, P70 and P80. These data show that sodium citrate buffer stabilized the formulation better than sodium phosphate buffer after shaking.

pH value.

The results of measurements of pH are shown in table. 6 below.

Table 6

Sample pH value THAT 5°C 40°C shaking, 2W 4W 8W 2W 4W 8W at 100 rpm, 25°C 5 days P40 4.293 4.233 4.273 4,380 4.618 4.537 4.606 4.296 P50 5.051 5.008 5.011 4.936 5.070 4,998 5.044 5.002 P60 6.053 6.025 5.927 5.927 6.030 5.911 5.949 6.017 R70 7.093 7.043 6.999 6.941 7.037 7.024 7.014 7.027 P80 7,864 7.823 7.749 7.767 7.834 7.834 7.767 7,883 P60 without NaCl 6.091 6.040 5.927 5.736 6.049 5,852 5,862 6.028 C50 5.069 5.034 4.986 4.971 5.030 4.997 5.016 5.011 C55 5,564 5.524 5.486 5.501 5.517 5.459 5.501 5.517 C60 6.071 6.044 6.020 5.983 6.038 6.038 5,998 6.035 C65 6.566 6.551 6.526 6.422 6.562 6.513 6.516 6.544

It can be seen from the data that both phosphate and citrate buffers with 50 mM NaCl, in the pH range of 5.0 to 6.5, were able to maintain the indicated

-29043224 pH value throughout the study. During buffer exchange, concentration adjustment, storage for 8 weeks at 5 and 40°C and shaking, no significant change in the pH of the samples was detected.

Conversely, the pH decreased in P60 without NaCl after 8 weeks storage at both 5°C and 40°C, while the pH increased in P40 after buffer change and 8 weeks storage at both temperatures. However, shaking did not lead to any change in pH in both P40 and P60 without NaCl.

protein concentration.

The results of measurements of protein concentration are shown in table. 7 below.

Table 7

Sample Concentration at UV, mg/ml THAT 5°C 40°С Shaking, at 100 rpm, 25°C, 5 days 2W 4W 8W 2W 4W 8W 8W* P40 1.72 1.75 1.72 1.71 1.88 1.70 1.69 1.50 1.73 P50 1.74 1.80 1.81 1.81 1.91 1.99 1.98 1.56 1.80 P60 1.90 1.92 1.88 1.87 1.96 2.10 2.04 1.31 1.87 R70 1.79 1.76 1.75 1.73 1.83 1.75 1.72 1.66 1.74 P80 2.20 2.25 2.21 2.20 2.25 2.21 2.23 2.19 2.25 P60 without NaCl 1.82 1.85 1.82 1.81 1.87 1.90 1.92 1.58 1.86 C50 1.83 1.80 1.81 1.81 1.80 1.76 1.72 1.71 1.80 C55 1.84 1.83 1.83 1.84 1.89 1.85 1.79 1.54 1.82 C60 2.00 1.98 1.97 1.97 2.08 2.21 2.14 1.19 1.96 C65 2.03 2.05 1.98 2.00 2.17 2.19 2.39 1.26 2.06

Storage for 8 weeks at 5°C and shaking for 5 days had no effect on protein concentration. No significant changes were noted among all formulations.

Conversely, during storage at 40°C, the protein concentration slightly increased in P50, P60, P60 without NaCl, C60 and C65, slightly decreased in C50, and remained in P40, P70, P80, C55. Due to the formation of visible particles in samples at 40°C, samples that were stored at 40°C/for 8 weeks, the samples were retested after centrifugation at 12000 rpm for 1 min. The results showed a drop in protein concentration after centrifugation in samples containing particles, indicating that the particles present in the samples had an effect on absorbance at 280 nm.

Enzyme concentration as determined by 4-MUG.

The results of measurements of the concentration of the enzyme 4-MUG are shown in table. 8 below.

Table 8

Sample name 4-MUG enzyme concentration, mg/ml THAT 5°С 40°С Shaking, at 100 rpm, 25°C, 5 days 2W 4W 8W 2W 4W 8W P40 0.86 0.91 0.91 0.76 0.34 0.16 0.04 1.02 P50 0.92 1.08 1.08 1.05 0.78 0.62 0.33 1.12 P60 0.89 1.10 1.13 1.13 0.92 0.87 0.58 1.14 R70 0.82 0.93 0.92 0.86 0.00 0.00 0.00 1.00 P80 0.27 0.00 0.00 0.00 0.00 BQL But. 0.00 P60 without NaCl 0.87 1.10 1.06 1.05 1.01 0.98 0.73 1.09 C50 0.87 1.04 1.06 1.05 0.89 0.68 0.33 1.14 C55 0.90 1.15 1,I 1.09 1.03 0.96 0.63 1.13 C60 1.02 1.22 1.33 1.23 1.08 0.92 0.53 1.26 C65 1.02 1.23 1.29 1.28 0.15 0.01 0.00 1.29

After buffer change, the concentration of the 4-MUG enzyme in P80 dropped sharply to 0.27 mg/ml. This indicated that pH 8.0 had a significant effect on enzymatic activity. After storage for 2 weeks at both 5°C and 40°C, the enzyme concentration dropped to zero. With the exception of P80, after storage for 8 weeks at 5°C, enzyme concentrations in most formulations were stable and dropped slightly at P40.

Conversely, storage at 40°C clearly affected the concentration of the enzyme. After 2 weeks, the enzyme was not detected in P70, dropped sharply in P40 and C65, and clearly decreased in P50. After 4 weeks, the enzyme concentration continued to decrease. As a result, after 8 weeks, the enzyme concentration values were close to zero in P40 and C65, dropped to 0.33 mg / ml in buffers with a pH of 5.0 (P50 and C50) and to 0.5-0.7

-30043224mg/ml in pH 5.5~6.0 buffers (P60, P60 without NaCl, C55 and C60). Among them, P60 without NaCl had the highest enzyme concentration (0.73 mg/mL) in the end. The results indicate that the enzyme concentration was the most maintained with respect to the 4-MUG enzyme concentration in the pH range of 5.5~6.0, but no significant difference was found between sodium phosphate buffer and sodium citrate buffer.

During the shaking study, with the exception of P80, the enzyme concentration did not change significantly.

Enzymatic activity against 4-MUG.

The results of measurements of enzymatic activity against 4-MUG are shown in table. 9 below.

Table 9

Sample name Enzymatic activity against 4-MUG. U/l THAT 5°C 40°C Shaking, with 100 rpm 25°C. 5 days 2W 4W 8W 2W 4W 8W P40 4703.4 5092.7 4153.7 3697.2 1822.3 712.2 211.5 5439.9 P50 5043.8 6021.5 4947.8 5101.4 4344.4 2836.9 1561.7 5953.8 P60 4915.3 6133.7 5193.7 5487.5 5170.4 4002.9 2790.4 6087.0 R70 4481.1 5233.1 4211.5 4195.4 0.2 0.4 oh oh 5360.5 P80 1402.6 1.8 1.6 0.7 0.2 BQL oh oh 0.6 P60 without NaCl 4763.7 6130.1 4862.1 5120.0 5662.4 4476.0 3549.7 5799.6 C50 4772.4 5811.8 4850.7 5110.8 4964.9 3121.6 1544.8 6057.4 C55 4957.3 6450.1 5091.7 5321.7 5740.9 4411.9 3054.1 6036.5 C60 5641.1 6829.9 6061.0 5999.2 6026.4 4243.2 2563.3 6662.8 C65 5639.3 6848.2 5892.8 6238.7 761.6 65.5 3.4 6827.8

The trend in enzyme activity for 4-MUG was consistent with the trend in enzyme concentration as measured by 4-MUG.

P80 showed the worst stability in terms of enzymatic activity as measured by 4-MUG under test conditions. P80 enzymatic activity was reduced by approximately 70% after buffer change. Later, the enzymatic activity was almost lost after storage for 2 weeks at 5°C and 40°C. The main condition significantly affected the enzymatic activity.

After storage for 8 weeks at 5°C, the enzymatic activity of PS80 was completely lost; a 20% decrease was found in P40; no significant changes were found in the other formulations.

Conversely, storage at 40° C. resulted in a clear decrease in enzymatic activity in all formulations. After 2 weeks, enzymatic activity was almost completely lost in P70, decreased sharply in P40 and C65, and clearly dropped in P50. Enzymatic activity continued to decline to varying degrees from week 2 to week 4. At the end of the study, enzymatic activity was almost lost in C65, falling to 211.5 U/L in P40, to ~1550 U/L in P50 and C50, and to 2500~3000 U/L in C60, P60 and C55. Among the formulations, P60 without NaCl retained the highest activity, amounting to 3549.7 U/l.

Based on the 4-MUG enzyme activity test results, the enzyme was most stable at pH in the pH range of 5.5~6.0, but no difference was found between sodium phosphate buffer and sodium citrate buffer.

Purity: SEC-HPLC.

The SEC measurement results are shown in Table 1 below. 10.

