Selection method for high-M6P recombinant proteins

Abstract

This invention provides a method for producing recombinant human lysosomal proteins. [Solution] A method for producing recombinant human lysosomal protein is provided, comprising: culturing host cells in a bioreactor that secretes recombinant human lysosomal protein; removing the culture medium from the bioreactor; filtering the culture medium to provide a filtrate; loading the filtrate into an anion exchange chromatography (AEX) column to capture the lysosomal protein; and eluting a first protein product from the AEX column. Such recombinant human lysosomal protein may have a high content of mannose-6-phosphate residues. Pharmaceutical compositions containing such recombinant human lysosomal protein, as well as methods for treating and using such recombinant human lysosomal protein, are also described.

Description

Technical Field

[0001] The principles and embodiments of the present invention generally relate to the production of recombinant proteins, particularly lysosomal enzymes having a high content of mannose-6-phosphate.

Background Art

[0002] Lysosomal storage diseases are a group of autosomal recessive genetic diseases characterized by the accumulation of cellular sphingolipids, glycogen, or mucopolysaccharides within an intracellular compartment called the lysosome. Individuals with these diseases carry a mutant gene encoding an enzyme that is defective in catalyzing the hydrolysis of one or more of these substances, and these substances accumulate in the lysosome. For example, Pompe disease, also known as acid maltase deficiency or type II glycogenosis, is one of several lysosomal storage diseases. Other examples of lysosomal disorders include Gaucher disease, GM1-gangliosidosis, fucosidosis, mucopolysaccharidosis, Hurler syndrome, Niemann-Pick A and B diseases, and Fabry disease. Pompe disease is also classified as a neuromuscular disease or a metabolic myopathy.

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

[0004] Pompe disease varies greatly depending on the degree of enzyme deficiency, the severity of onset, and age, with over 500 different mutations in the GAA gene identified, many of which cause disease symptoms of varying severity. The disease is classified into a broad range of types: early onset, infancy-onset, and late onset. Earlier onset and lower enzyme activity are generally associated with a more severe clinical course. Childhood Pompe disease is the most severe, resulting from complete or near-complete acid α-glucosidase deficiency and presenting with symptoms including severe muscle tone loss, weakness, hepatomegaly and cardiac hypertrophy, and cardiomyopathy. The tongue may be enlarged and protruding, and swallowing may be difficult. Most affected children die by the age of two from respiratory or cardiac complications. Late-onset Pompe disease can be present at any age after 12 months, is characterized by the absence of cardiac involvement, and has a better short-term prognosis. The symptoms are associated with progressive skeletal muscle dysfunction, accompanied by general weakness in the trunk, proximal lower extremities, and diaphragm, as well as respiratory muscle wasting. In adult patients, there may be no significant symptoms or limitations in movement. The prognosis generally depends on the degree of respiratory muscle involvement. The majority of individuals affected by Pompe disease experience progressive physical weakness requiring wheelchair use and assisted ventilation, often leading to premature death due to respiratory failure.

[0005] Recent treatment options for Pompe disease include enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (rhGAA). Conventional rhGAA products are known as alglucosidase alfa, myozyme®, or lumizyme® from Genzyme, Inc. ERT is a chronic treatment required for the patient's lifetime and involves administering the replacement enzyme by intravenous infusion. The replacement enzyme is then transported in circulation and enters intracellular lysosomes, where it acts to break down stored glycogen, compensating for the deficient activity of the endogenously deficient mutant enzyme and thus alleviating the symptoms of the disease. In infant-onset Pompe disease patients, treatment with alglucosidase alfa has been shown to significantly improve survival rates compared to past controls, and in late-onset Pompe disease, alglucosidase alfa has been shown to have a statistically significant effect, to the best of our knowledge, on the 6-minute walk test (6MWT) and forced vital capacity (FVC) results compared to placebo.

[0006] However, most subjects either remain stable or continue to worsen while receiving treatment with alglucosidase alpha. The reason for the apparent lower limit of efficacy of ERT with alglucosidase alpha is unclear, but it may be partly due to the progressive nature of the underlying muscle lesion or the inadequacy of tissue targeting of current ERT. For example, the injected enzyme is not stable at neutral pH, including plasma pH (approximately pH 7.4), and can be irreversibly inactivated in circulation. Furthermore, injected alglucosidase alpha shows inadequate uptake in key disease-associated muscles, possibly due to insufficient glycosylation by mannose-6-phosphate (M6P) residues. Such residues bind to cation-independent mannose-6-phosphate receptors (CIMPR) on the cell surface, allowing the enzyme to enter intracellular and lysosomes. Therefore, high doses of the enzyme may be required for effective treatment to ensure that sufficient amounts of active enzyme reach the lysosomes, which makes the treatment costly and time-consuming.

[0007] rhGAA has seven potential N-linked glycosylation sites. Because the glycosylation sites are heterogeneous among the types of N-linked oligosaccharides (N-glycans) present, rhGAA consists of a complex mixture of proteins and N-glycans with varying binding affinities to M6P receptors and other carbohydrate receptors. High-mannose rhGAA containing an N-glycan with one M6P group (mono-M6P) binds to CIMPR with low affinity (approximately 6,000 nM), while rhGAA containing two M6P groups on the same N-glycan (bis-M6P) binds with high affinity (approximately 2 nM). Representative structures of non-phosphorylated, mono-M6P, and bis-M6P glycans are shown in Figure 1A. The mannose-6-P group is shown in Figure 1B. Once inside the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAA has low total levels of glycans containing M6P and bis-M6P, resulting in poor delivery of rhGAA to lysosomes in target muscle cells. Productive drug targeting of rhGAA is shown in Figure 2A. The majority of rhGAA molecules in these conventional products lack phosphorylated N-glycans, thereby lacking affinity for CIMPR. Non-phosphorylated high-mannose glycans can also be removed by mannose receptors, resulting in unproductive clearance of ERT (Figure 2B).

[0008] Other types of N-glycans containing galactose and sialic acid, complex carbohydrates, are also present in rhGAA. Since complex N-glycans are not phosphorylated, they do not have affinity for CIMPR. However, complex N-glycans with exposed galactose residues have moderate to high affinity for asialoglycoprotein receptors on hepatocytes, leading to rapid nonproductive clearance of rhGAA (Figure 2B).

[0009] Current manufacturing methods used to produce conventional rhGAA, such as Myozyme®, Lumizyme®, or alglucosidase alpha, have not significantly increased the content of M6P or bis-M6P due to the inherent complexity and extreme difficulty of processing cellular carbohydrates. Therefore, further improvements in enzyme replacement therapy for the treatment of Pompe disease are needed, including new methods for the production, capture, and purification of rhGAA.

[0010] Similarly, other recombinant proteins that target lysosomes, such as other lysosomal enzymes, also bind to CIMPR. However, current manufacturing methods used to produce other conventional recombinant proteins that target lysosomes do not provide recombinant proteins with high M6P or bis-M6P content. Therefore, further improvements are needed in the methods for producing, capturing, and purifying these other recombinant proteins. [Overview of the project] [Means for solving the problem]

[0011] One aspect of the present invention relates to a method for producing recombinant human lysosomal protein. In various embodiments of this aspect, the method includes culturing host cells in a bioreactor that secretes recombinant human lysosomal protein, removing the culture medium from the bioreactor, filtering the culture medium to provide a filtrate, loading the filtrate into an anion exchange chromatography (AEX) column to capture the lysosomal protein, and eluting a first protein product from the AEX column.

[0012] In one or more embodiments, the recombinant human lysosomal protein is recombinant human α-glucosidase (rhGAA). In one or more embodiments, rhGAA contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 2.

[0013] In one or more embodiments, the method further comprises packing a first protein product into a chromatography column and eluting a second protein product from the column. In some embodiments, the column is an immobilized metal affinity chromatography (IMAC) column.

[0014] In one or more embodiments, the method further comprises packing a second protein product into a chromatography column and eluting a third protein product from the column. In some embodiments, the third chromatography column is selected from cation exchange chromatography (CEX) columns and size exclusion chromatography (SEC) columns.

[0015] In one or more embodiments, the filtration of the culture medium is selected from alternating tangential flow filtration (ATF) and tangential flow filtration (TFF).

[0016] In one or more embodiments, the method further comprises inactivating a virus in one or more of the first protein product, the second protein product, and the third protein product.

[0017] In one or more embodiments, the method further comprises filtering a second or third protein product to provide a filtered product, and filling a vial with the filtered product. In one or more embodiments, the method further comprises freeze-drying the filtered product.

[0018] In one or more embodiments, the host cells include Chinese hamster ovary (CHO) cells. In some embodiments, the host cells include the CHO cell line GA-ATB-200 or ATB-200-X5-14 or its subculture.

[0019] In one or more embodiments, (i) at least 90% of the first protein product, the second protein product, or the third protein product is bound to a cation-independent mannose-6-phosphate receptor (CIMPR), or (ii) at least 90% of the first protein product, the second protein product, or the third protein product contains an N-glycan having mono-mannose-6-phosphate (mono-M6P) or bis-mannose-6-phosphate (bis-M6P).

[0020] In one or more embodiments, the recombinant human lysosomal protein is an rhGAA comprising seven potential N-glucosylation sites, wherein at least 50% of the rhGAA molecule contains an N-glycan unit having two mannose-6-phosphate residues at the first site, at least 30% of the rhGAA molecule contains an N-glycan unit having one mannose-6-phosphate residue at the second site, at least 30% of the rhGAA molecule contains an N-glycan unit having two mannose-6-phosphate residues at the fourth site, and at least 20% of the rhGAA molecule contains an N-glycan unit having one mannose-6-phosphate residue at the fourth site.

[0021] Another aspect of the present invention relates to recombinant protein products prepared by any of the methods described herein.

[0022] Another aspect of the present invention relates to a pharmaceutical composition comprising a recombinant protein product and a pharmaceutically acceptable carrier.

[0023] A further aspect of the present invention relates to a method for treating lysosomal storage disorders, comprising administering a pharmaceutical composition to a patient in need thereof.

[0024] In one or more embodiments, the lysosomal storage disorder is Pompe disease and the recombinant protein is rhGAA. In one or more embodiments, within 4 hours of administration of a pharmaceutical composition comprising the rhGAA product, a pharmacological chaperone of α-glucosidase is co-administered to the patient. In some embodiments, the pharmacological chaperone is selected from 1-deoxynojirimycin and N-butyl-deoxynojirimycin. In some embodiments, the pharmacological chaperone is co-formulated with the rhGAA product.

[0025] Various embodiments are listed below. It will be understood that the embodiments listed below can be combined not only with those listed below, but also in other suitable combinations according to the scope of the present invention.

[0026] Further features of the present invention will become apparent from the following specification description and the accompanying drawings.