- 31 043224

Table 10

Sample name SEC TO 5°C 40°C Shaking at 100 rpm, 25°C, 5 days 2W 4W 8W 2W 4W 8W P40 % monomers 94.0 89.1 81.9 74.3 41.2 18.7 7.1 90.8 HMW, % 1.3 1.6 2.4 2.4 H.o. H.o. H.o. 0.7 LMW, % 4.7 9.4 15.7 23.4 58.8 81.3 93.0 8.5 P50 % monomers 99.3 98.1 95.4 92.8 81.1 66.1 42.0 96.6 HMW, % 0.4 0.4 0.7 1.4 0.2 H.o. 0.1 0.6 LMW, % 0.4 1.6 4.0 5.9 18.6 33.9 57.9 2.8 P60 % monomers 98.7 97.7 96.8 96.2 96.1 92.4 80.8 96.8 HMW, % 0.8 0.9 1.2 1.9 2.1 0.6 2.7 1.1 LMW, % 0.5 1.4 2.0 2.0 1.8 7.0 16.5 2.1 R70 % monomers 97.7 94.6 92.4 87.0 0.5 H.o. 0.1 93.5 HMW, % 1.8 3.3 5.4 11.0 96.9 96.4 96.7 4.1 LMW, % 0.5 2.1 2.3 1.9 2.7 3.6 3.2 2.4 P80 % monomers 44.9 35.3 34.4 26.7 H.o. H.o. 0.2 15.2 HMW, % 54.5 63.0 61.4 69.3 97.3 97.2 96.5 83.7 LMW, % 0.6 1.7 4.2 4.0 2.7 2.8 3.3 1.1 P60 without NaCl % monomers 96.6 98.0 97.0 96.8 94.95 92.4 83.6 95.6 HMW, % 0.5 0.5 1.0 1.2 1.44 0.7 3.0 ι, ο LMW, % 2.9 1.5 2.0 2.0 3.62 7.0 13.5 3.4 C50 % monomers 98.3 99.5 95.6 93.0 96.3 62.3 32.3 92.1 HMW, % 0.2 0.2 0.4 0.2 2.0 0.1 H.o. 0.4 LMW, % 1.5 0.4 4.0 6.8 1.7 37.6 67.7 7.6 C55 % monomers 98.5 99.4 99.3 95.9 94.81 88.52 68.5 92.6 HMW, % 0.3 0.2 0.2 1.4 0.29 0.19 0.2 0.3 LMW, % 1.3 0.4 0.5 2.7 4.9 11.3 31.3 7.1 C60 % monomers 98.7 99.2 97.8 97.6 98.0 95.8 77.5 92.8 HMW, % 0.3 0.3 0.8 1.1 1.1 1.0 5.5 0.8 LMW, % 1.0 0.6 1.4 1.3 0.9 3.2 17.0 6.4 C65 % monomers 98.8 98.9 97.6 97.9 22.4 10.0 H.o. 91.4 HMW, % 0.4 0.5 1.2 1.9 76.7 86.1 86.8 4.4 LMW, % 0.8 0.6 1.2 0.3 1.0 3.9 13.2 4.2

After buffer change, the SEC purity of some of the formulations was significantly reduced compared to the starting material (SEC percentage of monomers: 98.9%). In P80, SEC purity dropped to 44.9% and 54.5% aggregates formed; P40 showed a 4.9% decrease in percentage of monomers compared to pre-swap DS, with more LMW fragments formed than HMW molecules (4.7% vs. 1.3%); P70 showed a slight decrease (1.2%) in the percentage of monomers corresponding to an increase in the number of aggregates; in P60 without NaCl, a decrease of 2.3% in the percentage of monomers was observed.

After 8 weeks storage at 5°C, the SEC purity of formulations P60, P60 without NaCl and C65 was well maintained, but the SEC purity of the other formulations was significantly reduced compared to T0. At P40, SEC purity dropped sharply to 74.3% (T0: 94.0%) and 23.4% LMW fragments formed; in P50 and C50 showed a slight decrease (5~7%) in the percentage of monomers and an increase in the percentage (5~7%) of fragments with LMW. In P80, a decrease of 18.2% was found, which was mainly due to aggregation (14.8%). Thus, as in P70, a decrease of 10.7% in the percentage of monomers and an increase of 9.2% in the percentage of fragments with HMW were revealed. C55 showed little change in the percentage of monomers, HMW and LMW.

Storage at 40°C led to a sharp change in the percentage of monomers in all formulations. In P70 and P80, the percentage of monomers according to SEC dropped to 0~0.5% after 2 weeks, and in C65, 22.4% remained, and they mostly turned into aggregates after 8 weeks. In P40, P50 and C50, a significant drop (50~90%) in the percentage of monomers was detected, mainly due to the formation of fragments with LMW. In P60, P60 without NaCl and C60 after 8 weeks, the percentage of monomers according to SEC was reduced to 80.8, 83.6 and 77.5%, respectively.

The SEC results indicate that a pH of 6.0 is most appropriate for ATB200 stability, with no significant difference found between sodium phosphate buffer and sodium citrate buffer. In addition, the absence of sodium chloride in the formulations did not affect the stability of ATB200.

During the shaking study, SEC purity decreased slightly in P60 and P60 without NaCl. P40, P50, P70, P80, C50, C60 and C65 showed a clear decrease in the percentage of monomers.

Polydispersity: DLS.

The results of DLS measurements are shown in table. 11 below.

Table 11

Research condition Sample Z-medium, d.HM pdl Pk 1 average INT. Pk 2 average INT. Pk 3 average INT. Pk 1 INT. Pk 2 int. Peak 3 INT. area Percent area Percent Square di Proce NT ania d.HM d.HM d.HM THAT P40 10.9 0.183 11.8 4080 0 96.9 3.1 0 P50 10.7 0.181 11.4 4248 0 96.9 3.1 0 P60 10.2 0.186 10.9 4199 0 96.6 3.4 0 R70 9.6 0.144 11.2 0 0 100 0 0 P80 12.6 0.150 14.1 4421 0 98.8 1.2 0 P60 without NaCl 10.5 0.223 10.7 3952 0 93.8 6.2 0 C50 10.3 0.133 11.4 0 0 100 0 0 C55 9.9 0.129 P,1 0 0 100 0 0 C60 9.7 0.114 11.0 0 0 100 0 0 C65 9.7 0.103 10.7 0 0 100 0 0 5°С, 2 W P40 12.8 0.260 13.3 437 0 89.6 10.4 0 P50 11.8 0.266 11.7 500.7 0 88.3 I,7 0 P60 9.9 0.161 10.7 4352 0 97.8 2.2 0 R70 10.0 0.137 10.9 4560 0 98.9 1.1 0 P80 13.2 0.100 14.7 0 0 100 0 0 P60 without NaCl 7562.0 1,000 4099.0 0 0 100 0 0 C50 11.0 0.219 11.7 3459 0 94.1 5.9 0 C55 10.1 0.152 11.2 4409 0 98.7 1.3 0 C60 3506.0 0.271 3745.0 9.144 0 93.6 6.4 0 C65 355.5 1,000 4194.0 11.08 0 74.6 25.4 0 5°С, 4 W P40 12.2 0.220 13.4 2891 0 94.3 5.7 0 P50 11.8 0.255 12.0 1059 0 89.8 10.2 0 P60 9.9 0.126 I,1 0 0 100 0 0 R70 9.9 0.087 10.8 0 0 100 0 0 P80 13.5 0.176 14.7 4442 0 97.5 2.5 0 P60 without NaCl 9.6 0.102 10.6 0 0 100 0 0 C50 11.3 0.229 11.9 2913 0 92.9 7.1 0 C55 10.5 0.205 11.2 4067 0 95.6 4.4 0 C60 9.9 0.116 I,1 0 0 100 0 0

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C65 10.7 0.208 12.5 4042 0 97.4 2.6 0 5°С, 8 W P40 15.1 0.329 13.7 116.8 0 77.6 22.4 0 P50 14.1 0.335 14.0 113.7 0 79.2 20.8 0 P60 10.1 0.148 11.0 4395 0 98.3 1.7 0 R70 10.5 0.152 11.6 4397 0 98.4 1.6 0 P80 13.6 0.121 15.4 0 0 100 0 0 P60 without NaCl 10.3 0.187 11.0 4055 0 96.3 3.7 0 C50 11.8 0.281 12.3 486.6 0 88.4 11.6 0 C55 11.8 0.265 12.3 583.3 0 89.6 10.4 0 C60 11.8 0.261 12.2 3465 0 91.7 8.3 0 C65 9.7 0.105 10.8 0 0 100 0 0 40°С, 2 W P40 1768 0.316 2017 5272 0 96.7 0 P50 8330 0.418 7.2 0 0 100 0 0 P60 1318 0.952 741.5 5393 132.8 63.3 18.6 18.2 R70 66.72 0.627 39.52 4508 0 69.4 30.6 0 P80 2963 0.733 4504 33.27 0 87.8 12.2 0 P60 without NaCl 106.2 0.744 143 2912 9.298 71.5 21 7.5 C50 5874 1 4325 5.199 0 94.9 5.1 0 C55 30.84 0.721 704.9 9.816 0 59.1 40.9 0 C60 2574 1 168.9 5444 0 57.1 42.9 0 C65 96.59 0.31 124.4 4622 0 96.6 3.4 0 40°С, 4 W P40 2116 0.249 2178 5236 0 94.9 5.1 0 P50 1894 0.251 2385 0 0 100 0 0 P60 644.6 0.708 2879 475.1 79.89 58.8 34.1 7.1 R70 34.21 0.122 39.44 0 0 100 0 0 P80 22.64 0.096 25.18 0 0 100 0 0 P60 without NaCl 239.5 0.53 394.6 4499 0 95 5 0 C50 173.1 0.758 9.513 1373 5098 45.1 44.9 10 C55 561.3 1 1932 187.7 9.455 69.1 14 9.3 C60 419.3 0.706 1941 244.8 68.83 61.6 34.1 4.3 C65 138.3 0.248 192.7 25.24 0 98.6 1.4 0 40°С, 8 W P40 1177 0.419 1512 9.926 4.277 93.4 5 1.6 P50 1381 0.672 1723 9.313 0 92.7 7.3 0 P60 283 0.587 1028 191.9 37.93 62.2 34.9 2.9 R70 36.85 0.135 43.2 0 0 100 0 0 P80 23.2 0.114 26.46 0 0 100 0 0 P60 without NaCl 3215 0.445 1103 0 0 100 0 0 C50 2118 0.184 2711 5.565 0 92.1 7.9 0 C55 145.3 1 801.3 153.6 10.1 59.8 23 14 C60 462.7 0.918 912.6 175.8 4998 54 30.9 15.1 C65 196.2 0.309 279.6 45.86 5177 92.1 6.9 1 Shaking, at 100 rpm, 25°C, 5D P40 13.0 0.331 11.8 190.8 0 80.1 19.9 0 P50 11.8 0.290 11.3 267.5 0 85.2 14.8 0 P60 10.1 0.165 11.0 4383 0 97.9 2.1 0 R70 10.1 0.136 11.4 0 0 100 0 0 P80 51.6 0.657 21.1 3805 0 55.6 44.4 0 P60 without NaCl 9.7 0.118 10.8 0 0 100 0 0 C50 12.7 0.319 11.8 355.7 0 82.6 17.4 0 C55 9.8 0.140 10.9 4434 0 99 1 0 C60 10.1 0.174 11.0 4397 0 97.8 2.2 0 C65 9.6 0.084 10.5 0 0 100 0 0

DLS data reflect the hydrodynamic radius of protein molecules and the polydispersity of particles. Opalescence was observed in some samples, so DLS was used to analyze invisible particles. The DLS results were mostly in line with the looks.

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

During storage for 8 weeks at 40°C, there was a sharp change in hydrodynamic radius and PDI in all formulations. However, due to the complex profiles of the aggregates, it is difficult to compare these compositions based on the result.

In the shaking study, the hydrodynamic radius and PDI P80 were characterized by a sharp increase; in P40, P50 and C50 hydrodynamic radius and PDI were characterized by a slight increase; P60, P70, P60 without NaCl, C55, C60 and C65 showed no significant change.

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

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Particles: HIAC.

The results of HIAC measurements in the study with shaking are shown in table. 12 below.

Table 12

Sample Particle Size (µm) Differential Count/ml THAT Shaking, at 100 rpm, 25°C, 5 days P40 > 5 1704 2593 > yu 97 163 >25 8 0 P50 > 5 4600 7378 > yu 600 815 >25 15 0 P60 > 5 1052 1245 > yu 112 134 >25 0 0 R70 > 5 171 2860 > yu 15 341 >25 0 8 P80 > 5 15830 15104 > yu 5786 4104 >25 630 341 P60 without NaCl > 5 289 149 > yu 52 23 >25 0 0 C50 > 5 200 200 > yu 23 15 >25 0 0 C55 > 5 223 319 > yu 23 23 >25 0 0 C60 > 5 408 497 > yu 15 23 >25 0 0 C65 > 5 445 512 > yu 8 0 >25 0 0

During the shaking study, P70 had a significant increase in particle number after shaking at 25° C. for 5 days, and all other formulations had comparable particle numbers.

Free information.

Overall, P60 and C60 stood out as the most stable formulations compared to the others. Thus, it was concluded that ATB200 was the most stable in the range of 5.0-6.0. However, there was no difference between phosphate and citrate buffer. In addition, the absence of sodium chloride in the composition did not significantly affect the stability of ATB200.

Composition Example 2 Freeze-Thaw.

The three formulations were evaluated for stability during the freeze-thaw process. The three compositions are summarized in Table. 13 below. The target concentration of the ATB200 enzyme was 5 mg/mL.

Table 13

Sample Buffer pH value NaCl P60 25 mM phosphate 6.0 50mm C60 25 mM citrate СР60 25 mM phosphate- citrate buffer

Sample preparation.

In the ATB200 DS, the buffer was changed to 3 buffers for formulation with a dialysis cassette (20,000 MWCO). Dialyses were performed at 5°C with gentle agitation, each involving three buffer changes, once every 6~10 h.

After each dialysis, an appropriate volume of formulation buffer was added to bring the final concentration under UV to 5 mg/mL. The solutions were then sterile filtered with a 0.22 μm PES filter. Each composition was then aseptically filled into 2 ml glass vials in a bioshielded laminar flow cabinet with a filling volume of 500~1000 µl according to

- 35 043224 with the required amount of sample for analytical tests. The vials were stoppered and then rolled up with a crimp cap immediately after filling.

Prior to freeze-thaw, vials of each formulation were withdrawn and the remaining sample vials subjected to the freeze-thaw cycles provided. After freeze-thaw, sample vials were collected at predetermined sampling points.

Sample testing.

Tested using three freeze-thaw methods as indicated below.

Method 1: uncontrolled freezing and thawing. Samples were frozen in a -80°C freezer and thawed in a 25°C freezer.

Method 2: controlled freezing and uncontrolled thawing. The samples were placed in a Frosty container and frozen in a freezer at -80°C. The Frosty container uses isopropanol to achieve a controlled freezing rate of 1°C/min. The Frosty container was placed in a chamber at 25°C to thaw the samples. The rate of temperature rise was about 1°C/min.

Method 3: controlled freeze and thaw. The lyophilizer was used to freeze and thaw the samples. The lowest sample temperature reached during freezing was -47°C. The sample temperature was raised to 25°C. The rate of temperature change for both freezing and thawing was controlled at approximately 0.5° C./min. The results were confirmed by repeating the experiment.

Five or three freeze-thaw cycles were performed using each method. The following tests were performed on samples before and after freeze-thaw: appearance, concentration (in UV and enzyme) and SEC-HPLC. Summary data on the tested parameters are given in Table. 14 below.

Table 14

Equipment and condition Speed______and freezing time speed and heating time THAT 1 cycle F/T 3 F/T cycles 5 F/T cycles Refrigerator, 80°C to 25°C N. o., 40-60 min. N. o., 40-60 min. X X X X Freezer Nalgene Mg. Frosty Cryo 1°С, from -80°С to 25°С 1°C/min, 100-120 min ~1°С/min, 100-120 min X X X Lyophilizer, -47°С to 25°С 0.5°C/min, -164 min 0.5°C/min, -164 min X X X*

X: appearance, concentration (under UV and enzyme), enzymatic activity, SEC.

*: This experiment was repeated, 3 FT cycles for the first experiment and 5 FT cycles for the second experiment.

Results.

Appearance - 1st stage.

The results of the first stage of the appearance analyzes are shown in table. 15A and 15B below.

Table 15A

Sample name THAT cooling chamber 1FT 3ft 5 FT P60 Colorless, slightly opalescent, Colorless, slightly opalescent Colorless, slightly opalescent Colorless, slightly opalescent C60 free of visible particles shchy, small number of visible particles high, more visible particles than 1FT whistling, large number of visible particles СР60

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Table 15B

Name Lyophilizer Container Frosty sample 1FT 3FT 1FT 3FT 5 FT P60 Colorless, slightly opalescent, not containing visible particles Colorless, slightly opalescent, few visible particles Colorless, slightly opalescent, few visible particles Colorless, slightly opalescent, more visible particles than in 1FT Colorless, slightly opalescent, large number of visible particles C60 СР60

All formulations were found to be colorless, slightly opalescent and free of visible particles at RT (before freeze-thaw), with the appropriate formulation buffers used as reference.

It turned out that after 5 cycles of rapid freeze-thaw (method 1) all formulations contained more visible particles compared to their TO. CP60 contained the fewest particles of the three after 5 ft.

It turned out that after 5 cycles of slow freeze-thaw (method 2) all formulations contained more visible particles than in TO, although less than those treated by method 1. Fewer particles were observed in samples CP60 after 5 cycles of FT than in samples P60 and C60 after 1 FT cycle.

It turned out that after 3 cycles of slow freeze-thaw (method 3) all formulations contained more visible particles than in TO. There was no difference among the three formulations.

Appearance - 2nd stage.

The results of the second stage of the appearance analyzes are shown in table. 23 below.

Table 16

Sample THAT cooling chamber 1FT 3FT 5 FT P60 C60 Colorless, slightly opalescent, few visible particles Colorless, slightly opalescent, few visible particles (more than in TO) Colorless, slightly opalescent, more visible particles Colorless, opalescent, large number of visible protein particles СР60 Colorless, slightly opalescent, small amount of visible particles (according to compared to 3 ft.) Buffers (P60, C60 and SR60) Colorless, transparent, free of visible particles

In the 2nd stage of the study with slow freeze-thaw (method 3), more FT cycles were performed than in the first stage. There were a large number of visible particles in P60 after 5 FT compared to TO, while a small number of particles were observed in both C60 and CP60.

Concentration and enzymatic activity determined using 4-MUG as a substrate.

The results of measurements of the concentration and activity of the enzyme using 4-MUG as a substrate are shown in table. 17 (methods 1-2 and the first step of method 3) and in table. 18 (second step of method 3) below.

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Table 17

THAT cooling chamber Container Mg, Frosty Lyophilizer sample 5 FT 5 FT 5 FT Conc.. Activity. Conc.. Activity. Conc.. Activity. Conc.. Activity. mg/ml u/l mg/ml u/l mg/ml u/l mg/ml u/l P60 3.33 18711.6 3.09 17338.9 3.23 18145.9 2.77 15840.0 C60 3.45 19353.9 3.27 18335.3 3.35 18803.1 3.37 19565.6 СР60 3.32 18659.8 3.34 18748.2 3.23 18106.9 3.01 17349.1

Table 18

Name Lyophilizer THAT 1FT 3FT 5FT sample Conc.. Activity. Conc.. Activity. Conc.. Activity. Conc.. Activity. mg/ml u/l mg/ml u/l mg/ml u/l mg/ml u/l P60 3.09 17252.8 2.51 13954.9 1.45 7925.3 0.96 4973.7 C60 2.98 16608.8 3.36 18816.8 3.06 16931.2 3.80 20606.3 СР60 2.97 16561.6 2.93 16375.3 2.76 15261.8 0.99 5157.5

After 5 quick freeze-thaw cycles (method 1), a slight decrease in the enzyme concentration, determined using 4-MUG as a substrate, was observed in P60 when compared to the T0 sample, while no significant difference was found in C60 and CP60.

After 5 cycles of slow freeze-thaw using the Frosty container (method 2), no significant difference was observed in any of the 3 formulations compared to the T0 samples.

After 3 slow freeze-thaw cycles using a lyophilizer (method 3), a distinct decrease in enzyme concentration was observed in both P60 and CP60, but there was no significant change in C60 when compared to T0. Freeze-thaw using method 3 was repeated with 5 cycles and revealed the same trend. Enzyme concentration in P60 samples dropped by 18.8% after 1 F/T cycle and worsened after 3 cycles (53.1% decrease) and 5 cycles (68.9% decrease). In CP60, the enzyme concentration started to fall after 3 cycles, and eventually decreased to the same level as in P60 after 5 cycles. C60 showed virtually no change after 5 slow freeze-thaw cycles.

The results of the analysis of the enzyme concentration determined using 4-MUG as a substrate showed that the rate of freezing/heating, the number of F/T cycles and the type of buffer can affect the stability of ATB200. The citrate buffer (C60) system provided an acceptable stabilizing effect regardless of the freeze-thaw method used.

The trend in enzymatic activity for 4-MUG was the same as for the enzyme concentration determined with 4-MUG as a substrate.