Brief Description of the Drawings

[0027] [Figure 1A] Shows non-phosphorylated high-mannose glycans, mono-M6P glycans, and bis-M6P glycans. [Figure 1B] Shows the chemical structure of the M6P group. [Figure 2A] Describes the productive targeting of rhGAA via glycans having M6P to a target tissue (e.g., muscle tissue of a subject having Pompe disease). [Figure 2B] Explained by non-productive drug clearance to non-target tissues (e.g., liver and spleen) or binding of non-M6P glycans to non-target tissues. [Figure 3A] Graphically shows the CIMPR receptor (also known as the IGF2 receptor) and the domains of this receptor. [Figure 3B]This table shows the binding affinity (nanomolecular) of bis- and mono-M6P-containing glycans to CIMPR, the binding affinity of high-mannose glycans to mannose receptors, and the binding affinity of desialylated complex glycans to asialoglycoprotein receptors. RhGAA containing M6P and bis-M6P-containing glycans can productively bind to CIMPR on muscle. [Figure 4A] These graphs show the results of CIMPR affinity chromatography for Lumizyme® and Myozyme®, respectively. The dashed line indicates the M6P elution gradient. Elution with M6P replaced GAA molecules bound via M6P-containing glycans with CIMPR. As shown in Figure 4A, 78% of the GAA activity in Lumizyme® eluted before the addition of M6P. Figure 4B shows that 73% of the GAA Myozyme® activity eluted before the addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme® eluted with M6P. These figures indicate that the majority of rhGAA in these two conventional rhGAA products lacks the M6P-containing glycans necessary to target CIMPR in target muscle tissue. [Figure 4B] These graphs show the results of CIMPR affinity chromatography for Lumizyme® and Myozyme®, respectively. The dashed line indicates the M6P elution gradient. Elution with M6P replaced GAA molecules bound via M6P-containing glycans with CIMPR. As shown in Figure 4A, 78% of the GAA activity in Lumizyme® eluted before the addition of M6P. Figure 4B shows that 73% of the GAA Myozyme® activity eluted before the addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme® eluted with M6P. These figures indicate that the majority of rhGAA in these two conventional rhGAA products lacks the M6P-containing glycans necessary to target CIMPR in target muscle tissue. [Figure 5]This shows a DNA construct for transforming CHO cells with rhGAA-encoding DNA. CHO cells were transformed with the rhGAA-encoding DNA construct. [Figure 6] This is a schematic diagram illustrating exemplary methods for the production, capture, and purification of recombinant lysosomal proteins. [Figure 7A] These graphs show the results of CIMPR affinity chromatography for myozyme® and ATB200 rhGAA, respectively. As is clear from Figure 7B, approximately 70% of the rhGAA in ATB200 rhGAA contained M6P. [Figure 7B] These graphs show the results of CIMPR affinity chromatography for myozyme® and ATB200 rhGAA, respectively. As is clear from Figure 7B, approximately 70% of the rhGAA in ATB200 rhGAA contained M6P. [Figure 8] This graph shows the results of CIMPR affinity chromatography of ATB200 rhGAA with and without capture by anion exchange (AEX) column. [Figure 9] This graph shows the polywax elution profiles of Lumizyme® and ATB200 rhGAA. [Figure 10] This table outlines the N-glycan structure of Lumizyme® compared to three different preparations of ATB200 rhGAA, identified as BP-rhGAA, ATB200-1, and ATB200-2. [Figure 11A] The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 11B] The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 11C] The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 11D] The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 11E]The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 11F] The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 11G] The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 11H] The results of site-specific N-glycosylation analysis of ATB200 rhGAA are shown. [Figure 12A] Figure 12A is a graph comparing the CIMPR binding affinity of ATB200 rhGAA (left trace) and Lumizyme (right trace). [Figure 12B] Figure 12B is a table comparing the bis-M6P content of Lumizyme® and ATB200 rhGAA. [Figure 13A] Figure 13A is a graph comparing ATB200 rhGAA activity (left trace) and Lumizyme® rhGAA activity (right trace) in normal fibroblasts at various GAA concentrations. [Figure 13B] Figure 13B is a table comparing ATB200 rhGAA activity (left trace) and Lumizyme® rhGAA activity (right trace) in fibroblasts derived from subjects with Pompe disease at various GAA concentrations. [Figure 13C] Figure 13C is a table comparing potassium uptake in fibroblasts from healthy subjects and subjects with Pompe disease. [Figure 14A] Figure 14A is a graph showing the amount of glycogen in mouse myocardium in relation to the dose of recombinant human acid α-glucosidase after contact with a solvent (negative control), 20 mg / ml alglucosidase alpha (Lumizyme®), or 5, 10, or 20 mg / kg of ATB200. [Figure 14B]Figure 14B is a graph showing the amount of glycogen in mouse quadriceps muscle in relation to the dose of recombinant human acid α-glucosidase after contact with a solvent (negative control), 20 mg / ml alglucosidase alpha (Lumizyme®), or 5, 10, or 20 mg / kg of ATB200. [Figure 14C] Figure 14C is a graph showing the amount of glycogen in mouse triceps muscle in relation to the dose of recombinant human acid α-glucosidase after contact with a solvent (negative control), 20 mg / ml alglucosidase alpha (Lumizyme®), or 5, 10, or 20 mg / kg of ATB200. [Figure 15] This table shows that the combination of ATB200 rhGAA and the chaperone miglustat provided significantly better glycogen clearance in GAA knockout mice than treatment with Lumizyme® or ATB200 rhGAA without the miglustat chaperone. [Figure 16] A series of electron micrographs of the heart, diaphragm, and soleus muscle from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200 in the presence and absence of miglustat, showing the levels of lysosome-associated membrane protein (LAMP-1). [Figure 17] These are a series of electron micrographs of the heart and soleus muscle from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200 in the presence and absence of miglustat, showing glycogen levels by staining with Schiff periodate reagent (PAS). [Figure 18] A series of electron micrographs (1000x) of quadriceps muscles from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200 in the presence and absence of miglustat, stained with methylene blue to show vacuoles (indicated by arrows). [Figure 19]A series of electron micrographs (40x) of quadriceps muscles from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200 in the presence and absence of miglustat, showing the levels of the autophagy markers microtubule-associated protein 1A / 1B light chain 3-phosphatidylethanolamine conjugate (LC3A II) and p62, insulin-dependent glucose transporter GLUT4, and insulin-independent glucose transporter GLUT1. [Figure 20] This graph shows the results of CIMPR affinity chromatography of recombinant α-galactosidase A (rhα-Gal A) enzyme before and after capture and purification using an anion exchange (AEX) column. [Figure 21A] This graph shows wire-hand and grip strength muscle data from wild-type and Gaa knockout mice treated with a solvent, alglucosidase alpha, and ATB200 in the presence of miglustat. [Figure 21B] This graph shows wire-hand and grip strength muscle data from wild-type and Gaa knockout mice treated with a solvent, alglucosidase alpha, and ATB200 in the presence of miglustat. [Figure 22A] This graph shows glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200, in and without miglustat. [Figure 22B] This graph shows glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200, in and without miglustat. [Figure 22C] This graph shows glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200, in and without miglustat. [Figure 22D]This graph shows glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200, in and without miglustat. [Figure 22E] This graph shows glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200, in and without miglustat. [Figure 22F] This graph shows glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200, in and without miglustat. [Figure 22G] This graph shows glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200, in and without miglustat. [Figure 23] A series of micrographs (100x and 200x) of vastus lateralis (VL) muscle fibers from wild-type and Gaa knockout mice treated with solvent, alglucosidase alpha, and ATB200 in the presence and absence of miglustat, showing dystrophin signaling. [Figure 24] This paper describes the experimental design of an open-label, constant-sequence, dose-escalation, human first-stage, Phase 1 / 2 study to evaluate the safety, tolerability, pharmacokinetics, disease progression, and efficacy of intravenous infusion of ATB200 co-administered with oral miglustat in adults with Pompe disease. [Figure 25A] This graph shows the concentration-time profile of total GAA protein in plasma in human subjects after administration of 5, 10, or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 25B]This graph shows the concentration-time profile of total GAA protein in plasma in human subjects after administration of 5, 10, or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 25C] This graph shows the AUC of total GAA protein in plasma in human subjects after administration of 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 25D] This graph shows the plasma total GAA protein concentration-time profiles in two separate human subjects after administration of 20 mg / kg ATB200 and 260 mg miglustat. [Figure 26] This graph shows the concentration-time profile of miglustat in plasma in human subjects after administration of 130 mg or 260 mg of miglustat. [Figure 27A] This graph shows the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), and hexose tetrasaccharide (Hex4) levels in human patients after administration of escalating doses of ATB200 (5, 10, and 20 mg / kg), followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 27B] This graph shows the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), and hexose tetrasaccharide (Hex4) levels in human patients after administration of escalating doses of ATB200 (5, 10, and 20 mg / kg), followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 27C]This graph shows the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), and hexose tetrasaccharide (Hex4) levels in human patients after administration of escalating doses of ATB200 (5, 10, and 20 mg / kg), followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 27D] This graph shows the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), and hexose tetrasaccharide (Hex4) levels in human patients after administration of escalating doses of ATB200 (5, 10, and 20 mg / kg), followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Modes for carrying out the invention]

[0028] Before describing some exemplary embodiments of the present invention, it should be understood that the present invention is not limited to the details of the configuration or process steps described below. Other embodiments of the present invention are possible and can be carried out or performed in a variety of ways.

[0029] While GAA is specifically mentioned, it will be understood by those skilled in the art that the methods described herein can be used to generate, capture, and purify other recombinant proteins that target lysosomes, including but not limited to the lysosomal enzyme α-galactosidase A.

[0030] Various aspects of the present invention relate to novel methods for generating, capturing, and purifying recombinant human lysosomal proteins, such as recombinant human acid α-glucosidase (rhGAA). Other aspects of the present invention relate to recombinant proteins produced by the methods described herein, as well as pharmaceutical compositions, treatment methods, and uses of such recombinant proteins.

[0031] The terms used herein generally have the common meaning in the art within the context of the present invention and in the specific context in which each term is used. Certain terms are described below or elsewhere in this specification to provide additional expert guidance in describing the compositions and methods of the present invention, as well as the methods of their manufacture and use.

[0032] In this specification, unless the context requires otherwise, the word “comprises,” or variations such as “comprises” or “comprising,” is used to indicate a comprehensive meaning, i.e., the presence of the described features, and does not preclude the presence or addition of further features in various embodiments of the invention.

[0033] As used herein, the term “lysosomal protein” refers to any protein that targets lysosomes, such as lysosomal enzymes. Examples of lysosomal enzymes and related diseases are shown in Table 1 below:

[0034] JPEG2026136113000002.jpg161154

[0035] As used herein, the term “Pompe disease,” also known as acid maltase deficiency, type II glycosomal storage disorder (GSDII), and type II glycosomal storage disorder, is intended to refer to a genetic lysosomal storage disorder characterized by mutations in the GAA gene, which encodes the human acid alpha-glucosidase enzyme. This term includes, but is not limited to, early and late-onset forms of Pompe disease, including, but not limited to, infancy, adolescence, and adulthood.

[0036] As used herein, the term “acid α-glucosidase” is intended to refer to a lysosomal enzyme that hydrolyzes the α-1,4 linkage between D-glucose units in glycogen, maltose, and isomaltose. Other names include, but are not limited to, lysosomal α-glucosidase (EC:3.2.1.20); glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase and exo-1,4-α-glucosidase. Human acid α-glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information (NCBI) Gene ID 2548), which is mapped to the long arm of chromosome 17 (positions 17q25.2-q25.3). More than 500 mutations have been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations that result in misfolding or misprocessing of the acid α-glucosidase enzyme include T1064C(Leu355Pro) and C2104T(Arg702Cys). Furthermore, GAA mutations that affect enzyme maturation and processing include Leu405Pro and Met519Thr. The activity of the acid α-glucosidase protein requires the conserved hexapeptide WIDMNE at amino acid residues 516-521. As used herein, the abbreviation "GAA" is intended to refer to the acid α-glucosidase enzyme, and the italicized abbreviation "GAA" is intended to refer to the human gene encoding the human acid α-glucosidase enzyme. The italicized abbreviation "Gaa" is intended to refer to a non-human gene encoding a non-human acid α-glucosidase enzyme, including but not limited to rat or mouse genes, and the abbreviation "Gaa" is intended to refer to the non-human acid α-glucosidase enzyme. Therefore, the abbreviation "rhGAA" is intended to refer to recombinant human acid α-glucosidase enzyme.

[0037] As used herein, the term “alglucosidase alpha” is intended to refer to recombinant human acid α-glucosidase identified as [199-arginine,223-histidine]prepro-α-glucosidase (human); Chemical Abstracts registration number 420794-05-0. Alglucosidase alpha is approved for commercial sale in the United States by Genzyme as Lumizyme® and Myozyme® as of January 2016.

[0038] As used herein, the term "ATB200" is intended to refer to the recombinant human acid α-glucosidase described in the concurrently pending patent application PCT / U.S. Patent Application Publication No. 2015 / 053252, which is incorporated herein by reference.

[0039] As used herein, the term “glycan” is intended to refer to a polysaccharide chain covalently bonded to an amino acid residue on a protein or polypeptide. As used herein, the term “N-glycan” or “N-linked glycan” is intended to refer to a polysaccharide chain covalently bonded to an amino acid residue on a protein or polypeptide by a nitrogen bond to the nitrogen atom of the amino acid residue. For example, an N-glycan may be covalently bonded to the side-chain nitrogen atom of an asparagine residue. A glycan may contain one or more monosaccharide units, which may be covalently bonded to form a linear or branched chain. In at least one embodiment, the N-glycan unit bonded to ATB200 may contain one or more monosaccharide units independently selected from N-acetylglucosamine, mannose, galactose, or sialic acid. The N-glycan unit on a protein may be determined by any suitable analytical technique, such as mass spectrometry. In some embodiments, N-glycan units can be determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using instruments such as a Thermo Scientific Orbitrap Velos Pro® mass spectrometer, a Thermo Scientific Orbitrap Fusion Lumos Tribid® mass spectrometer, or a WatersXevo® G2-XS QTof mass spectrometer.