During the rapid freeze-thaw study (method 1), there was a slight decrease in enzymatic activity in P60 after 5 cycles, no significant change was detected in C60 and CP60.

There was no obvious change in any sample during the slow freeze-thaw study at a temperature change of 1° C./min (method 2).

In a slow freeze-thaw study using a lyophilizer (method 3), the enzymatic activity in P60 showed a clear decrease and in CP60 showed a slight decrease. However, in the C60, there were practically no changes. During the Phase 2 slow freeze-thaw study according to Method 3, enzymatic activity in P60 was reduced by 19.1% after 1 cycle, by 54.1% after 3 cycles, and by 71.2% after 5 cycles. In CP60, enzymatic activity began to fall after 3 cycles, falling to a level similar to P60 after 5 cycles. In C60 after 5 cycles the change was not significant.

Purity: SEC-HPLC.

The results of measurements of purity are shown below in table. 20 (methods 1-2 and the first step of method 3) and table. 21 (second step of method 3).

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Table 20

Sample SEC THAT cooling chamber Container Mr, Frosty Lyophilizer 1FT 3FT 5 FT 1FT 3FT 5 FT 1FT 3ft P60 % monomers 99.7 99.7 99.6 99.5 99.7 99.7 99.7 93.4 92.1 HMW, % 0.2 0.2 0.2 0.2 0.3 0.2 0.2 5.8 7.6 LMW, % 0.0 0.2 0.2 oh, s 0.1 0.1 0.1 0.9 oh, s C60 % monomers 99.8 99.9 99.9 99.9 99.8 99.8 99.8 99.7 99.9 HMW, % 0.2 0.2 0.1 0.1 0.2 0.2 0.2 0.2 ο,ι LMW, % But. But. But. But. But. But. But. 0.2 But. СР60 % monomers 99.8 99.7 99.7 99.7 99.8 99.8 99.8 99.6 99.9 HMW, % 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 ο,ι LMW, % But. 0.1 0.1 0.1 But. But. But. 0.2 But.

Table 21

Sample name SEC THAT Lyophilizer 1FT 3ft 5 FT P60 % monomers 99.8 87.8 54.4 31.8 HMW, % 0.2 12.2 45.6 68.2 LMW, % But. But. ο,ι <0.1 C60 % monomers 99.8 99.8 99.8 99.7 HMW, % 0.2 0.2 0.2 0.3 LMW, % But. But. But. But. СР60 % monomers 99.8 99.6 98.3 31.3 HMW, % 0.2 0.4 1.7 68.7 LMW, % But. But. <0.1 <0.1

No change in SEC percentage of monomers was detected in any sample after no more than 5 cycles of fast F/T (method 1) or slow F/T (method 2).

In the first fast F/T study (method 3), P60 samples showed a clear decrease in SEC percentage of monomers (primarily due to mold formation with HMW) after 3 cycles. At the same time, there was no obvious change in the C60 and CP60. In the 2nd study with slow F/T according to method 3 (0.5°C/min), the percentage of monomers in the P60 samples began to fall after 1 cycle and eventually fell by 68.1% after 5 cycles. And in CP60, the percentage of monomers began to fall after 3 cycles, but to a much lesser extent than in P60; however, it reached the same level as in P60 after 5 cycles. There was no change in the percentage of monomers in C60 after no more than 5 cycles.

The SEC purity results were consistent with enzyme concentration and activity, confirming that the freeze/warm rate, number of F/T cycles, and buffer type can affect the stability of ATB200 during the freeze-thaw process. Based on the results of the SEC, it was concluded that buffers containing sodium phosphate also did not protect ATB200 during freeze-thaw cycles.

Free information.

ATB200 formulated in citrate buffer (C60) withstood multiple freeze-thaw cycles better than the other two buffers (P60 and CP60). Regardless of the freeze-thaw method, ATB200 remained stable in citrate buffer.

Composition example 3. Auxiliary substance.

Prepared eight compositions (E1-8) using different excipients. The ATB200 enzyme concentration in the excipient evaluation study was 5 mg/mL. Three buffers, two stabilizers, and one surfactant were selected to evaluate protein stability in the formulations described in Table 1. 22 below.

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Table 22

Sample Buffer Index NaCl Trehalose Mannitol Polysorbate VN 80 E1 25 mM phosphate 6.0 50mm 2% / 0.05% E2 / 2% 0.05% E3 25 mM citrate 2% / 0.05% E4 / 2% 0.05% E5 25 mM phosphate / 2% / E6 25 mM citrate / 2% / E7 25 mM phosphate citrate combined buffer 2% / 0.05% E8 / 2% 0.05%

Sample preparation.

Separately, 25 mM sodium phosphate buffer containing 50 mM NaCl at pH 6.0, 25 mM sodium citrate buffer containing 50 mM NaCl at pH 6.0, and 25 mM sodium phosphate-citrate combined buffer containing 50 mM NaCl at pH 6.0. In the ATB200 enzyme solution, the buffer was changed to three buffers using a dialysis cassette. Dialyses were performed at 5°C with gentle agitation, each involving 3 buffer changes, once every 6~10 h.

After dialysis, trehalose, mannitol, or PS80 was added to the dialysate to obtain the formulations listed in Table 1. 22. Finally, an appropriate volume of each of the formulation buffers was added to bring the final concentration of ATB200 to 5 mg/mL. The solutions were aseptically filtered using a 0.22 μm PES filter.

Each formulation was aseptically filled into 2 ml glass vials in a bioshielded laminar flow hood with a fill volume of approximately 1 ml. The flasks were stoppered and then immediately rolled up after filling.

Sample testing.

Formulations E1-8 were tested under 4 different conditions. Vials with each formulation were stored at 5°C for 12 weeks (12W) and at 40°C for 8 weeks (8W), subjected to freeze-thaw (0.5°C/min) for 5 cycles and shaken at 100 rpm. /min for 5 days at 25°C. The testing and sampling plan is described in Table 1. 23. Samples were tested at baseline (T0), 2 weeks (2W), 4 weeks (4W), 8 weeks (8W) and 12 weeks (12W).

Table 23

Processing condition THAT 2W 4W 8W 12W Storage at 5 °C X X X X X Storage at 40°C X X X / Freeze/Thaw, -80°C to 25°C in freezer and -47°C to 25°C with lyophilizer 1 cycle 3 cycles 5 cycles X X X Shaking, at 100 rpm, 25°С 5 days / x,(Z)

X: appearance analysis. Results.

Appearance.

The results of the appearance analyzes for the freeze-thaw study are shown in Table 1. 24 (using a refrigerator) and table. 25 (using lyophilizer) below.

Table 24

THAT Freezer 1FT 3FT|5FT E1 E2 E3 E4 Colorless, slightly opalescent, free of visible particles Colorless, slightly opalescent, free of visible particles E5 Colorless, slightly opalescent, numerous small visible Colorless, opalescent, numerous visible particles Colorless, opalescent, numerous visible particles

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particles E6 Colorless, transparent, free of visible particles Colorless, opalescent, numerous small visible particles Colorless, opalescent, numerous small visible particles E7 E8 Colorless, slightly opalescent, free of visible particles

Table 25

THAT Lyophilizer 1FT [3FT|5FT E1 Colorless, slightly opalescent, not containing visible particles Colorless, slightly opalescent, free of visible particles E2 E3 E4 E5 Colorless, opalescent, numerous visible particles Colorless, opalescent, numerous visible particles Colorless, opalescent, numerous visible particles E6 Colorless, opalescent, numerous small visible particles Colorless, opalescent, numerous small visible particles Colorless, opalescent, numerous small visible particles E7 Colorless, slightly opalescent, free of visible particles E8

During the freeze-thaw study, the number of visible particles slightly increased in F5 and F6, which did not contain PS80, with increasing number of freeze-thaw cycles. There were no differences in other compositions.

Free information.

Formulations without PS80 (F5 and F6) did not perform as well as formulations with PS80 in the freeze-thaw study, as evidenced by the formation of visible particles after several freeze-thaw cycles.

Formulation Example 4 Hemolysis in Human Whole Blood.

Blood dilution series (1:2, 1:3, 1:4, 1:5 and 1:10) from a human donor were prepared in saline. When mixed with deionized water, the dilution that resulted in an OD at 540 nm of 0.8 to 1.2 was used in the assay and is referred to as blood substrate. Four types of samples were tested: test drug, placebo, positive control and negative control. The test formulation was rhGAA ATB200 formulated with 25 mM sodium citrate, 2% mannitol and 0.05% polysorbate 80 at pH 6.0. The placebo was the same as the test drug except that it lacked the rhGAA ATB200. The positive control of the formulation was sterile water for injection and had a pH of 5. The negative control of the formulation was saline (0.9 NaCl) and had a pH of 5.

The test preparation (ATB200) at 300, 600 and 1000 μg/ml with saline, placebo with saline, negative control (saline) and positive control (water) were mixed with a blood substrate from a human donor. Samples were incubated without shaking for 1 h at 37°C. After incubation, the tubes were centrifuged for 10 min at about 100xg at room temperature. The amount of hemoglobin in the supernatant of each sample was analyzed spectrophotometrically at 540 nm.

The percentage of hemolysis for the test drug was determined using the formula:

% hemolysis = absorb. TA/placebo with blood - abs. saline solution with blood - absorption. TA/placebo.

Absorption water with blood - absorb. saline solution with blood.

The percentage of hemolysis of water plus blood is 100%. Saline solution was the negative control. Hemolysis equal to or less than 10% was considered insignificant. The percentage of hemolysis was calculated for each concentration of the test drug and for placebo.

In table. 26 below shows the results of the tested samples.

- 41 043224

Table 26

Treatment Concentration (µg/ml) OD ₅₄ o no blood OD540 with blood % hemolysis* Salt solution ³ - 0.016 Water ^b - 1.002 100 ATV200 300 0.001 0.016 -0.10 ATV200 600 0.002 0.003 -1.52 ATV200 1000 0.003 0.004 -1.52 Placebo for ATV200 _ ^with 0.000 0.003 -1.32 Placebo for ATV200 ^_d 0.000 0.003 -1.32 Placebo for ATV200 _ ^e 0.003 0.028 0.91

a = negative control for hemolysis b = positive control for hemolysis.

c = placebo diluted in saline at the same ratio as 300 µg/mL of test drug.

d = placebo diluted in saline at the same ratio as 600 µg/mL of test drug.

e = placebo diluted in saline at the same ratio as 1000 µg/mL of test drug.