[0040] As used herein, the term “high mannose N-glycan” is intended to refer to an N-glycan having 1 to 6 or more mannose units. In at least one embodiment, the high mannose N-glycan unit contains a bis(N-acetylglucosamine) chain bonded to an asparagine residue and may further bond to a branched polymannose chain. As used interchangeably herein, the term “M6P” or “mannose-6-phosphate” is intended to refer to a mannose unit having a phosphorylated 6-position, i.e., a phosphate group bonded to a hydroxyl group at the 6-position. In at least one embodiment, one or more mannose units of one or more N-glycan units are phosphorylated at the 6-position to form a mannose-6-phosphate unit. In at least one embodiment, the term “M6P” or “mannose-6-phosphate” refers to both mannose phosphodiesters having N-acetylglucosamine (GlcNAc) as a “cap” on the phosphate group, and mannose units having an exposed phosphate group lacking the GlcNAc cap. In at least one embodiment, the protein N-glycan may have a plurality of M6P groups, each having at least one M6P group with a GlcNAc cap and at least one other M6P group lacking a GlcNAc cap.

[0041] As used herein, the term “complex N-glycan” is intended to mean an N-glycan comprising one or more galactose and / or sialic acid units. In at least one embodiment, the complex N-glycan may be a high-mannose N-glycan, in which one or more monosaccharide units, independently selected from N-acetylglucosamine, galactose, and sialic acid, are further bonded with one or more mannose units.

[0042] When used herein, the compound miglustat, also known as N-butyl-1-deoxynojirimycin NB-DNJ or (2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: JPEG2026136113000003.jpg30154

[0043] One formulation of miglustat is marketed under the trade name Zavesca® as a monotherapy for type 1 Gaucher disease.

[0044] As will be described later, pharmaceutically acceptable salts of miglustat may also be used in this invention. When using a salt of miglustat, the dose of the salt is adjusted so that the dose of miglustat received by the patient is equal to the dose that the patient would receive if the free base of miglustat were used.

[0045] When used herein, the compound duvoglustat, also known as 1-deoxynojirimycin, DNJ, or (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: JPEG2026136113000004.jpg30154

[0046] When using a salt of duvoglustat, the salt dose is adjusted so that the dose of duvoglustat the patient receives is equal to the dose they would receive if the free base of duvoglustat were used.

[0047] As used herein, the term “pharmacological chaperone” or, in some cases, simply “chaperone” is intended to refer to a substance that specifically binds to a lysosomal protein and has one or more of the following effects: • Promotes the formation of stable molecular three-dimensional structures of proteins; • To prevent endoplasmic reticulum-associated degradation of proteins, promote the proper transport of proteins from the endoplasmic reticulum to another cellular location, preferably a native cellular location; • Prevents aggregation of structurally unstable or misfolded proteins; • To restore and / or improve the wild-type function, stability and / or activity of at least some of the proteins; and / or • Improves the phenotype or function of cells containing proteins.

[0048] Therefore, a pharmacological chaperone of acid α-glucosidase is a molecule that binds to acid α-glucosidase, resulting in proper folding, transport, non-aggregation, and activity of acid α-glucosidase. As used herein, this term includes, but is not limited to, active-site-specific chaperones (ASSCs), inhibitors or antagonists, and agonists that bind to the active site of an enzyme. In at least one embodiment, the pharmacological chaperone may be an inhibitor or antagonist of acid α-glucosidase. As used herein, the term “antagonist” is intended to refer to any molecule that binds to acid α-glucosidase and partially or completely block, inhibit, reduce, or neutralize the activity of acid α-glucosidase. In at least one embodiment, the pharmacological chaperone is miglustat. Another non-limiting pharmacological chaperone of acid α-glucosidase is duvoglustat.

[0049] As used herein, the term “active site” is intended to refer to a region of a protein that is relevant to and necessary for the protein’s specific biological activity. In at least one embodiment, the active site may be a site that contributes to an amino acid residue that binds to a substrate or other binding partner and is directly involved in the formation and cleavage of chemical bonds.

[0050] As used herein, “therapeutic effective dose” and “effective amount” are intended to refer to the amount of recombinant human lysosomal protein (e.g., rhGAA) and / or chaperone and / or combination thereof that produces a therapeutic response in a subject. The therapeutic response may be any response that a user (e.g., a clinician) recognizes as an effective response to treatment, including any surrogate clinical markers or symptoms described herein and known in the art. Thus, in at least one embodiment, the therapeutic response may be improvement or suppression of one or more symptoms or markers of Pompe disease, such as those known in the art. Symptoms or markers of Pompe disease include, but are not limited to, cardiomyopathy, cardiac hypertrophy, progressive muscle weakness, particularly in the trunk or lower extremities, severe hypotension, macroglossia (and sometimes tongue protrusion), difficulty swallowing, sucking, and / or feeding, respiratory failure, moderate hepatomegaly, facial muscle relaxation, loss of reflexes, exercise intolerance, exertional dyspnea, orthopnea, sleep apnea, morning headache, somnolence, lordosis and / or scoliosis, decreased deep tendon reflexes, low back pain, and failure to achieve developmental motor milestones. It should be noted that the concentration of a chaperone (e.g., miglustat) having an inhibitory effect on acid α-glucosidase may constitute an "effective amount" for the purposes of this invention due to dilution (and consequently changes in binding due to changes in equilibrium), bioavailability, and the metabolism of the chaperone during in vivo administration.

[0051] As used herein, the terms “enzyme replacement therapy” or “ERT” are intended to refer to the introduction of a non-naturally occurring purified enzyme into an individual having a deficiency of such enzyme. The administered protein may be obtained from a natural source or by recombinant expression. The term also refers to the introduction of a purified enzyme into an individual that otherwise requires or would benefit from the administration of a purified enzyme. In at least one embodiment, such an individual suffers from enzyme deficiency. The introduced enzyme may be a purified recombinant enzyme produced in vitro, or a protein purified from an isolated tissue or fluid such as placenta or animal milk, or from a plant.

[0052] As used herein, the term “combination therapy” is intended to mean any therapy in which two or more individual therapies are administered simultaneously or sequentially. In at least one embodiment, the outcome of combination therapy is enhanced compared to the effect of each therapy as administered individually. Enhancement can include any improvement in the effects of the various therapies that results in a more favorable outcome compared to the outcome achieved by the therapy administered individually. Enhanced effects or outcomes can include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy administered individually; additive enhancement, where the enhanced effect is substantially equal to the additive effect of each therapy administered individually; or less than synergistic enhancement, where the enhanced effect is lower than the additive effect of each therapy administered individually, but better than the effect of each therapy administered individually. Enhanced effects can be measured by any means known in the art that can measure the effect or outcome of a treatment.

[0053] As used herein, the term “pharmaceutically acceptable” is intended to mean molecular entities and compositions that are physiologically acceptable and do not typically produce adverse reactions when administered to humans. Preferably, as used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state government regulatory authority or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, particularly humans.

[0054] As used herein, the term “carrier” is intended to refer to a compound and the diluent, adjuvant, excipient, or solvent administered to it. Suitable pharmaceutically acceptable carriers are known in the art and are described in at least one embodiment in “Remington's Pharmaceutical Sciences” 18th edition or other editions by E.W. Martin.

[0055] As used herein, the terms “subject” or “patient” are intended to refer to a human or a non-human animal. In at least one embodiment, the subject is a mammal. In at least one embodiment, the subject is a human.

[0056] As used herein, the term “anti-drug antibody” is intended to mean an antibody that specifically binds to a drug administered to a subject, and to the drug produced by the subject as at least part of a humoral immune response to the administration of the drug to the subject. In at least one embodiment, the drug is a therapeutic protein drug product. The presence of anti-drug antibodies in a subject may cause an immune response ranging from mild to severe, including, but not limited to, life-threatening immune responses such as anaphylaxis, cytokine release syndrome, and cross-reactivity neutralization of endogenous proteins that mediate important functions. Furthermore, or or otherwise, the presence of anti-drug antibodies in a subject may reduce the efficacy of the drug.

[0057] As used herein, the term “neutralizing antibody” is intended to refer to an anti-drug antibody that acts to neutralize the function of a drug. In at least one embodiment, a therapeutic protein-drug product is a counterpart to an endogenous protein whose expression is reduced or absent in the subject. In at least one embodiment, a neutralizing antibody may act to neutralize the function of the endogenous protein.

[0058] As used herein, the terms “about” and “approximately” are intended to refer to an acceptable degree of error in a measured quantity, taking into account the nature or precision of the measurement. For example, the degree of error may be indicated by the number of significant figures provided with respect to the measurement, as understood in the art, including, but not limited to, a change of ±1 from the most accurate significant figures reported with respect to the measurement. A typical exemplary degree of error is within 20 percent (%) of a given value or range of values, preferably within 10 percent, and more preferably within 5 percent. Alternatively, particularly in biological systems, the terms “about” and “approximately” may mean a value within one order of magnitude of a given value, preferably within five times, and more preferably within two times. The numerical values ​​described herein are approximate unless otherwise specified, and the terms “about” or “approximately” mean that they can be inferred unless explicitly stated.

[0059] Where used herein, the term “simultaneously” is intended to mean, as understood by those skilled in the art, at the same time or within a reasonable short time before or after. For example, when two treatments are administered simultaneously, one treatment may be administered before or after the other to allow time for preparation of the latter of the two treatments. Thus, “simultaneous administration” of two treatments includes, but is not limited to, one treatment following the other within 20 minutes or less, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, or less than 1 minute.

[0060] The term “pharmaceutically acceptable salt,” as used herein, is intended to mean a salt that, within the bounds of sound medical judgment, is suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions, is generally water-soluble or oil-soluble or dispersible in proportion to a reasonable benefit / risk ratio, and is effective for its intended use. This term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. A list of suitable salts can be found, for example, in SMBirge et al., J.Pharm.Sci., 1977, 66, pp. 1-19, which is incorporated herein by reference.

[0061] The term “pharmaceutically acceptable acid addition salt” as used herein means an inorganic acid, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphate, hemisulfic acid, and hemisulfic acid. This term is intended to refer to salts formed from organic acids, including but not limited to xanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, and undecanoic acid.

[0062] The term “pharmaceutically acceptable base addition salt” as used herein is intended to mean a salt formed with ammonia or an inorganic base, including but not limited to ammonium or metal cation hydroxides, carbonates, or bicarbonates such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum, that retains the biological efficacy and properties of the free acid and is not biologically or otherwise undesirable. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, quaternary amine compounds, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, and hydraba. This includes, but is not limited to, salts of mine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, polyamine resins, etc.

[0063] ATB200 rhGAA In at least one embodiment, recombinant human lysosomal protein (e.g., rhGAA) is expressed in Chinese hamster ovary (CHO) cells and contains an increased content of one or more N-glycan units having one or more mannose-6-phosphate residues compared to the content of conventional recombinant human lysosomal protein such as alglucosidase alpha. In at least one embodiment, the acid α-glucosidase is a recombinant human acid α-glucosidase referred to herein as ATB200, as described in concurrently pending international patent application PCT / US Patent Application Publication 2015 / 053252. ATB200 has high affinity (K D It binds to the cation-independent mannose-6-phosphate receptor (CIMPR) at ~2~4nM and to Pompe fibroblasts and skeletal myoblasts (K 取り込み It has been shown to be efficiently internalized by alglucosidase alpha(t)(~7~14nM). ATB200 was characterized in vivo and is linked to alglucosidase alpha(t) 1 / 2 (t) Apparent plasma half-life (t) shorter than ~60 minutes 1 / 2 It was shown to have a duration of approximately 45 minutes.

[0064] In at least one embodiment, recombinant human acid α-glucosidase is an enzyme having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. JPEG2026136113000005.jpg41154JPEG2026136113000006.jpg210154JPEG20261361130 00007.jpg209154JPEG2026136113000008.jpg209154JPEG2026136113000009.jpg93154

[0065] In at least one embodiment, recombinant human acid α-glucosidase has the wild-type GAA amino acid sequence described in Sequence ID No. 1, as described in U.S. Patent No. 8,592,362, and has GenBank accession number AHE24104.1 (GI:568760974). In at least one embodiment, recombinant human acid α-glucosidase is glucosidase alpha, which is a human acid α-glucosidase enzyme encoded by the most dominant of the nine observed haplotypes of the GAA gene.

[0066] In at least one embodiment, recombinant human acid α-glucosidase is initially expressed as having the full-length 952-amino acid sequence of wild-type GAA described in SEQ ID NO: 1, and the recombinant human acid α-glucosidase then undergoes intracellular processing to remove some of the amino acids, e.g., the first 56 amino acids. Thus, recombinant human acid α-glucosidase secreted by a host cell may have a shorter amino acid sequence than the recombinant human acid α-glucosidase initially expressed intracellularly. In at least one embodiment, the shorter protein may have the amino acid sequence described in SEQ ID NO: 2, differing from SEQ ID NO: 1 only in that the first 56 amino acids, including the signal peptide and precursor peptide, are removed, resulting in a protein with 896 amino acids. Other modifications in the number of amino acids are also possible to the amino acid sequences described in SEQ ID NO: 1 or SEQ ID NO: 2, such as having deletions, substitutions, and / or insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. In some embodiments, the rhGAA product comprises a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0067] In at least one embodiment, recombinant human acid α-glucosidase undergoes posttranslational and / or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, N-terminal glutamine may form pyroglutamic acid. As yet another example, asparagine residues may undergo deamination to aspartic acid. As yet another example, aspartic acid residues may undergo isomerization to isoaspartic acid. As yet another example, unpaired cysteine ​​residues in the protein may form disulfide bonds with free glutathione and / or cysteine. Thus, in some embodiments, the enzyme is initially expressed having the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, and the enzyme undergoes one or more of these posttranslational and / or chemical modifications. Such modified forms are also within the scope of this disclosure.

[0068] Polynucleotide sequences encoding GAA and such mutant human GAA are also intended and may be used for recombinant expression of rhGAA according to the present invention.

[0069] Preferably, 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10, or less than 5% of the total recombinant human lysosomal protein (e.g., rhGAA) molecules lack an N-glycan unit having one or more mannose-6-phosphate residues, or lack the ability to bind to the cation-independent mannose-6-phosphate receptor (CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, or <100% or more of the recombinant human lysosomal protein (e.g., rhGAA) molecules contain at least one N-glycan unit having one or more mannose-6-phosphate residues, or have the ability to bind to CIMPR.

[0070] A recombinant human lysosomal protein (e.g., rhGAA) molecule may have one, two, three, or four mannose-6-phosphate (M6P) groups on its glycan. For example, only one N-glycan in a recombinant human lysosomal protein molecule may have M6P (mono-phosphorylated), a single N-glycan may have two M6P groups (bis-phosphorylated), or two different N-glycans in the same recombinant human lysosomal protein molecule may each have a single M6P group. A recombinant human lysosomal protein molecule may also have N-glycans that do not have M6P groups. In another embodiment, the N-glycan contains, on average, more than 3 moles / mol of M6P and more than 4 moles / mol of sialic acid, resulting in the human lysosomal protein containing, on average, at least 3 moles of mannose-6-phosphate residues and at least 4 moles of sialic acid per mole of recombinant human lysosomal protein. On average, at least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycans in recombinant human lysosomal proteins may be in the form of mono-M6P glycans, for example, about 6.25% of the total glycans may have a single M6P group, on average at least about 0.5, 1, 1.5, 2.0, 2.5, or 3.0% of the total glycans in recombinant human lysosomal proteins may be in the form of bis-M6P glycans, and on average less than 25% of the total recombinant human lysosomal proteins do not contain phosphorylated glycans bound to CIMPR.

[0071] Recombinant human lysosomal proteins (e.g., rhGAA) may have an average M6P-carrying N-glycan content in the range of 0.5–7.0 mol / mol recombinant human lysosomal protein, or any intermediate value within a subrange containing 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 lysosomal protein. Recombinant human lysosomal proteins can be fractionated to provide recombinant human lysosomal protein preparations having different average numbers of M6P-containing or bis-M6P-containing glycans, thereby allowing for further customization of recombinant human lysosomal proteins for targeting lysosomes in target tissues by selecting specific fractions or by selectively combining different fractions.

[0072] In some embodiments, recombinant human lysosomal protein (e.g., rhGAA) has an average of 2.0 to 8.0 moles of M6P per mole of recombinant human lysosomal protein (e.g., rhGAA). This range includes all intermediate and partial ranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 moles of M6P / mol recombinant human lysosomal protein (e.g., rhGAA).

[0073] Up to 60% of the N-glycans in recombinant human lysosomal protein (e.g., rhGAA) may be completely sialylated, for example, up to 10%, 20%, 30%, 40%, 50%, or 60% of the N-glycans may be completely sialylated. In some embodiments, 4–20% of the total N-glycans are completely sialylated. In other embodiments, 5%, 10%, 20%, or 30% or less of the N-glycans in recombinant human lysosomal protein (e.g., rhGAA) have sialic acid and terminal galactose residues (Gal). This range includes all intermediate and partial ranges, for example, 7–30% of the total N-glycans in recombinant human lysosomal protein can carry sialic acid and terminal galactose. In yet another embodiment, 5%, 10%, 15%, 16%, 17%, 18%, 19%, or 20% or less of the N-glycans in recombinant human lysosomal protein have terminal galactose only and do not contain sialic acid. This range includes all intermediate and partial values, and for example, 8-19% of the total N-glycans in recombinant human lysosomal proteins in a composition may contain only terminal galactose and no sialic acid.

[0074] In other embodiments of the present invention, 40, 45, 50, 55-60% of the total N-glycans in recombinant human lysosomal protein (e.g., rhGAA) are complex N-glycans; or 1, 2, 3, 4, 5, 6, 7% or less of the total N-glycans in recombinant human lysosomal protein (e.g., rhGAA) are hybrid N-glycans; 5, 10, or 15% or less of the high-mannose N-glycans in recombinant human lysosomal protein (e.g., rhGAA) are unphosphorylated; at least 5% or 10% of the high-mannose N-glycans in recombinant human lysosomal protein (e.g., rhGAA) are phosphorylated mono-M6P; and / or at least 1 or 2% of the high-mannose N-glycans in recombinant human lysosomal protein (e.g., rhGAA) are phosphorylated bis-M6P. These values ​​include all intermediate and partial ranges. Recombinant human lysosomal proteins (e.g., rhGAA) may satisfy one or more of the above content ranges.

[0075] In some embodiments, recombinant human lysosomal protein (e.g., rhGAA) has an average of 2.0 to 8.0 moles of sialic acid residues per mole of recombinant human lysosomal protein (e.g., rhGAA). This range includes all intermediate and partial ranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 mole residues / mol of recombinant human lysosomal protein (e.g., rhGAA). While not bound by theory, the presence of N-glycan units containing sialic acid residues is thought to prevent unproductive clearance of recombinant human lysosomal protein (e.g., rhGAA) by the asialoglycoprotein receptor.

[0076] In one or more embodiments, recombinant human lysosomal protein (e.g., rhGAA) has M6P and / or sialic acid units at certain N-glycosylation sites of the recombinant human lysosomal protein. For example, as described above, rhGAA has seven potential N-linked glycosylation sites. These potential glycosylation sites are located at the following positions in SEQ ID NO: 2: N84, N177, N334, N414, N596, N826, and N869. Similarly, with respect to the full-length amino acid sequence of SEQ ID NO: 1, these potential glycosylation sites are located at the following positions: N140, N233, N390, N470, N652, N882, and N925. Other variants of rhGAA may have similar glycosylation sites depending on the position of the asparagine residue. Generally, the ASN-X-SER or ASN-X-THR sequences in protein amino acid sequences indicate potential glycosylation sites, with the exception that X cannot be HIS or PRO.

[0077] In various embodiments, rhGAA has a specific N-glycosylation profile. In one or more embodiments, at least 20% of rhGAA is phosphorylated at a first N-glycosylation site (e.g., 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 mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have mono-M6P units at the first N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have bis-M6P units at the first N-glycosylation site.

[0078] In one or more embodiments, at least 20% of rhGAA is phosphorylated at a second N-glycosylation site (e.g., 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 a second N-glycosylation site. This phosphorylation may result in mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have mono-M6P units at the second N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have bis-M6P units at the second N-glycosylation site. In one or more embodiments, at least 5% of rhGAA is phosphorylated at a third N-glycosylation site (e.g., 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 is phosphorylated at a third N-glycosylation site. For example, the third N-glycosylation site may be a mixture of unphosphorylated high-mannose glycans, di-, tri-, and tetra-antennari complex glycans, and hybrid glycans as the dominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of rhGAA is sialylated at a third N-glycosylation site.

[0079] In one or more embodiments, at least 20% of rhGAA is phosphorylated at a fourth N-glycosylation site (e.g., 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 a fourth N-glycosylation site. This phosphorylation may result in mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have mono-M6P units at the fourth N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have bis-M6P units at the fourth N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of rhGAA is sialylated at a fourth N-glycosylation site.

[0080] In one or more embodiments, at least 5% of rhGAA is phosphorylated at a fifth N-glycosylation site (e.g., 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 is phosphorylated at a fifth N-glycosylation site. For example, the fifth N-glycosylation site may have fucosylated dianthenthri complex glycans as the primary species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA is sialylated at a fifth N-glycosylation site.

[0081] In one or more embodiments, at least 5% of rhGAA is phosphorylated at a sixth N-glycosylation site (e.g., 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 is phosphorylated at a sixth N-glycosylation site. For example, the sixth N-glycosylation site may have a mixture of di-, tri-, and tetra-antennatriglycans as the main species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA is sialylated at a sixth N-glycosylation site.

[0082] In one or more embodiments, at least 5% of rhGAA is phosphorylated at the seventh N-glycosylation site (e.g., 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 is phosphorylated at the seventh N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60%, or 65% of rhGAA has an arbitrary glycan at the seventh N-glycosylation site. In some embodiments, at least 30%, 35%, or 40% of rhGAA has a glycan at the seventh N-glycosylation site.

[0083] In various embodiments, rhGAA has an average fucose content of 0 to 5 moles per mole of rhGAA, a GlcNAc content of 10 to 30 moles per mole of rhGAA, a galactose content of 5 to 20 moles per mole of rhGAA, a mannose content of 10 to 40 moles per mole of rhGAA, an M6P content of 2 to 8 moles per mole of rhGAA, and a sialic acid content of 2 to 8 moles per mole of rhGAA. In various embodiments, rhGAA has an average fucose content of 2 to 3 moles per mole of rhGAA, a GlcNAc content of 20 to 25 moles per mole of rhGAA, a galactose content of 8 to 12 moles per mole of rhGAA, a mannose content of 22 to 27 moles per mole of rhGAA, an M6P content of 3 to 5 moles per mole of rhGAA, and a sialic acid content of 4 to 7 moles per mole of rhGAA.

[0084] Recombinant human lysosomal proteins (e.g., rhGAA) are preferably produced by Chinese hamster ovary (CHO) cells such as the CHO cell line GA-ATB-200 or ATB-200-001-X5-14, or by subcultures or derivatives of such CHO cell cultures. DNA constructs expressing allelic variants of acid α-glucosidase or other mutant acid α-glucosidase amino acid sequences, such as those that are at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2, can be constructed and expressed in CHO cells. These mutant acid α-glucosidase amino acid sequences may contain deletions, substitutions, and / or insertions relative to SEQ ID NO: 1, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions, and / or insertions relative to the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. Those skilled in the art may select alternative vectors suitable for transforming CHO cells for the production of such DNA constructs.

[0085] Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, Wisconsin), and can be used, for example, with default settings. For example, polypeptides having at least 90%, 95%, 98%, or 99% identity to a particular polypeptide described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are intended. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When BLASTP is used, the similarity percentage is based on the BLASTP positive score, and the sequence identity percentage is based on the BLASTP identity score. "Identity" in BLASTP indicates the number and percentage of total residues in identical high-scoring sequence pairs; and "positive" in BLASTP indicates the number and percentage of residues that are similar to each other and have a positive alignment score. Amino acid sequences having any degree of identity or similarity or any moderate identity to the amino acid sequences disclosed herein are intended and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides can be predicted using the genetic code and obtained by conventional means, particularly by backtranslating their amino acid sequences using the genetic code.

[0086] In this invention, we have found that recombinant human acid α-glucosidases with superior ability to target cation-independent mannose-6-phosphate receptors (CIMPR) and cellular lysosomes, as well as glycosylation patterns that reduce unproductive clearance in vivo, can be produced using Chinese hamster ovary (CHO) cells. These cells can be induced to express recombinant human acid α-glucosidases with significantly higher levels of N-glycan units containing one or more mannose-6-phosphate residues than conventional recombinant human acid α-glucosidase products such as alglucosidase alpha. The recombinant human acid α-glucosidases produced by these cells have significantly more muscle cell-targeting mannose-6-phosphate (M6P) and bis-mannose-6-phosphate N-glycan residues than conventional acid α-glucosidases such as Lumizyme®, as exemplified by ATB200. While not bound by theory, this extensive glycosylation suggests that the ATB200 enzyme can be more effectively taken up by target cells and therefore more efficiently removed from circulation than other recombinant human acid α-glucosidases, such as alglucosidase alpha, which has much lower M6P and bis-M6P content. ATB200 has been shown to efficiently bind to CIMPR, be efficiently taken up by skeletal and cardiac muscle, and possess a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces unproductive clearance in vivo.