*% hemolysis was determined using the OD of water as a positive control.

Incubation of human blood substrate with three placebo samples diluted in saline at the same ratio as the doses of the three test drugs did not cause any significant hemolysis of human blood. The percent hemolysis of the diluted placebo samples was calculated as -1.3, -1.3 and 0.9%, respectively. Incubation of human blood substrate with ATB200 at concentrations of 300, 600 and 1000 μg/ml did not cause any significant hemolysis of human blood. The percent hemolysis of the test formulation samples was calculated as -0.1%, 1.5%, and -1.5%, respectively.

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

Formulation example 5. Flocculation in human plasma and serum.

Dosed solutions of test drug (3 concentrations) and placebo (3 concentrations) were mixed with equal volumes of human plasma and serum from a donor. The test formulation was rhGAA ATB200 formulated with 25 mM sodium citrate, 2% mannitol and 0.05% polysorbate 80 at pH 6.0. The placebo was the same as the test drug except that it lacked the rhGAA ATB200.

One ml of each dose of test drug or placebo was mixed with an equal volume of plasma, serum and saline. Samples were incubated for 30 min at room temperature. After incubation, the tubes were examined macroscopically and microscopically for precipitation or coagulation. An aliquot from each tube was centrifuged at 14,000 rpm in a microcentrifuge for 10 min.

Each tube was examined for the presence or absence of a precipitate.

Precipitation/coagulation and precipitation were evaluated as follows:

= negative;

= minor precipitation or settling;

= minimal precipitation or settling;

= moderate precipitation or settling;

= significant precipitation or settling.

In table. 27 below shows the results of the tested samples.

-42043224

Table 27

Treatment Final concentration (µg/ml) blood plasma Serum saline solution Macro Micro Sediment Macro Micro Sediment Macro Micro Sediment Placebo for ATV200 - ^a 0 0 0 0 0 0 - Placebo for ATV200 - ^b 0 0 0 0 0 0 - Placebo for ATV200 - ^with 0 0 0 0 0 0 - ATV200 300 0 0 0 0 0 0 0 0 0 ATV200 600 0 0 0 0 0 0 0 0 0 ATV200 1000 0 0 0 0 0 0 0 0 0

a = Placebo diluted in saline at the same ratio as the test drug dosage solution at a final concentration of 300 µg/mL.

b = placebo diluted in saline at the same ratio as the test drug dosage solution at a final concentration of 600 µg/mL.

c = placebo diluted in saline at the same ratio as the test drug dosage solution at a final concentration of 1000 µg/mL.

No precipitation was observed macroscopically or microscopically in human plasma or serum when mixed with each concentration of ATB200. No precipitation was detected in placebo samples. When all placebo or ATB200 samples were centrifuged, no precipitation was observed.

Based on the results of this study, the ATB200 formulation was found to be compatible with human plasma and serum up to and including a final concentration of 1000 µg/mL.

Example. Pharmacokinetic and safety data for atb200 recombinant acid aglucosidase. Administered with miglustat in ert-treated and ert-naive patients with Pompe disease.

This study was primarily designed to evaluate the safety, tolerability, and pharmacokinetics (PK) of ATB200 co-administered with miglustat. Using a translational PK/pharmacodynamics (PD) model in Gaa knockout mice, the combination of 20mg/kg ATB200 with a high dose (eg 260mg) of miglustat in humans was predicted to provide optimal glycogen reduction.

In the description below, a high dose of miglustat is a dose of approximately 260 mg and a low dose of miglustat is a dose of approximately 130 mg.

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

This is an open-label, Phase 1/2, fixed-sequence, escalating-dose, escalation study, the first in humans, to evaluate the safety, tolerability, PK, PD, and efficacy of intravenous infusions of ATB200 given in combination with oral miglustat in adults with Pompe disease (Fig. 21). Mean values of total GAA protein and PK data for miglustat were assessed in the first 8 patients of cohort 1 during visit 9 and in the first 2 patients of cohort 3.

^a Analyzed safety data from 2 indicator patients from cohort 1 at each dose level before dosing in cohorts 2 and 3.

^b During stages 2 and 3, miglustat was administered orally before the start of intravenous infusions of ATB200. All doses of ATB200 were administered by intravenous infusion over 4 hours.

^c The first 2 patients in cohorts 2 and 3 served as indicator patients for their respective cohorts.

Key inclusion criteria:

men and women aged 18-65 who have been diagnosed with Pompe disease based on documented GAA enzyme deficiency or GAA genotyping.

Received ERT using alglucosidase-alpha for 2-6 years (or >2 years for cohort 2) before the start of the trial (cohort 1).

Currently receiving biweekly alglucosidase-alpha who received 2 infusions with no drug-related adverse events leading to dosing interruption (cohorts 1 and 2).

Should be able to walk 200 to 500 m on a 6-minute walk test (cohorts 1 and 3).

The forced standing vital capacity should be 30-80% of the predicted normal value (cohorts 1 and 3).

- 43 043224

Must be wheelchair bound and unable to walk unaided (Cohort 2).

PK analysis.

Blood samples were taken to analyze the concentration and activity of total GAA protein in blood plasma.

Stage 1: Before starting ATB200 infusion and 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, and 24 hours after starting the 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 determination of miglustat plasma concentrations were taken immediately before oral administration of miglustat (time 0) and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 9, 11 and 25 hours after oral administration. miglustat. The amount of miglustat in plasma was determined by validated LC-MS/MS analysis.

Plasma total GAA protein concentrations for 5, 10, and 20 mg/kg ATB200 were determined by validated LC-MS/MS quantitation of the rhGAA-specific signature peptide(s).

A preliminary analysis was completed in 8 patients in cohort 1 who completed stages 1 and 2 and in 2 patients in cohort 3 who started stage 3.

Initial patients transitioning from ERT are representative of the Pompe disease group, with a median of 5.02 years on ERT (Table 28).

Character! ERT Duration (Lumizyme®/Myozyme®) Years Mean (STDV) Age Years Mean (Range) Gender (m/f) % 6MWT Meters Mean (STDV) Table 28 Sticks at Baseline 5.02 (1.20) 47.7 (8.19) 80/20 398.4 (95.92) I e N/A 33.0 (12.73) 0/100 432.1 (67.81) Standing FVC Mean % Predicted (STDV) 51.9(13.84) 51.0 (26.87)

6MWT = 6-minute walk test; FVC = forced vital capacity; N/A = no data; STDV = standard deviation.

^a n=10 from cohort 1 (outpatients transferred from ERT) of the interim data analysis; n=2 from cohort 3 (not treated).

General protein GAA.

When administered alone, ATB200 increases slightly in a dose-dependent fashion (Table 29 and FIGS. 22A-22D). Variability appears to increase with dose of miglustat (Fig. 22C). Co-administration of 20mg/kg ATB200 with a high dose of miglustat (260mg) increased total GAA protein (AUC) exposure by approximately 25% relative to ATB200 alone at 20mg/kg. The half-life (α-phase) was increased by 45%, suggesting that the high dose of miglustat stabilizes ATB200 in plasma. An increase in the half-life is accompanied by an increase in AUC from time to maximum plasma concentration up to about 12 hours after dosing. An increase in AUC and half-life can be observed on a logarithmic scale throughout the terminal elimination phase (FIG. 22B). ATB200 showed a relatively large volume of distribution. Plasma distribution of total GAA protein appears similar between ERT-naive patients (cohort 3) and ERT-treated patients (cohort 1) (FIGS. 22A and 22D).

- 44 043224

Table 29

Total GAA Protein

1 5 mg/kg alone ⁰ 61 (18.1) 3.8 (3.04.0) 215 (16.7) 218(16.4) 1.9 (16.7) 1.1 (Yu.2) 2.1 (16.9) 4.62 (12.7) 1 10 mg/kg alone ⁰ 144 (16.6) 4.0 (3.54.0) 578 (20.3) 584 (20.4) 1.6 (46.1) 1.3 (10.5) 1.59 (25.4) 3.87 (16.5) 1 20 mg/kg alone ⁰ 345 (10D) 4.0 (3.54.0) 1508 (14.5) 1512 (14.4) 2.1 (29.7) 1.5 (6.5) 1.22 (21.7) 3.52 (12.4) 1 ATB200 20 mg/kg + miglustat, SD ^d , low dose 334 (16.2) 4.0 (3.54.0) 1694 (17.7) 1701 (17.5) 2.4 (16.6) 1.8 (10.2) 1.09 (22.9) 3.76 (13.3) 1 ATB200 20 mg/kg + miglustat, MD ^d , low dose 353 (13.7) 4.0 (3.55.0) 1804 (15.7) 1808 (15.8) 2.5 (8.1) 1.9 (21.8) 1.02 (21.4) 3.73 (12.3) 1 ATB200 20 mg/kg + miglustat, SD ^e , high dose 349 (13.9) 4.0 (3.54.0) 1878 (17.5) 1886 (17.5) 2.7(13.1) 2.3 (18.9) 0.98 (26.5) 3.74 (12.3) 1 ATB200 20 mg/kg + miglustat, MD ^d , high dose 356 (20.2) 4.0 (3.54.0) 1886 (21.3) 1901 (21.7) 2.5 (20.5) 2.1 (16.1) 0.98 (27.3) 3.6 (18.7) uh ATB200 20 mg/kg + miglustat, MD, high dose 291 (21.6) 4.3 (4.04.5) 1597 (34.8) 1600 (34.9) 2.4 (5.4) 2 (14.5) 0.69 (28.9) 2.61 (17.3) 3 ATB200 20 mg/kg + miglustat, MD ^f , high dose 330 (27.5) 4.0 (4.04.0) 1672 (32.7) 1676 (32.6) 2.6 (8.7) 1.9 (9.0) 0.66 (26.6) 2.33 (23.2) AUC = area under the curve; CL _T = total elimination from the body; C _max = maximum drug concentration; CV = coefficient of variation; MD = multiple doses; SD = single dose; 1½ = half-life; t _max = time to reach maximum drug concentration; V _ss = apparent volume of distribution at steady state. ^a Geometric mean (CV, %), ^b median value (min. - max.), Harmful arithmetic value, (CV, %), ^d n = 8, ^e n = 7, ^f n = 2.