[0087] The broad glycosylation of ATB200 may, for example, contribute to a decrease in the immunogenicity of ATB200 compared to alglucosidase alpha. As will be understood by those skilled in the art, glycosylation of proteins with conserved mammalian sugars generally increases the solubility of the product and reduces its aggregation and immunogenicity. Glycosylation indirectly alters protein immunogenicity by minimizing protein aggregation and blocking protein immunogenic epitopes from the immune system (Guidance for Industry-Immunogenicity Assessment for Therapeutic Protein Products, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, August 2014). Therefore, in at least one embodiment, administration of recombinant human acid α-glucosidase does not induce anti-drug antibodies. In at least one embodiment, administration of recombinant human acid α-glucosidase reduces the incidence of anti-drug antibodies in a subject to a level higher than that induced by administration of alglucosidase alpha.

[0088] As described in concurrently pending international patent application PCT / U.S. Patent Application Publication No. 2015 / 053252, cells such as CHO cells can be used to produce the rhGAA described in the said document, and this rhGAA can be used in the present invention. Examples of such CHO cell lines are GA-ATB-200 or ATB-200-001-X5-14, or their subcultures that produce rhGAA compositions as described therein. Such CHO cell lines may contain multiple copies of the gene, such as 5, 10, 15, 20 or more copies per polynucleotide encoding the GAA.

[0089] High-M6P and bis-M6P rhGAAs, such as ATB200 rhGAA, transform CHO cells with DNA constructs encoding GAA. While CHO cells have been previously used to produce rhGAAs, transformed CHO cells have not been observed to be cultured and selected to produce rhGAAs with high content of CIMPR-targeting M6P and bis-M6P glycans.

[0090] Surprisingly, it has been found that it is possible to transform CHO cell lines, select transformants that produce rhGAA containing a high content of glycans having M6P or bis-M6P that target CIMPR, and stably express this high-M6P rhGAA. Therefore, a method for producing these CHO cell lines is also described in concurrently pending international patent application PCT / US Patent Application Publication 2015 / 053252. The method comprises transforming CHO cells with DNA encoding GAA or a GAA variant, stably incorporating the DNA encoding GAA into its chromosome and selecting CHO cells that stably express GAA, selecting CHO cells that express GAA with a high content of glycans having M6P or bis-M6P, and optionally selecting CHO cells having N-glycans with a high sialic acid content and / or a low non-phosphorylated high-mannose content.

[0091] These CHO cell lines can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell lines and recovering the compositions from the CHO cell cultures.

[0092] Generation, capture, and purification of recombinant human lysosomal proteins Various embodiments of the present invention relate to methods for generating and / or capturing and / or purifying recombinant human lysosomal proteins (e.g., rhGAA). An exemplary method 600 for generating, capturing and purifying recombinant human lysosomal proteins is shown in Figure 6.

[0093] In Figure 6, arrows indicate the direction of movement of various liquid phases containing recombinant human lysosomal protein. Bioreactor 601 contains a culture of cells, such as CHO cells, that express recombinant human lysosomal protein (e.g., rhGAA) and secrete it into the surrounding liquid medium. Bioreactor 601 may be any suitable bioreactor for culturing cells, such as a perfusion, batch, or fed-batch culture bioreactor. In various embodiments, the bioreactor has a volume from about 1 L to about 20,000 L. Exemplary bioreactor volumes are about 1 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 150 L, about 200 L, about 250 L, about 300 L, about 350 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about Includes 900L, approximately 1,000L, approximately 1,500L, approximately 2,000L, approximately 2,500L, approximately 3,000L, approximately 3,500L, approximately 4,000L, approximately 5,000L, approximately 6,000L, approximately 7,000L, approximately 8,000L, approximately 9,000L, approximately 10,000L, approximately 15,000L, and approximately 20,000L.

[0094] As shown in Figure 6, the culture medium can be removed from the bioreactor. Such removal of the medium may be continuous for perfusion bioreactors and batch for batch or fed-batch reactors. The medium is filtered by the filtration system 603 to remove the cells. In some embodiments, the cells removed from the medium can be reintroduced into the bioreactor and the medium containing secreted recombinant human lysosomal protein can be further processed. The filtration system 603 can be any suitable filtration system, including alternating tangential flow filtration (ATF) systems, tangential flow filtration (TFF) systems, centrifugal filtration systems, etc. In various embodiments, the filtration system utilizes filters having pore sizes of about 10 nanometers to about 2 micrometers. Typical filter pore sizes include approximately 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 1.5 μm, and 2 μm.

[0095] In various embodiments, the culture medium removal rate is approximately 1 L / day to approximately 20,000 L / day. Exceptional culture medium removal rates are approximately 1 L / day, 10 L / day, 20 L / day, 30 L / day, 40 L / day, 50 L / day, 60 L / day, 70 L / day, 80 L / day, 90 L / day, 100 L / day, 150 L / day, 200 L / day, 250 L / day, 300 L / day, 350 L / day, 400 L / day, 500 L / day, 600 L / day, 700 L / day, and 800 L / day. The amounts are approximately 900 L / day, 1,000 L / day, 1,500 L / day, 2,000 L / day, 2,500 L / day, 3,000 L / day, 3,500 L / day, 4,000 L / day, 5,000 L / day, 6,000 L / day, 7,000 L / day, 8,000 L / day, 9,000 L / day, 10,000 L / day, 15,000 L / day, and 20,000 L / day. Alternatively, the culture medium removal rate can be expressed as a function of bioreactor volume, such as approximately 0.1 to 3 reactor volumes / day. Exemplary media removal rates include reactor volumes of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, and 3.

[0096] For continuous or fed-batch processing, the rate at which fresh culture medium is supplied to the bioreactor can be approximately 1 L / day to approximately 20,000 L / day. Exceptional culture medium introduction rates are approximately 1 L / day, 10 L / day, 20 L / day, 30 L / day, 40 L / day, 50 L / day, 60 L / day, 70 L / day, 80 L / day, 90 L / day, 100 L / day, 150 L / day, 200 L / day, 250 L / day, 300 L / day, 350 L / day, 400 L / day, 500 L / day, 600 L / day, 700 L / day, and 800 L / day. The daily rates are approximately 900 L / day, 1,000 L / day, 1,500 L / day, 2,000 L / day, 2,500 L / day, 3,000 L / day, 3,500 L / day, 4,000 L / day, 5,000 L / day, 6,000 L / day, 7,000 L / day, 8,000 L / day, 9,000 L / day, 10,000 L / day, 15,000 L / day, and 20,000 L / day. Alternatively, the culture medium introduction rate can be expressed as a function of bioreactor volume, such as approximately 0.1 to 3 reactor volumes / day. Exemplary media introduction rates include reactor volumes of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, and 3.

[0097] After filtration, the filtrate is packed into the protein capture system 605. The protein capture system 605 may contain one or more chromatography columns. If two or more chromatography columns are used, the columns may be arranged in series so that the next column is packed after the first column is packed. Alternatively, the medium removal method can be paused while switching columns.

[0098] In various embodiments, the protein capture system 605 includes one or more anion exchange (AEX) columns for direct product capture of recombinant human lysosomal proteins, particularly lysosomal proteins with high M6P content. While we do not wish to be bound by any particular theory, it is thought that using AEX chromatography to capture recombinant human lysosomal proteins from filtered medium ensures that the captured recombinant protein product has a higher M6P content due to the greater negative charge of recombinant proteins having one or more M6P groups. As a result, non-phosphorylated recombinant proteins and host cell impurities do not bind to the column resin and highly phosphorylated recombinant proteins, while non-phosphorylated recombinant proteins and host cell impurities pass through the column. Therefore, AEX chromatography can be used to enrich the M6P content of protein products (i.e., select proteins with more M6P) due to the high affinity of M6P-containing proteins to AEX resin.

[0099] While we do not wish to be bound by any particular theory, it is believed that direct product capture of recombinant proteins using AEX chromatography reliably removes recombinant proteins with high M6P content from media containing proteases, as well as other enzymes capable of degrading and / or dephosphorylating proteins. As a result, high-quality products are preserved.

[0100] A suitable AEX chromatography column has chemical functional groups that bind to negatively charged proteins. Exemplary functional groups include, but are not limited to, primary, secondary, tertiary, and quaternary ammonium or amine groups. These functional groups may be bound to a membrane (e.g., a cellulose membrane) or a conventional chromatography resin. Exemplary column media include GE Healthcare Lifesciences' SP, CM, Q, and DEAE Sepharose® Fast Flow media.

[0101] The volume of the AEX chromatographic column can be any appropriate volume, such as 1 L to 1,000 L. Exemplary column volumes include approximately 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 500 L, 600 L, 700 L, 800 L, 90 L, and 1,000 L.

[0102] Table 2 below shows exemplary conditions for anion exchange columns:

[0103] JPEG2026136113000010.jpg154154

[0104] After recombinant human lysosomal proteins are loaded into the protein capture system 605, the recombinant human lysosomal proteins are eluted from the column(s) by altering the pH and / or salt content in the column.

[0105] The eluted recombinant human lysosomal proteins may be subjected to further purification and / or quality assurance steps. For example, as shown in Figure 6, the eluted recombinant human lysosomal proteins may be subjected to a virus killing step 607. Such virus killing 607 may include low pH killing, decontamination, or one or more other techniques known in the art.

[0106] The recombinant protein product from the virus killing step 607 can be further purified by introducing it into a second chromatography system 609. Alternatively, the recombinant protein eluted from the protein capture system 605 can be directly supplied to the second chromatography system 609. In various embodiments, the second chromatography system 609 includes one or more immobilized metal affinity chromatography (IMAC) columns for further removal of impurities. Exemplary metal ions include cobalt, nickel, copper, iron, zinc, or gallium.

[0107] The volume of the second chromatographic column (e.g., IMAC column) can be any suitable volume, such as 0.1 L to 100 L. Exemplary column volumes include approximately 0.1 L, 0.2 L, 0.3 L, 0.4 L, 0.5 L, 0.6 L, 0.7 L, 0.8 L, 0.9 L, 1 L, 1.5 L, 2 L, 2.5 L, 3 L, 3.5 L, 4 L, 4.5 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, and 50 L, 60 L, 70 L, 80 L, 90 L, and 100 L.

[0108] Table 3 below shows example conditions for the IMAC column:

[0109] JPEG2026136113000011.jpg111154

[0110] JPEG2026136113000012.jpg217154

[0111] After the recombinant protein is loaded into the second chromatography system 609, the recombinant protein is eluted from the column. As shown in Figure 6, the eluted recombinant protein can be subjected to a virus killing step 611. As with virus killing 607, virus killing 611 may include low pH killing, detergent killing, or one or more other techniques known in the art. In some embodiments, only one of virus killing 607 or 611 is used, or multiple virus killings are performed at the same stage in the purification method.

[0112] As shown in Figure 6, the recombinant protein product from the virus killing step 611 can be further purified by introducing it into a third chromatography system 613. Alternatively, the recombinant protein eluted from the second chromatography system 609 can be directly supplied to the third chromatography system 613. In various embodiments, the third chromatography system 613 includes one or more cation exchange chromatography (CEX) columns and / or size exclusion chromatography (SEC) columns for further removal of impurities. The recombinant protein product is then eluted from the third chromatography system 613.

[0113] The volume of the third chromatographic column (e.g., CEX or SEC column) can be any suitable volume, such as 0.1 L to 200 L. Exemplary column volumes include approximately 0.1 L, 0.2 L, 0.3 L, 0.4 L, 0.5 L, 0.6 L, 0.7 L, 0.8 L, 0.9 L, 1 L, 1.5 L, 2 L, 2.5 L, 3 L, 3.5 L, 4 L, 4.5 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, and 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, and 200 L.

[0114] Table 4 below shows exemplary conditions for a CEX column:

[0115] JPEG2026136113000013.jpg162154

[0116] The recombinant protein product may also be subjected to further processing. For example, another filtration system 615 may be used to remove viruses. In some embodiments, such filtration may utilize a filter having a pore size of 5 to 50 μm. Other product processing may include a product preparation step 617 in which the recombinant protein product is sterilized, filtered, concentrated, stored, and / or has further components to be added to the formulation of the final product. For example, the recombinant protein product can be concentrated by a factor of 2 to 10, such as from an initial protein concentration of about 2 to about 20 mg / ml to a final protein concentration of about 4 to about 200 mg / ml. This final product can be used to fill vials and may be lyophilized for future use.

[0117] Administration of Recombinant Proteins Recombinant human lysosomal proteins (e.g., rhGAA) or pharmaceutically acceptable salts thereof can be formulated according to standard procedures as pharmaceutical compositions suitable for administration to humans. For example, in a preferred embodiment, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic to relieve pain at the injection site. Generally, the components are supplied separately or mixed together in a unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of the active substance. When the composition is administered by infusion, it may be dispensed in an infusion bottle containing pharmaceutical-grade sterile water, saline, or dextrose / water. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the components can be mixed before administration.

[0118] Recombinant human lysosomal protein (e.g., rhGAA) (or a composition or pharmaceutical containing recombinant human lysosomal protein) is administered via an appropriate route. In one embodiment, recombinant human lysosomal protein is administered intravenously. In other embodiments, recombinant human lysosomal protein (e.g., rhGAA) is administered by direct injection into target tissue such as the heart or skeletal muscle (e.g., intramuscular) or the nervous system (e.g., direct injection into the brain; intraventricular; intrathecal). Multiple routes may be used simultaneously as needed.

[0119] Recombinant human lysosomal protein (e.g., rhGAA) (or compositions or pharmaceuticals containing recombinant human lysosomal protein) is administered in a therapeutically effective dose (e.g., a dose sufficient to treat the disease by improving disease-related symptoms, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of disease symptoms when administered regularly). The therapeutically effective dose for treating the disease depends on its properties and the extent of the disease's effects and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may be used, at the discretion of the practitioner, to help identify the optimal dose range. The exact dose used also depends on the route of administration and the severity of the disease and should be determined according to the practitioner's judgment and the circumstances of each patient. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems. In at least one embodiment, recombinant human acid α-glucosidase is administered by intravenous infusion at doses of approximately 1 mg / kg to approximately 100 mg / kg, for example, approximately 5 mg / kg to approximately 30 mg / kg, typically approximately 5 mg / kg to approximately 20 mg / kg. In at least one embodiment, recombinant human acid α-glucosidase is administered by intravenous infusion at doses of approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 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 acid α-glucosidase is administered by intravenous infusion at a dose of approximately 20 mg / kg. In at least one embodiment, recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of approximately 20 mg / kg. The effective dose for a particular individual can be varied over time (e.g., increased or decreased) depending on the individual's needs. For example, the dose may be increased when the individual has a physical illness or stress, or when acid-fast α-glucosidase antibodies become present or increase, or when the symptoms of the disease worsen.

[0120] The therapeutically effective dose of recombinant human acid α-glucosidase (or a composition or pharmaceutical containing recombinant human acid α-glucosidase) is administered regularly and continuously, depending on its properties and the severity of the disease. As used herein, “regular” administration refers to the periodic administration of the therapeutically effective dose (distinguished from a single dose). The interval can be determined by standard clinical techniques. In preferred embodiments, recombinant human acid α-glucosidase is administered monthly, every other month; weekly; twice weekly; or daily. The administration interval for a single individual does not need to be constant and can be varied over time according to the individual’s needs. For example, the administration interval can be reduced when there is a physical illness or stress, when anti-recombinant human acid α-glucosidase antibodies become present or increase, or when the symptoms of the disease worsen. In some embodiments, a therapeutically effective dose of 5, 10, 20, 50, 100, or 200 mg of enzyme / kg body weight is administered twice weekly, weekly, or every other week, with or without a chaperone.

[0121] Recombinant human lysosomal protein (e.g., rhGAA) may be prepared for later use, such as in unit dose vials or syringes, or in vials or pouches for intravenous administration. Kits containing recombinant human lysosomal protein (e.g., rhGAA), along with other active ingredients such as optionally selected excipients or chaperones or other drugs, may be enclosed in packaging material and accompanied by instructions for reconstitution, dilution, or administration to subjects requiring treatment, such as patients with Pompe disease.

[0122] Combination therapy with rhGAA and pharmacological chaperones In various embodiments, rhGAA produced by the method described herein (e.g., ATB200) can be used in combination therapy with pharmacological chaperones such as miglustat or duvoglustat.

[0123] In at least one embodiment, the pharmacological chaperone (e.g., miglustat) is administered orally. In at least one embodiment, miglustat is administered in oral doses of about 200 to about 400 mg, or in oral doses 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 in oral doses of about 233 mg to about 400 mg. In at least one embodiment, miglustat is administered in oral doses of about 250 to about 270 mg, or in oral doses of about 250 mg, about 255 mg, about 260 mg, about 265 mg, or about 270 mg. In at least one embodiment, miglustat is administered as an oral dose of about 260 mg.

[0124] Those skilled in the art will understand that for adult patients with an average weight of approximately 70 kg, oral doses of miglustat in the range of approximately 200 mg to 400 mg or any smaller range may be appropriate. For patients with a weight significantly lower than approximately 70 kg, including but not limited to infants, children, or underweight adults, a lower dose may be considered appropriate by a physician. Therefore, in at least one embodiment, miglustat is administered as an oral dose of approximately 50 mg to approximately 200 mg, or as an oral dose of approximately 50 mg, approximately 75 mg, approximately 100 mg, 125 mg, approximately 150 mg, approximately 175 mg, or approximately 200 mg. In at least one embodiment, miglustat is administered as an oral dose of approximately 65 mg to approximately 195 mg, or as an oral dose of approximately 65 mg, approximately 130 mg, or approximately 195 mg.

[0125] In at least one embodiment, miglustat is administered as a pharmaceutically acceptable dosage form suitable for oral administration, which includes, but is not limited to, tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders for reconstitution with water or other suitable solvents before use, optionally including flavorings and colorings, for immediate release, delayed release, deformed release, sustained release, pulsed release, or controlled release applications. Solid compositions such as tablets, capsules, lozenges, troches, pills, boluses, powders, pastes, granules, bullets, sugar-coated tablets, or premix preparations may also be used. In at least one embodiment, miglustat is administered as a tablet. In at least one embodiment, miglustat is administered as a capsule. In at least one embodiment, the dosage form contains about 50 mg to about 300 mg of miglustat. In at least one embodiment, the dosage form contains about 65 mg of miglustat. In at least one embodiment, the dosage form contains about 130 mg of miglustat. In at least one embodiment, the dosage form contains about 260 mg of miglustat. When the dosage form contains about 65 mg of miglustat, it is intended that miglustat may be administered as a four-dosage-form dose or a total dose of 260 mg of miglustat. However, in patients with a weight significantly lower than the mean adult weight of 70 kg, including but not limited to infants, children, or underweight adults, miglustat may be administered as a one-dosage-form dose (total dose of 65 mg of miglustat), a two-dosage-form dose (total dose of 130 mg of miglustat), or a three-dosage-form dose (total dose of 195 mg of miglustat).

[0126] Solid and liquid compositions for oral use may be prepared according to methods well known in the art. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients, which may be in solid or liquid form. Tablets or capsules may be prepared by conventional means using pharmaceutically acceptable excipients, including but not limited to binders, fillers, lubricants, disintegrants, or wetting agents. Suitable pharmaceutically acceptable excipients are well known in the art and include, but are not limited to, pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC), hydroxypropylcellulose (HPC), sucrose, gelatin, acacia gum, lactose, crystalline cellulose, calcium hydrogen phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, dicalcium phosphate, sodium glycine croscarmellose, and silicate complexes. The tablets can be coated by methods well known in the art. In at least one embodiment, miglustat is administered as a formulation marketed as Zavesca® (Actelion Pharmaceuticals).

[0127] In at least one embodiment, miglustat and recombinant human acid 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, miglustat is administered before the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 3 hours prior to the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered approximately 2 hours prior to the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 2 hours prior to the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered approximately 1.5 hours prior to the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered approximately 1 hour prior to the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 50 to 70 minutes before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 55 to 65 minutes before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 25 to 35 minutes before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 27 to 33 minutes before administration of recombinant human acid α-glucosidase.

[0128] In at least one embodiment, miglustat is administered simultaneously with the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered before or within 20 minutes after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered before or within 15 minutes after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered before or within 10 minutes after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered before or within 5 minutes after the administration of recombinant human acid α-glucosidase.

[0129] In at least one embodiment, miglustat is administered after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 2 hours after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1 hour after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1.5 hours after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 2 hours after administration of recombinant human acid α-glucosidase.

[0130] Another aspect of the present invention provides a kit for the adjunctive treatment of Pompe disease in patients in need thereof. The kit includes a pharmaceutically acceptable dosage form containing miglustat, a pharmaceutically acceptable dosage form containing recombinant human acid α-glucosidase as defined herein, and instructions for administering the pharmaceutically acceptable dosage form containing miglustat and the pharmaceutically acceptable dosage form containing recombinant human acid α-glucosidase to a patient in need thereof. In at least one embodiment, the pharmaceutically acceptable dosage form containing miglustat is an oral dosage form as described herein, including but not limited to tablets or capsules. In at least one embodiment, the pharmaceutically acceptable dosage form containing recombinant human acid α-glucosidase is a sterile solution suitable for injection as described herein. In at least one embodiment, the instructions for administering the dosage forms include instructions for administering the pharmaceutically acceptable dosage form containing miglustat before administering the pharmaceutically acceptable dosage form containing recombinant human acid α-glucosidase by intravenous infusion, as described herein.

[0131] While not theoretically bound, miglustat is thought to act as a pharmacological chaperone for recombinant human acid α-glucosidase ATB200, binding to its active site. For example, miglustat has been found to reduce the proportion of unfolded ATB200 protein, stabilize the active conformation of ATB200, prevent denaturation and irreversible inactivation at neutral plasma pH, and allow for survival conditions long enough to reach and be taken up by tissues in circulation. However, the binding of miglustat to the active site of ATB200 may also result in inhibition of ATB200's enzymatic activity by preventing its natural substrate, glycogen, from accessing the active site. When miglustat and recombinant human acid α-glucosidase are administered to a patient under the conditions described herein, the concentrations of miglustat and ATB200 in plasma and tissues are stabilized to the extent that ATB200 is taken up by the tissue and targets lysosomes. However, due to the rapid clearance of miglustat, the hydrolysis of glycogen by ATB200 in lysosomes is not excessively inhibited by the presence of miglustat, and the enzyme is thought to retain sufficient therapeutically useful activity.

[0132] All of the above embodiments may be combined. This includes specific embodiments relating to: • The properties of pharmacological chaperones, e.g., miglustat; and the active site to which they are specific; • Dosage, route of administration of pharmacological chaperone (e.g., miglustat), and type of pharmaceutical composition, including the properties of the carrier and the use of commercially available compositions; The therapeutic protein drug product may be a counterpart to an endogenous protein whose expression is reduced or absent in the target, preferably recombinant human lysosomal protein (e.g., rhGAA), for example recombinant human acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and containing an increased amount of one or more N-glycan units having alpha-mannose-6-phosphate residues compared to the amount of one or more N-glycan units having alpha-mannose-6-phosphate residues of alglucosidase; and preferably having the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2; The number and type of N-glycan units on recombinant human lysosomal proteins (e.g., rhGAA), such as complex N-glycans formed from N-acetylglucosamine, galactose, sialic acid, or combinations thereof, bound to recombinant human lysosomal proteins; The degree of phosphorylation of mannose units on recombinant human lysosomal proteins (e.g., rhGAA) that form mannose-6-phosphate and / or bis-mannose-6-phosphate; • The type of formulation including the dosage and route of administration (e.g., intravenous administration, particularly intravenous infusion or direct administration to target tissue) of the replacement enzyme (e.g., recombinant human acid α-glucosidase), the carrier, and the therapeutically effective dose; • Dosage intervals for the pharmacological chaperone (miglustat) and recombinant human acid α-glucosidase; • The nature of the treatment response and the results of combination therapy (e.g., enhanced results compared to the effects of each individual treatment); • Timing of administration of combination therapy, for example, simultaneous or sequential administration of miglustat and recombinant human acid α-glucosidase, for example, when miglustat is administered before recombinant human acid α-glucosidase, or after recombinant human acid α-glucosidase, or within a certain time before or after administration; and • The nature of the patient being treated (e.g., a mammal such as a human) and the medical condition the individual is suffering from (e.g., enzyme deficiency).

[0133] Any embodiment in the above list can be combined with one or more other embodiments in the list. [Examples]

[0134] Further features of the present invention will become apparent, for example, from the following non-limiting embodiments illustrating the principles of the present invention.

[0135] Example 1: Limitations of existing myozyme® and lumizyme® rhGAA products To evaluate the rhGAA activity in Myozyme® and Lumizyme®, the only currently approved treatments for Pompe disease, these rhGAA preparations were injected into a CIMPR column (binding to rhGAA with an M6P group) and subsequently eluted with a free M6 gradient. The fractions were collected in 96-well plates, and GAA activity was assayed with a 4MU-α-glucose substrate. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a percentage of the total enzyme.