PK - data for miglustat.

Miglustat showed a dose-proportional dependence of kinetic parameters (Table 30 and Fig. 23). Plasma levels for miglustat appear to be similar between single and multiple doses.

Table 30 Summary of pharmacokinetic data for miglustat

SD, low dose I486 (29.9) 3.5 (1.5-3.5) And 807 (25.6) 12,565 (26.8) 5.6 (I.7) 10.6 (23) 85.8 (23.4) MD, low dose 1518 (27.6) 3.0 (1.5-3.5) 12,254 (26.4) 13,094 (28.3) 5.9 (32.1) 10.2 (23.9) 86.7 (43.9) SD, high dose 3059 (36.1) 3.5 (1.5-5) 23 999 (35) 25,859 (34.4) 5.7 (29.9) 10.6 (33) 86.3 (45.7) MD high dose 3569 (25.5) 3.0 (1.0-4.0) 24,970 (24.1) 25 972 (23) 5.3 (15.6) 10.3 (26.4) 81 (41.8) V _z \u003d apparent volume of distribution in the terminal state, ^and the geometric mean value (CV, %), ^b median value (min, - max.), ^C the arithmetic mean value, (CV, %).

Pharmacodynamics.

At visit 11, ERT-treated patients from cohort 1 (FIGS. 24A and 24B):

alanine aminotransferase (ALT) levels decreased in 5 of 8 patients; normalized in 4/4 patients with elevated baseline levels;

aspartate aminotransferase (AST) levels decreased in 6 of 8 patients; normalized in 3/4 patients with elevated baseline levels;

creatine phosphokinase (CPK) levels decreased in 6 of 8 patients; normalized in 2/6 patients with elevated baseline levels;

urinary tetrasaccharide glucose (HEX4) levels decreased in 8 of 8 patients.

By week 4, levels of all 4 biomarkers decreased in 2 patients in the untreated cohort (cohort 3) (FIGS. 24C and 24D).

In FIG. 24A-24D, data are presented as mean ± standard deviation.

Safety.

After a total of 155+ infusions, no serious adverse events (AE) or infusion-related reactions were reported in all patients.

Post-treatment AE, which occurred in 11/13 (84%) patients, was generally

- 45 043224 light and transient.

Treatment-related AE 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).

Conclusions.

ATB200 alone and in combination with miglustat was safe and well tolerated, with no infusion-related reactions to date.

ATB200 alone showed a superproportional dose-dependent increase in exposure, which was further enhanced by miglustat, suggesting a stabilizing effect of the chaperone on ATB200.

After switching from standard treatment to treatment with the ATB200/miglustat combination, patients generally showed improvement in biomarkers of muscle damage, with many patients showing normalization by week 18.

The original untreated patients treated with the ATB200/miglustat combination showed a sustained reduction in all biomarkers of muscle damage.

Sequences

SEQ ID NO: Met Gly Vai Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Vai 1 Cys Ala Leu Vai Ser Leu Ala Thr Ala Ala Leu Leu Gly His He

Leu Leu His Asp Phe Leu Leu Vai Pro Arg Glu Leu Ser Gly Ser Ser Pro Vai Leu Glu Glu Thr His Pro Ala His Gin Gin Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gin Ala His Pro Gly Arg Pro Arg Ala Vai Pro Thr Gin Cys Asp Vai Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala He Thr Gin Glu Gin Cys Glu Ala Arg Gly Cys Cys Tyr He Pro Ala Lys Gin Gly Leu Gin Gly Ala Gin Met Gly Gin Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp He Leu Thr Leu Arg Leu Asp Vai Met Met Met Glu Thr Glu Asn Arg Leu His Phe Thr He Lys Asp Pro Ala Asn Arg Arg Tyr Glu Vai Pro Leu Glu Thr Pro Arg Vai His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Vai Glu Phe Ser Glu Glu Pro Phe Gly Vai He Vai His Arg Gin Leu Asp Gly Arg Vai Leu Leu Asn Thr Vai Ala Pro Leu Phe Phe Ala Asp Gin Phe Leu Gin Leu Ser Thr Ser Leu Pro Ser Gin Tyr He Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg He Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Vai Phe Leu Leu Asn Ser Asn Ala Met Asp Vai Vai Leu Gin Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly He Leu Asp Vai Tyr He Phe Leu Gly Pro Glu Pro Lys Ser Vai Vai Gin Gin Tyr Leu Asp Vai Vai Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His

- 46 043224

Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala He Thr Arg Gin Vai Vai Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Vai Gin Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Vai Gin Glu Leu His Gin Gly Gly Arg Arg Tyr Met Met He Vai Asp Pro Ala He Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Vai Phe He Thr Asn Glu Thr Gly Gin Pro Leu He Gly Lys Vai Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Vai Ala Glu Phe His Asp Gin Vai Pro Phe Asp Gly Met Trp He Asp Met Asn Glu Pro Ser Asn Phe He Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Vai Pro Gly Vai Vai Gly Gly Thr Leu Gin Ala Ala Thr He Cys Ala Ser His Gin Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala He Ala Ser His Arg Ala Leu Vai Lys Ala Arg Gly Thr Arg Pro Phe Vai He Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Vai Trp Ser Ser Trp Glu Gin Leu Ala Ser Ser Vai Pro Glu He Leu Gin Phe Asn Leu Leu Gly Vai Pro Leu Vai Gly Ala Asp Vai Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Vai Arg Trp Thr Gin Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gin Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gin Gin Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His Gin Ala His Vai Ala Gly Glu Thr Vai Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Vai Asp His Gin Leu Leu Trp Gly Glu Ala Leu Leu He Thr Pro Vai Leu Gin Ala Gly Lys Ala Glu Vai Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gin Thr Vai Pro He Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala He His Ser Glu Gly Gin Trp Vai Thr Leu Pro Ala Pro Leu Asp Thr He Asn Vai His Leu Arg Ala Gly Tyr He lie Pro Leu Gin Gly

- 47 043224

Pro Gly Leu Thr Thr Glu Ser Arg Gin Gin Pro Met Ala Leu Ala Vai Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Vai Leu Glu Arg Gly Ala Tyr Thr Gin Vai He Phe Leu Ala Arg Asn Thr He Vai Asn Glu Leu Vai Arg Vai Thr Ser Glu Gly Ala Gly Leu Gin Leu Gin Lys Vai Thr Vai Leu Gly Vai Ala Thr Ala Pro Gin Gin Vai Leu Ser Asn Gly Vai Pro Vai Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Vai Leu Asp He Cys Vai Ser Leu Leu Met Gly Glu Gin Phe Leu Vai Ser Trp Cys

SEQ ID NO: Gin Gin Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gin Ala His

Pro Gly Arg Pro Arg Ala Vai Pro Thr Gin Cys Asp Vai Pro Pro

Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala He Thr Gin Glu Gin Cys Glu Ala Arg Gly Cys Cys Tyr He Pro Ala Lys Gin Gly

Leu Gin Gly Ala Gin Met Gly Gin Pro Trp Cys Phe Phe Pro Pro

Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp He Leu Thr Leu Arg Leu Asp Vai Met Met Glu Thr Glu Asn Arg Leu His Phe Thr He Lys Asp Pro Ala Asn Arg Arg Tyr Glu Vai Pro Leu Glu Thr Pro Arg Vai His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Vai Glu Phe Ser Glu Glu Pro Phe Gly Vai He Vai His Arg Gin Leu Asp Gly Arg Vai Leu Leu Asn Thr Vai Ala Pro Leu Phe Phe Ala Asp Gin Phe Leu Gin Leu Ser Thr Ser Leu Pro Ser Gin Tyr He Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg He Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Vai Phe Leu Leu Asn Ser Asn Ala Met Asp Vai Vai Leu Gin Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly He Leu Asp Vai Tyr He Phe Leu Gly Pro Glu Pro Lys Ser Vai Vai Gin Gin Tyr Leu Asp Vai Vai Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu

- 48 043224

Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala He Thr Arg Gin Vai Vai Glu Asn Met Thr Arg Ala His Phe Pro Leu

Asp Vai Gin Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg

Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala

Met Vai Gin Glu Leu His Gin Gly Gly Arg Arg Tyr Met Met

He Vai Asp Pro Ala He Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Vai Phe He Thr Asn Glu Thr Gly Gin Pro Leu He Gly Lys Vai Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Vai Ala Glu Phe His Asp Gin Vai Pro Phe Asp Gly Met Trp He Asp Met Asn Glu Pro Ser Asn Phe He Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Vai Pro Gly Vai Vai Gly Gly Thr Leu Gin Ala Ala Thr He Cys Ala Ser Ser His Gin Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala He Ala Ser His Arg Ala Leu Vai Lys Ala Arg Gly Thr Arg Pro Phe Vai He Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Vai Trp Ser Ser Trp Glu Gin Leu Ala Ser Ser Vai Pro Glu He Leu Gin Phe Asn Leu Leu Gly Vai Pro Leu Vai Gly Ala Asp Vai Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Vai Arg Trp Thr Gin Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gin Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gin Gin Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His Gin Ala His Vai Ala Gly Glu Thr Vai Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Vai Asp His Gin Leu Leu Trp Gly Glu Ala Leu Leu He Thr Pro Vai Leu Gin Ala Gly Lys Ala Glu Vai Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gin Thr Vai Pro He Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala He His Ser Glu Gly Gin Trp Vai Thr Leu Pro Ala Pro Leu Asp Thr He Asn Vai His Leu Arg Ala Gly Tyr He He Pro Leu Gin Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gin Gin Pro Met Ala Leu Ala Vai Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Vai Leu Glu Arg Gly Ala Tyr Thr Gin Vai He Phe Leu Ala Arg Asn Asn Thr He Vai Asn Glu Leu Vai Arg Vai Thr Ser Glu Gly Ala Gly Leu Gin Leu Gin Lys Vai Thr Vai Leu Gly Vai Ala Thr Ala Pro Gin Gin Vai Leu Ser Asn Gly Vai Pro Vai Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Vai Leu Asp He Cys Vai Ser Leu Leu Met Gly Glu Gin Phe Leu Vai Ser Trp Cys

- 49 043224

<110> Sequence Listing AMIKUS THERAPUTICS, INC.

<12 0> COMPOSITIONS CONTAINING RECOMBINANT ACID ALPHA-GLUCOSIDASE <130> AT-P8700-PCT <160>2 <170> PatentIn version 3.5 <210>1 <211>952 <212> PROTEIN <213> Homo sapiens <400> 1

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys 1 5 1015

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

2530

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val 35 4045

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly 50 5560

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 7580

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 9095

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155160

Pro Lys Asp Ile Leu Thr Leu Arg

165

Leu Asp Val Met Met Glu Thr Glu

170 175

- 50 043224

Asn

Val

Tyr

Glin 225

Phe

ile

trp

Ala

Gly 305

Val

Leu

Gln

Leu

Arg 385

Gln

Asn

Arg Leu His 180 Phe Thr ile Lys asp 185 Pro Ala Asn Arg Arg 190 Tyr Pro Leu 195 Glu Thr Pro Arg Val 200 His Ser Arg Ala Pro 205 Ser Pro Ser 210 Val Glu Phe Ser Glu 215 Glu Pro Phe gly Val 220 ile Val His Leu asp gly Arg Val 230 Leu Leu Asn Thr Thr 235 Val Ala Pro Leu Ala asp Gln Phe 245 Leu Gln Leu Ser Thr 250 Ser Leu Pro Ser Gln 255 Thr gly Leu 260 Ala Glu His Leu Ser 265 Pro Leu Met Leu Ser 270 Thr Thr Arg 275 ile Thr Leu trp Asn 280 Arg asp Leu Ala Pro 285 Thr Pro Asn 290 Leu Tyr gly Ser His 295 Pro Phe Tyr Leu Ala 300 Leu Glu asp Ser Ala His gly Val 310 Phe Leu Leu Asn Ser 315 Asn Ala Met asp Leu Gln Pro Ser 325 Pro Ala Leu Ser Trp 330 Arg Ser Thr gly Gly 335 asp Val Tyr 340 ile Phe Leu gly Pro 345 Glu Pro Lys Ser Val 350 Val Tyr Leu 355 asp Val Val gly Tyr 360 Pro Phe Met Pro Pro 365 Tyr trp Gly 370 Phe His Leu Cys Arg 375 trp gly Tyr Ser Ser 380 Thr Ala ile Gln Val Val Glu Asn 390 Met Thr Arg Ala His 395 Phe Pro Leu asp trp Asn asp Leu 405 asp Tyr Met asp Ser 410 Arg Arg asp Phe Thr 415 Lys asp Gly 420 Phe Arg asp Phe Pro 425 Ala Met Val Gln Glu 430 Leu