[0136] Figures 4A and 4B illustrate the problems associated with conventional ERTs (Myozyme® and Lumizyme®): 73% of rhGAA from Myozyme® (Figure 4B) and 78% of rhGAA from Lumizyme® (Figure 4A) did not bind to CIMPR (see the leftmost peak in each figure). Only 27% of rhGAA from Myozyme® and 22% of rhGAA from Lumizyme® contained M6P that could target CIMPR on muscle cells.

[0137] The effective doses of Myozyme® and Lumizyme® correspond to the amount of rhGAA containing M6P that targets CIMPR on muscle cells. However, the majority of rhGAA in these two conventional products does not target CIMPR receptors on target muscle cells. Administration of conventional rhGAA, in which the majority is not targeted to muscle cells, increases the risk of allergic reactions or immune induction to untargeted rhGAA.

[0138] Example 2: Preparation of CHO cells producing ATB200 rhGAA with high content mono- or bis-M6P-containing N-glycans CHO cells were transfected with DNA expressing rhGAA, and then transformants that produce rhGAA were selected. The DNA construct for transforming CHO cells with rhGAA-encoding DNA is shown in Figure 5. CHO cells were transfected with DNA expressing rhGAA, and then transformants that produce rhGAA were selected.

[0139] After transfection, DG44 CHO(DHFR-) cells containing stably incorporated GAA genes were selected in hypoxanthine / thymidine-deficient (-HT) medium.

[0140] Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Cell pools expressing large amounts of GAA were identified by GAA enzyme activity assays and used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid medium plates, harvested using the ClonePix system, and transferred to 24-deep-well plates. Individual clones were assayed for GAA enzyme activity to identify clones expressing high levels of GAA. The conditional medium for determining GAA activity was a 4-MU-α-glucosidase substrate. Clones producing higher levels of GAA, as measured by GAA enzyme assays, were further evaluated for viability, growth capacity, GAA productivity, N-glycan structure, and stable protein expression. CHO cell lines, including the GA-ATB-200 cell line expressing rhGAA with enhanced mono-M6P or bis-M6P N-glycans, were isolated using this procedure.

[0141] Example 3: Capture and purification of ATB200 rhGAA Multiple batches of rhGAA according to the present invention were produced in shaking flasks and perfusion bioreactors using the CHO cell line GA-ATB-200, and CIMPR binding was measured. Similar CIMPR receptor binding (approximately 70%) was observed for purified ATB200 rhGAA from different production batches, as shown in Figures 7B and 8, indicating that ATB200 rhGAA can be consistently produced. As shown in Figures 4A, 4B, 7A, and 7B, myozyme® and lumizyme® rhGAA showed significantly lower CIMPR binding than ATB200 rhGAA.

[0142] Example 4: Analytical comparison of ATB200 against Lumizyme® ATB200 rhGAA was fractionated by terminal phosphate using weak anion exchange ("WAX") liquid chromatography. Elution profiles were generated by eluting ERT with increasing amounts of salt. Profiles were monitored under UV (A 280 nm). ATB200 rhGAA 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. ATB200 rhGAA showed four prominent peaks eluting to the right of Lumizyme® (Figure 9). This confirms that ATB200 rhGAA is significantly more phosphorylated than Lumizyme®, as this evaluation is based on terminal charge rather than CIMPR affinity.

[0143] Example 5: Oligosaccharide properties of ATB200 rhGAA Purified ATB200 rhGAA and Lumizyme® glycans were evaluated by MALDI-TOF to determine the individual glycan structures found on each ERT (Figure 10). ATB200 samples were found to contain lower amounts of non-phosphorylated high-mannose N-glycans than Lumizyme®. A higher M6P glycan content in ATB200 compared to Lumizyme® indicates that ATB200 rhGAA targets muscle cells more effectively. The high percentages of mono-phosphorylated and bis-phosphorylated structures determined by MALDI are consistent with the CIMPR profile, which indicates significantly greater binding of ATB200 to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contains at least one native bis-M6P N-glycan structure. Higher content of this bis-M6P N-glycan on ATB200 rhGAA directly correlated with high affinity binding to CIMPR in an M6P receptor plate binding assay (KD approximately 2-4 nM), Figure 12A.

[0144] ATB200 rhGAA was also analyzed for site-specific N-glycan profiles using two different LC-MS / MS analytical techniques. In the first analysis, the protein was denatured, reduced, alkylated, and digested before LC-MS / MS analysis. During protein denaturation and reduction, 200 μg of protein sample, 5 μl of 1 mol / L Tris-HCl (final concentration 50 mM), 75 μl of 8 mol / L guanidine HCl (final concentration 6 M), 1 μl of 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 provide a total volume of 100 μL. The sample was mixed and incubated in a dry bath at 56°C for 30 minutes. During alkylation, denatured and reduced protein samples were mixed with 5 μL of 1 mol / L iodoacetamide (IAM, final concentration 50 mM) and incubated in the dark for 30 minutes. After alkylation, 400 μL of pre-cooled acetone was added to the sample, and the mixture was frozen at -80°C for 4 hours. The sample was then centrifuged at 4°C and 13000 rpm for 5 minutes, and the supernatant was removed. 400 μL of pre-cooled acetone was added to the pellet, and this was then centrifuged at 4°C and 13000 rpm for 5 minutes, and the supernatant was removed. The sample was air-dried on ice in the dark to remove acetone residue. 40 μL of 8 M urea and 160 μL of 100 μM NH4HCO3 were added to the sample to dissolve the protein. During trypsin digestion, 50 μg of protein was added to trypsin digestion buffer to a final volume of 100 μl, and 5 μL of 0.5 μg / mL trypsin (protein-to-enzyme ratio 20 / 1 w / w) was added. The solution was thoroughly mixed and incubated at 37°C overnight (16 ± 2 hours). 2.5 μL of 20% TFA (final concentration 0.5%) was added to stop the reaction. Next, the sample was analyzed using a Thermo Scientific Orbitrap Velos Pro® mass spectrometer.

[0145] In the second LC-MS / MS analysis, ATB200 samples were prepared following the same denaturation, reduction, alkylation, and digestion procedures, except that iodoacetic acid (IAA) was used as the alkylating reagent instead of IAM, and analyzed using a Thermo Scientific Orbitrap Fusion Lumos Tribid® mass spectrometer.

[0146] The results of the first and second analyses are shown in Figures 11A to 11H. In Figures 11A to 11H, the results of the first analysis are represented by the left bar (dark gray), and the results of the second analysis are represented by the right bar (light gray). In Figures 11B to 11G, the nomenclature for glycans follows Varki, A., Cummings, RD, Esko JD, et al., Essentials of Glycobiology, 2nd edition (2009).

[0147] As can be seen from Figures 8A-8I, the two analyses showed similar results, but there were some differences between them. This variation can be attributed to many factors, including the equipment used and the completeness of the N-glycan analysis. For example, if some species of phosphorylated glycans are not identified and / or quantified, the total number of phosphorylated glycans may be underestimated, and the percentage of rhGAA with phosphorylated glycans at those sites may be underestimated. As another example, if some species of non-phosphorylated glycans are not identified and / or quantified, the total number of non-phosphorylated glycans may be overestimated, and the percentage of rhGAA with phosphorylated glycans at those sites may be overestimated. Figure 11A shows the N-glycosylation site occupancy of ATB200. As can be seen from Figure 11A, the first, second, third, fourth, fifth, and sixth N-glycosylation sites are largely occupied by both analyses, which detect over 90% and up to approximately 100% of the ATB200 enzyme containing glycans detected at their respective potential sites. However, the seventh potential N-glycosylation site is glycosylated in about half the time.

[0148] Figure 11B shows the N-glycosylation profile of the first site, N84. As can be seen from Figure 11B, the dominant glycan species is bis-M6P glycan. In both the first and second analyses, it was detected that more than 75% of ATB200 had bis-M6P glycan at the first site.

[0149] Figure 11C shows the N-glycosylation profile of the second site, N177. As can be seen from Figure 11C, the major glycan species are mono-M6P glycan and non-phosphorylated high-mannose glycan. In both the first and second analyses, it was detected that more than 40% of ATB200 had mono-M6P glycan at the second site.

[0150] Figure 11D shows the N-glycosylation profile of the third site, N334. As can be seen from Figure 11D, the major glycan species are non-phosphorylated high-mannose glycans, di-, tri-, and tetra-antennari complex glycans, as well as hybrid glycans. In both the first and second analyses, it was detected that more than 20% of ATB200 had a sialic acid residue at the third site.

[0151] Figure 11E shows the N-glycosylation profile of the fourth site, N414. As can be seen from Figure 11E, the major glycan species are bis-M6P and mono-MGP glycans. In both the first and second analyses, it was detected that more than 40% of ATB200 had bis-M6P glycans at the fourth site. In both the first and second analyses, it was also detected that more than 25% of ATB200 had mono-M6P glycans at the fourth site.

[0152] Figure 11F shows the N-glycosylation profile of the fifth site, N596. As can be seen from Figure 11F, the major glycan species is the fucosylated dianthenthri complex glycan. In both the first and second analyses, it was detected that more than 70% of ATB200 had a sialic acid residue at the fifth site.

[0153] Figure 11G shows the N-glycosylation profile of the sixth site, N826. As can be seen from Figure 11F, the major glycan species are di-, tri-, and tetra-antennari complex glycans. In both the first and second analyses, it was detected that more than 80% of ATB200 had a sialic acid residue at the sixth site.

[0154] Analysis of glycosylation at site 7, N869, showed approximately 40% glycosylation, with the most common glycans being A4S3S3GF (12%), A5S3G2F (10%), A4S2G2F (8%), and A6S3G3F (8%).

[0155] Figure 11H shows an overview of phosphorylation at each of the seven potential N-glycosylation sites. As can be seen from Figure 11G, high phosphorylation levels were detected at sites 1, 2, and 4 in both the first and second analyses. Both analyses detected that more than 80% of ATB200 was mono- or di-phosphorylated at site 1, more than 40% of ATB200 was mono-phosphorylated at site 2, and more than 80% of ATB200 was mono- or di-phosphorylated at site 4.

[0156] Another glycan analysis of ATB200 was performed according to hydrophilic interaction liquid chromatography-fluorescence detection-mass spectrometry (HILIC-FLD-MS).

[0157] The results of the HILIC-FLD-MS analysis are shown in Table 5 below. In Table 5, the first digit of the three-digit number indicates the number of glycan branches, the second digit indicates the number of core fucose units, and the third digit indicates the number of terminal sialic acid units. Using this nomenclature, "303" represents a tribranched glycan (first 3) with 0 core fucose units (second 0) and 3 terminal sialic acids (last 3), "212" represents a bibranched glycan with 1 core fucose unit and 2 terminal sialic acids, "404" represents a tetrabranched glycan containing 0 core fucose units and 4 terminal sialic acids, and so on.

[0158] JPEG2026136113000014.jpg109154

[0159] JPEG2026136113000015.jpg220154

[0160] JPEG2026136113000016.jpg209154

[0161] 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.

[0162] Example 6: CIMPR affinity characteristics of ATB200 In addition to the higher proportion of rhGAA capable of binding to CIMPR, understanding the quality of the interaction is crucial. Lumizyme® and ATB200 rhGAA receptor binding were measured using a CIMPR plate binding assay. Briefly, GAA was captured using a CIMPR-coated plate. Various concentrations of rhGAA were applied to immobilized receptors, and unbound rhGAA was washed away. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 12A, ATB200 rhGAA bound to CIMPR significantly better than Lumizyme®.

[0163] Figure 12B shows the relative content of bis-M6P glycan in Lumizyme®, conventional rhGAA, and ATB200 according to the present invention. In Lumizyme®, on average only 10% of molecules contain bis-phosphorylated glycan. This is compared to ATB200, in which on average all rhGAA molecules contain at least one bis-phosphorylated glycan.

[0164] Example 7: ATB200 rhGAA was internalized more efficiently by fibroblasts than Lumizyme. The relative cellular uptake of ATB200 and Lumizyme® rhGAA was compared using normal and Pompe fibroblast cell lines. The comparison included comparing 5–100 nM ATB200 rhGAA according to the present invention with 10–500 nM conventional rhGAA Lumizyme®. After 16 hours of incubation, external rhGAA was inactivated with TRIS bases, and cells were washed three times with PBS before collection. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and graphed against total cellular protein, the results of which are shown in Figures 13A–B.

[0165] ATB200 rhGAA was also shown to be efficiently internalized into cells (Figures 13A and 13B), internalizing into both normal and Pompe fibroblasts, and exhibiting higher internalization than conventional Lumizyme® rhGAA. ATB200 rhGAA saturates the cell receptor at approximately 20 nM, while Lumizyme® requires approximately 250 nM. The uptake efficiency constant (K) can be extrapolated from these results. 取り込み As shown in Figure 13C, the rhGAA is 2-3 nm for ATB200 and 56 nM for Lumizyme®. These results suggest that ATB200 rhGAA is a well-targeted treatment for Pompe disease.