Glu

Leu

Arg

Phe 240

Tyr

Ser

gly

gly

Val 320

ile

Gln

gly

Thr

Val 400

Phe

His

- 51 043224

Gln Gly Gly Arg Arg

435

Tyr Met Met Ile Val

440

Asp Pro Ala Ile Ser 445

Ser Gly Pro Ala Gly

450

Ser Tyr Arg Pro Tyr

455

Asp Glu Gly Leu Arg 460

Gly Val Phe Ile Thr 465

Asn Glu Thr Gly Gln 470

Pro Leu Ile Gly Lys 475

Trp Pro Gly Ser Thr

485

Ala Phe Pro Asp Phe

490

Thr Asn Pro Thr Ala

495

Ala Trp Trp Glu Asp

500

Met Val Ala Glu Phe

505

His Asp Gln Val Pro

510

Asp Gly Met Trp Ile 515

Asp Met Asn Glu Pro 520

Ser Asn Phe Ile Arg

525

Ser Glu Asp Gly Cys

530

Pro Asn Asn Glu Leu

535

Glu Asn Pro Pro Tyr 540

Pro Gly Val Val Gly 545

Gly Thr Leu Gln Ala 550

Ala Thr Ile Cys Ala 555

Ser His Gln Phe Leu

565

Ser Thr His Tyr Asn

570

Leu His Asn Leu Tyr

575

Leu Thr Glu Ala Ile

580

Ala Ser His Arg Ala

585

Leu Val Lys Ala Arg

590

Thr Arg Pro Phe Val

595

Ile Ser Arg Ser Thr 600

Phe Ala Gly His Gly 605

Tyr Ala Gly His Trp 610

Thr Gly Asp Val Trp

615

Ser Ser Trp Glu Gln 620

Ala Ser Ser Val Pro 625

Glu Ile Leu Gln Phe 630

Asn Leu Leu Gly Val 635

Leu Val Gly Ala Asp

645

Val Cys Gly Phe Leu

650

Gly Asn Thr Ser Glu

655

Leu Cys Val Arg Trp

660

Thr Gln Leu Gly Ala 6 65

Phe Tyr Pro Phe Met

670

Asn His Asn Ser Leu

675

Leu Ser Leu Pro Gln

680

Glu Pro Tyr Ser Phe 685

Ser

Arg

Val 480

Leu

Phe

gly

Val

Ser 560

gly

gly

Arg

Leu

Pro 640

Glu

Arg

Ser

- 52 043224

Glu

Leu 705

Glu

Thr

Thr

Leu

Ser 785

Glu

His

Thr

Lys

Leu 865

Arg

Ala

Pro

Ser

Pro Ala Gln 690

Leu Pro His

Thr Val Ala

Trp Thr Val

740

Pro Val Leu

755

Gly Thr Trp 770

Leu Pro Pro

Gly Gln Trp

Leu Arg Ala

820

Thr Glu Ser

835

Gly Gly Glu 850

Glu Val Leu

Asn Asn Thr

Gly Leu Gln

900

Gln Gln Val

915

Gln Ala Met

695

Leu Tyr Thr

710

Arg Pro Leu 725

Asp His Gln

Gln Ala Gly

Tyr Asp Leu

775

Pro Pro Ala

790

Val Thr Leu 805

Gly Tyr Ile

Arg Gln Gln

Ala Arg Gly

855

Glu Arg Gly

870

Ile Val Asn 885

Leu Gln Lys

Leu Ser Asn

Arg Lys Ala

Leu Phe His

Phe Leu Glu

730

Leu Leu Trp

745

Lys Ala Glu 760

Gln Thr Val

Ala Pro Arg

Pro Ala Pro

810

Ile Pro Leu

825

Pro Met Ala 840

Glu Leu Phe

Ala Tyr Thr

Glu Leu Val

890

Val Thr Val

905

Gly Val Pro 920

Leu Thr Leu

700

Gln Ala His 715

Phe Pro Lys

Gly Glu Ala

Val Thr Gly

765

Pro Ile Glu

780

Glu Pro Ala 795

Leu AspThr

Gln Gly Pro

Leu Ala Val

845

Trp Asp Asp

860

Gln Val Ile 875

Arg Val Thr

Leu Gly Val

Val Ser Asn

925

Arg Tyr Ala

Val Ala Gly

720

Asp Ser Ser

735

Leu Leu Ile 750

Tyr Phe Pro

Ala Leu Gly

Ile His Ser

800

Ile Asn Val

815

Gly Leu Thr 830

Ala Leu Thr

Gly Glu Ser

Phe Leu Ala

880

Ser Glu Gly

895

Ala Thr Ala 910

Phe Thr Tyr

Pro Asp Thr

Lys Val Leu

Asp Ile Cys

Val Ser Leu

Leu Met Gly

- 53 043224

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945

950 <210>2 <211>896 <212> PROTEIN <213> Homo sapiens <400>2

Gln Gln Gly Ala Ser 1 5

Arg Pro Gly Pro Arg

Asp Ala Gln Ala His 15

Gly Arg Pro Arg Ala

Val Pro Thr Gln Cys

Asp Val Pro Pro Asn 30

Arg Phe Asp Cys Ala 35

Pro Asp Lys Ala Ile 40

Thr Gln Glu Gln Cys 45

Ala Arg Gly Cys Cys 50

Tyr Ile Pro Ala Lys 55

Gln Gly Leu Gln Gly 60

Gln Met Gly Gln Pro 65

Trp Cys Phe Phe Pro 70

Pro Ser Tyr Pro Ser 75

Lys Leu Glu Asn Leu 85

Ser Ser Ser Glu Met

Gly Tyr Thr Ala Thr 95

Thr Arg Thr Thr Pro

100

Thr Phe Phe Pro Lys

105

Asp Ile Leu Thr Leu

110

Leu Asp Val Met Met 115

Glu Thr Glu Asn Arg 120

Leu His Phe Thr Ile

125

Asp Pro Ala Asn Arg 130

Arg Tyr Glu Val Pro

135

Leu Glu Thr Pro Arg 140

His Ser Arg Ala Pro 145

Ser Pro Leu Tyr Ser 150

Val Glu Phe Ser Glu 155

Pro Phe Gly Val Ile

165

Val His Arg Gln Leu

170

Asp Gly Arg Val Leu

175

Asn Thr Thr Val Ala

180

Pro Leu Phe Phe Ala

185

Asp Gln Phe Leu Gln

190

Pro

Ser

Glu

Ala

Tyr 80

Leu

Arg

Lys

Val

Glu 160

Leu

Leu

Leu

Ser Thr Ser Leu Pro

Ser Gln Tyr Ile Thr

Gly Leu Ala Glu His

- 54 043224

195

200

205

Ser Pro Leu Met Leu

210

Ser Thr Ser Trp Thr

215

Arg Ile Thr Leu Trp 220

Arg Asp Leu Ala Pro 225

Thr Pro Gly Ala Asn 230

Leu Tyr Gly Ser His 235

Phe Tyr Leu Ala Leu

245

Glu Asp Gly Gly Ser

250

Ala His Gly Val Phe

255

Leu Asn Ser Asn Ala

260

Met Asp Val Val Leu

265

Gln Pro Ser Pro Ala

270

Ser Trp Arg Ser Thr

275

Gly Gly Ile Leu Asp

280

Val Tyr Ile Phe Leu

285

Pro Glu Pro Lys Ser 290

Val Val Gln Gln Tyr

295

Leu Asp Val Val Gly 300

Pro Phe Met Pro Pro 305

Tyr Trp Gly Leu Gly 310

Phe His Leu Cys Arg 315

Gly Tyr Ser Ser Thr

325

Ala Ile Thr Arg Gln

330

Val Val Glu Asn Met

335

Arg Ala His Phe Pro

340

Leu Asp Val Gln Trp

345

Asn Asp Leu Asp Tyr

350

Asp Ser Arg Arg Asp

355

Phe Thr Phe Asn Lys

360

Asp Gly Phe Arg Asp 365

Pro Ala Met Val Gln

370

Glu Leu His Gln Gly

375

Gly Arg Arg Tyr Met

380

Ile Val Asp Pro Ala 385

Ile Ser Ser Ser Gly 390

Pro Ala Gly Ser Tyr 395

Pro Tyr Asp Glu Gly

405

Leu Arg Arg Gly Val

410

Phe Ile Thr Asn Glu

415

Gly Gln Pro Leu Ile

420

Gly Lys Val Trp Pro

425

Gly Ser Thr Ala Phe

430

Asp Phe Thr Asn Pro 435

Thr Ala Leu Ala Trp

440

Trp Glu Asp Met Val 445

Asn

Pro 240

Leu

Leu

gly

Tyr

TRP 320

Thr

Met

Phe

Met

Arg 400

Thr

Pro

Ala

- 55 043224

Glu Phe His Asp Gln 450

Val Pro Phe Asp Gly

455

Met Trp Ile Asp Met 460

Glu Pro Ser Asn Phe 465

Ile Arg Gly Ser Glu 470

Asp Gly Cys Pro Asn 475

Glu Leu Glu Asn Pro

485

Pro Tyr Val Pro Gly

490

Val Val Gly Gly Thr

495

Gln Ala Ala Thr Ile

500

Cys Ala Ser Ser His

505

Gln Phe Leu Ser Thr

510

Tyr Asn Leu His Asn 515

Leu Tyr Gly Leu Thr

520

Glu Ala Ile Ala Ser

525

Arg Ala Leu Val Lys

530

Ala Arg Gly Thr Arg

535

Pro Phe Val Ile Ser

540

Ser Thr Phe Ala Gly 545

His Gly Arg Tyr Ala 550

Gly His Trp Thr Gly 555

Val Trp Ser Ser Trp

565

Glu Gln Leu Ala Ser

570

Ser Val Pro Glu Ile

575

Gln Phe Asn Leu Leu

580

Gly Val Pro Leu Val

585

Gly Ala Asp Val Cys

590

Phe Leu Gly Asn Thr 595

Ser Glu Glu Leu Cys 600

Val Arg Trp Thr Gln

605

Gly Ala Phe Tyr Pro 610

Phe Met Arg Asn His 615

Asn Ser Leu Leu Ser

620

Pro Gln Glu Pro Tyr 625

Ser Phe Ser Glu Pro 630

Ala Gln Gln Ala Met 635

Lys Ala Leu Thr Leu

645

Arg Tyr Ala Leu Leu

650

Pro His Leu Tyr Thr

655

Phe His Gln Ala His

660

Val Ala Gly Glu Thr 6 65

Val Ala Arg Pro Leu

670

Leu Glu Phe Pro Lys

675

Asp Ser Ser Thr Trp 680

Thr Val Asp His Gln 685

Leu Trp Gly Glu Ala 690

Leu Leu Ile Thr Pro

695

Val Leu Gln Ala Gly

700

Asn

Asn 480

Leu

His

His

Arg

Asp 560

Leu

gly

Leu

Leu

Arg 640

Leu

Phe

Leu

Lys

- 56 043224

Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln

705 710 715720

Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala

725 730735

Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro

740 745750

Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile

755 760765

Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro

770 775780

Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu

785 790 795800

Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala

805 810815

Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu

820 825830

Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val

835 840845

Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly

850 855860

Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp

865 870 875880

Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

885 890895

Claims (27)

1. Pharmaceutical composition containing: (a) recombinant human α-glucosidase (rhGAA) molecules, wherein the rhGAA molecules are derived from expression in Chinese Hamster Ovary (CHO) cells, wherein 40-60% of the N-glycans in the rhGAA molecules are complex-type N-glycans, where rhGAA molecules contain the first, second, third, fourth, fifth, sixth and seventh potential N-glycosylation sites in the amino acids corresponding to N84, N177, N334, N414, N596, N826 and N869 in SEQ ID NO: 2, respectively; where at least 50% of the rhGAA molecules contain a glycan bearing bis-mannose-6-phosphate (6c-M6P) at the first potential N-glycosylation site, and where the rhGAA molecules contain 3-5 moles of mannose-6-phosphate (M6P) per mole rhGAA; (b) a nitrate buffer; and (c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, - 57 043224 where the composition is characterized by a pH of 5.0 to 7.0. 2. The pharmaceutical composition according to claim 1, wherein the rhGAA is present at a concentration of 5 mg/mL to 50 mg/mL. 3. Pharmaceutical formulation according to claim 1 or 2, wherein the rhGAA is present at a concentration of 12 mg/mL to 18 mg/mL. 4. Pharmaceutical composition according to any one of claims 1 to 3, wherein rhGAA is present at a concentration of 15 mg/ml. 5. Pharmaceutical composition according to any one of claims 1 to 4, wherein the composition has a pH of 5.5 to 6.0. 6. Pharmaceutical composition according to any one of claims 1 to 5, wherein the composition has a pH value of 6.0. 7. Pharmaceutical composition according to any one of claims 1 to 6, wherein the citrate buffer contains a potassium, sodium or ammonium salt. 8. Pharmaceutical formulation according to any one of claims 1 to 7, wherein the mannitol is present at a concentration of 10 mg/ml to 50 mg/ml. 9. Pharmaceutical composition according to any one of claims 1 to 7, wherein polysorbate 80 is present at a concentration of 0.2 mg/ml to 0.5 mg/ml. 10. A pharmaceutical composition according to any one of claims 1 to 9, wherein the mannitol is present at a concentration of 20 mg/ml and polysorbate 80 is present at a concentration of 0.5 mg/ml. 11. Pharmaceutical composition according to any one of claims 1 to 10, further comprising: (d) an alkalizing agent and/or (e) an acidifying agent, wherein the alkalizing agent and the acidifying agent are present in amounts sufficient to maintain the pH of the pharmaceutical formulation in the range of 5.0 to 6.0. 12. A pharmaceutical composition according to any one of claims 1 to 11, wherein the rhGAA molecules contain at least 4 moles of sialic acid residues per mole of rhGAA. 13. A pharmaceutical formulation according to any one of claims 1 to 11, wherein the rhGAA molecules contain 2.0 to 8.0 moles of sialic acid residues per mole of rhGAA. 14. Pharmaceutical composition according to any one of claims 1-13, where the rhGAA molecules contain seven potential N-glycosylation sites and where: (a) at least 50% of rhGAA contains an N-glycan unit bearing 6c-M6P at the first potential N-glycosylation site; or (b) no at least 30% of rhGAA contains an N-glycan unit bearing mono-M6P at a second potential N-glycosylation site; or (c) at least 30% of rhGAA contains an N-glycan unit bearing 6c-M6P at a fourth potential N-glycosylation site; or (d) at least 20% of rhGAA contains an N-glycan unit bearing mono-M6P at a fourth potential N-glycosylation site. 15. Pharmaceutical composition according to any one of claims 1 to 14, where the pharmaceutical composition consists essentially of: (a) rhGAA molecules; (b) sodium citrate; (c) citric acid monohydrate; (d) mannitol; (e) polysorbate 80; (f) water; (g) optionally an acidifying agent; and (h) optionally an alkalizing agent, wherein the composition has a pH of 5.0 to 6.0. 16. Pharmaceutical composition according to any one of claims 1 to 15, where the pharmaceutical composition consists essentially of: (a) rhGAA molecules present at a concentration of 15 mg/ml; (b) sodium citrate buffer present at a concentration of 25 mM; (c) mannitol present at a concentration of 20 mg/ml; (d) polysorbate 80 present at a concentration of 0.5 mg/ml; (e) water; (f) optionally an acidifying agent; and (g) optionally an alkalizing agent, wherein the composition has a pH of 5.0 to 6.0. 17. A pharmaceutical composition comprising a pharmaceutical composition according to any one of claims 1 to 16, wherein the pharmaceutical composition has been lyophilized. 18. The use of a pharmaceutical composition according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17 for the treatment of Pompe disease in a patient. - 58 043224 19. Use according to claim 18, wherein the pharmaceutical composition is diluted prior to administration to a patient, or where the pharmaceutical composition is reconstituted prior to administration to a patient. 20. Use according to claim 18 or 19, wherein the pharmaceutical composition is administered in combination with miglustat. 21. A method for preparing a pharmaceutical composition according to any one of claims 1 to 16, wherein the method includes: (a) obtaining a solution containing a nitrate buffer, at least one excipient selected from the group consisting of mannitol, polysorbate 80 and combinations thereof, and a rhGAA molecule; (b) adjusting the pH of the solution; and (c) adding an additional volume of water to the solution; where rhGAA molecules are derived from expression in Chinese hamster ovary (CHO) cells, where 40-60% of the N-glycans in rhGAA molecules are complex-type N-glycans, where rhGAA molecules contain the first, second, third, fourth, fifth, sixth and a seventh potential N-glycosylation site at the amino acids corresponding to N84, N177, N334, N414, N596, N826 and N869 in SEQ ID NO: 2, respectively; where at least 50% of the rhGAA molecules contain a glycan bearing bis-mannose-6-phosphate (6c-M6P) at the first potential N-glycosylation site, and where the rhGAA molecules contain 3-5 moles of mannose-6-phosphate (M6P) per mole of rhGAA. 22. The method of claim 21, further comprising filtering the solution. 23. The method of claim 21 or 22, further comprising storing the solution. 24. A method for preparing a pharmaceutical composition, the method comprising lyophilizing the pharmaceutical composition according to any one of claims 1-16. 25. A method of treating Pompe disease, the method comprising administering to a patient a pharmaceutical composition according to any one of claims 1-16. 26. A method of treating Pompe disease, the method comprising reconstituting the pharmaceutical composition of claim 17 and administering the reconstituted pharmaceutical composition to a patient. 27. The method of claim 25 or 26, wherein the method further comprises administering miglustat to the patient.