[0166] Example 8: Glycogen reduction in Gaa knockout mice Figures 14A–14C show the effects of administering alglucosidase alpha (Lumizyme®) and ATB200 to Gaa knockout mice. The animals received two IV bolus doses (every other week). Tissue was collected two weeks after the final dose and analyzed for acid α-glucosidase activity and glycogen content.

[0167] As can be seen in Figures 14A-14C, ATB200 was found to dose-dependently reduce tissue glycogen in acid α-glucosidase (Gaa) knockout mice. A 20 mg / kg dose of ATB200 consistently removed stored glycogen at a greater rate in Gaa knockout mice than at the 5 mg / kg and 10 mg / kg dose levels. However, as seen in Figures 14A-14C, ATB200 administered at 5 mg / kg showed a similar reduction in glycogen in mouse cardiac and skeletal muscle (quadriceps and triceps) as Lumizyme® administered at 20 mg / kg, while ATB200 administered at 10 and 20 mg / kg showed a significantly better reduction in glycogen levels in skeletal muscle than Lumizyme®.

[0168] Figure 15 shows the effects of administering alglucosidase alpha (Lumizyme®) and ATB200 in Gaa knockout mice, as well as the effects of co-administration of ATB200 and miglustat on glycogen clearance. 12-week-old GAA KO mice were injected with Lumizyme® or ATB200, 20 mg / kg IV every four weeks; as shown, miglustat was co-administered at 10 mg / kg PO 30 minutes prior to rhGAA. Tissue was collected 14 days after the last enzyme dose for glycogen measurement. Figure 15 shows the relative decrease in glycogen in quadriceps and triceps skeletal muscle, with ATB200 resulting in a greater decrease in glycogen than Lumizyme®, and ATB200 / miglustat resulting in an even greater decrease in glycogen.

[0169] Example 9: Muscle physiology and morphology in Gaa knockout mice Gaa knockout mice were administered two IV bolus doses of recombinant human acid α-glucosidase (alglucosidase alpha or ATB200) at 20 mg / kg every other week. Miglustat was orally administered at a dose of 10 mg / kg to a subset of animals treated with ATB200 30 minutes prior to ATB200 administration. Control mice were treated with solvent only. Soleus, quadriceps, and septal tissues were collected two weeks after the last administration of recombinant human acid α-glucosidase. Soleus and septal tissues were analyzed for glycogen levels by staining with Schiff periodate reagent (PAS) and for lysosomal proliferation by measuring the levels of lysosomal-associated membrane protein (LAMP1) markers upregulated in Pompe disease. Semithin sections of quadriceps muscle embedded in epoxy resin (Epon) were stained with methylene blue and observed under an electron microscope (1000×) to determine the extent of vacuole presence. Immunohistochemical analysis of the quadriceps muscle samples determined the levels of the autophagy marker microtubule-associated protein 1A / 1B light chain 3-phosphatidylethanolamine conjugate (LC3A II) and p62, as well as the insulin-dependent glucose transporter GLUT4 and the insulin-independent glucose transporter GLUT1.

[0170] In a similar experiment, Gaa knockout mice were given four IV bolus doses of recombinant human acid α-glucosidase (alglucosidase alpha or ATB200) at a dose of 20 mg / kg every other week. Miglustat was orally administered at a dose of 10 mg / kg to a subset of animals treated with ATB200 30 minutes prior to ATB200 administration. Control mice were treated with solvent only. Cardiac muscle tissue was collected two weeks after the last administration of recombinant human acid α-glucosidase and analyzed by staining with periodate Schiff's reagent (PAS) for glycogen levels and by measuring LAMP1 levels for lysosomal proliferation.

[0171] As shown in Figure 16, administration of ATB200 showed a reduction in lysosomal proliferation in cardiac and skeletal muscle (soleus muscle) tissue compared to conventional treatment with alglucosidase alfa, and co-administration of miglustat with ATB200 showed that lysosomal proliferation approached levels seen in wild-type (WT) mice. Furthermore, as shown in Figure 17, administration of ATB200 showed a reduction in punctate glycogen levels in cardiac and skeletal muscle (soleus muscle) tissue compared to conventional treatment with alglucosidase alfa, and co-administration of miglustat with ATB200 again showed that levels approached levels seen in wild-type (WT) mice.

[0172] Similarly, as shown in Figure 18, co-administration of miglustat and ATB200 significantly reduced the number of vacuoles in muscle fibers in the quadriceps of Gaa knockout mice compared to untreated mice and mice treated with alglucosidase alpha. As shown in Figure 19, levels of both LC3II and p62 were increased in Gaa knockout mice compared to wild-type mice, but significantly decreased with treatment with ATB200 and miglustat, suggesting that the increased autophagy associated with acid α-glucosidase deficiency is reduced with co-administration of ATB200 and miglustat. Furthermore, levels of insulin-dependent glucose transporter GLUT4 and insulin-independent glucose transporter GLUT1 were increased in Gaa knockout mice compared to wild-type mice, but again significantly decreased with treatment with ATB200 and miglustat. Elevated GLUT4 and GLUT1 levels associated with acid α-glucosidase deficiency may contribute to increased glucose uptake into muscle fibers, leading to increased glycogen synthesis both basally and after food intake. Therefore, combined treatment with ATB200 and miglustat was found to improve skeletal muscle morphology and physiology in a mouse model of Pompe disease.

[0173] Example 10: Muscle function in Gaa knockout mice In a long-term experiment with 12 bi-weekly doses, 20 mg / kg ATB200 + 10 mg / kg miglustat progressively increased functional muscle strength in GaaKO mice from baseline, as measured by both grip strength and wire hang tests (Figures 21A-21B). Mice treated with alglucosidase alfa (Lumizyme®) at the same ERT dose (20 mg / kg) were observed to be reduced under the same conditions for most of the experiment (Figures 21A-21B). Similar to the short-term experiment, ATB200 / miglustat resulted in substantially better glycogen clearance than alglucosidase alfa at 3 months (Figures 22A-22C) and 6 months (Figures 22D-22G) after treatment. ATB200 / miglustat also reduced autophagy and intracellular accumulation of LAMP1 and dysferrin after 3 months of treatment compared to alglucosidase alfa (Figure 23). In Figure 21A, * indicates statistically significant results compared to Lumizyme alone (p<0.05, two-tailed t-test). In Figures 22A-22G, * indicates statistically significant results compared to Lumizyme® alone (p<0.05, multiple comparisons using Dunnett's method under one-way ANOVA analysis).

[0174] In summary, these data indicate that ATB200 / miglustat efficiently targeted muscle, reversed cellular dysfunction, and improved muscle function. Importantly, the apparent improvement in muscle structure, along with reduced autophagy and intracellular accumulation of LAMP1 and dyspherin, may be a good alternative for improved muscle physiology correlated with improved functional muscle strength. These results suggest that monitoring autophagy and these key muscle proteins is a reasonable and practical method for evaluating the effectiveness of therapeutic treatments for Pompe disease in GaaKO mice, which may prove to be useful biomarkers from muscle biopsies in clinical trials.

[0175] Figure 23 shows that 6 months of ATB200 administration with or without miglustat reduces intracellular dystrophin accumulation in GaaKO mice. Dystrophin accumulation was significantly reduced with ATB200 ± miglustat compared to Lumizyme®.

[0176] Example 11: Capture of rhα-Gal A The CIMPR binding profile of recombinant human α-galactosidase A (rhα-Gal A) in used cell culture medium was measured before product capture using AEX chromatography (Figure 20A) and after product capture using AEX chromatography (Figure 20B). The dashed lines in both graphs represent the M6P elution gradient. Before AEX product capture, 80% of rhα-Gal A could bind to CIMPR. After AEX product capture, total rhα-Gal A binding increased to 96%.

[0177] Example 12: Pharmacokinetic and safety data of recombinant acid α-glucosidase ATB200 co-administered with miglustat in patients with Pompe disease who have previously undergone ERT and those who have not. This experiment was designed primarily to evaluate the safety, tolerability, and pharmacokinetics (PK) of ATB200 administered in combination with miglustat. Based on PK / pharmacodynamic (PD) translation models in Gaa knockout mice, it is predicted that the combination of 20 mg / kg of ATB200 with a high dose (e.g., 260 mg) of miglustat in humans will provide optimal glycogen reduction.

[0178] In the following explanation, "high dose" of miglustat refers to a dose of approximately 260 mg, and "low dose" refers to a dose of approximately 130 mg.

[0179] The objective of this Phase 1 / 2 trial was to evaluate preliminary total GAA protein, ATB200, and miglustat PK data, as well as safety markers, from 10 patients.

[0180] This shows the experimental design of an open-label, constant-sequence, dose-escalation, human first-instance, phase 1 / 2 study to evaluate the safety, tolerability, pharmacokinetics (PK), disease progression (PD), and efficacy of intravenous infusion of ATB200 co-administered with oral miglustat in adults with Pompe disease (Figure 24). Mean total GAA protein and miglustat PK results were evaluated from the first 8 patients in Cohort 1 and the first 2 patients in Cohort 3, presented through 9 visits. a Prior to administration in cohorts 2 and 3, safety data from two sentinel lymph node patients in cohort 1 were reviewed at each dose level. b In stages 2 and 3, miglustat was administered orally before initiating intravenous infusion of ATB200. For all doses, ATB200 was infused intravenously over 4 hours. c The first two patients in cohorts 2 and 3 became sentinel lymph node patients for their respective cohorts.

[0181] Key participation criteria Men and women aged 18-65 diagnosed with Pompe disease based on reported GAA enzyme activity deficiency or GAA genotyping. • Prior to the start of the trial, participants in Cohort 1 had received ERT using alglucosidase alpha for 2-6 years (more than 2 years for Cohort 2). • Currently, patients are receiving alglucosidase alfa every other week and have completed the last two infusions without any drug-related adverse events that resulted in discontinuation of medication (Cohorts 1 and 2). • Candidates must be able to walk 200-500 meters in the 6-minute walk test (Cohorts 1 and 3). • Standing forced vital capacity should be between 30% and 80% of the predicted normal value (Cohorts 1 and 3). • Must be wheelchair-bound and unable to walk without assistance (Cohort 2) PK analysis: • Blood samples were collected for plasma total GAA protein and activity levels. Stage 1: Before the start of ATB200 injection, and 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12 and 24 hours after injection. Stages 2 and 3: 1, 2, 3, 4, 4.5, 5, 6, 7, 9, 11, 13, and 25 hours after oral administration of miglustat. Blood samples for plasma miglustat concentration were collected immediately before oral administration of miglustat (time 0) and at 1, 1.5, 2, 2.5, 3, 4, 5, 6, 9, 11, and 25 hours after oral administration of miglustat. Plasma miglustat was determined by a valid LC-MS / MS assay. • Plasma total GAA protein concentrations of 5, 10, and 20 mg / kg for ATB200 were determined by effective LC-MS / MS quantification of rhGAA-specific "signature" peptides (one or more).

[0182] A preliminary analysis was completed for eight patients from Cohort 1 who completed Stages 1 and 2, and for two patients from Cohort 3 who initiated Stage 3. • The first patients switched to ERT represented the Pompe disease population and had received an average of 5.02 years of ERT (Table 6).

[0183] JPEG2026136113000017.jpg63154

[0184] Total GAA protein When administered alone, ATB200 increased slightly above dose-proportional (Table 7 and Figures 25A-25D). Variability appeared to increase with miglustat dose (Figure 25C). Co-administration of 20 mg / kg ATB200 with a high dose of miglustat (260 mg) increased total GAA protein exposure (AUC) by approximately 25% compared to 20 mg / kg ATB200 alone. The distribution half-life (α-phase) increased by 45%, suggesting that the high dose of miglustat stabilized ATB200 in plasma. The increase in distribution half-life was accompanied by an increase in AUC from time to maximum plasma concentration up to approximately 12 hours post-administration. The increases in AUC and half-life could be observed on a logarithmic scale during the terminal elimination phase (Figure 25B). ATB200 showed a relatively high volume of distribution. The properties of total plasma GAA protein appear to be similar between patients who have not undergone ERT (Cohort 3) and patients who have undergone ERT (Cohort 1) (Figures 25A and 25D).

[0185] JPEG2026136113000018.jpg123154

[0186] Miglustat PK Miglustat showed dose-proportional pharmacokinetics (Table 8 and Figure 26). Plasma miglustat appeared similar between single and multiple doses.

[0187] JPEG2026136113000019.jpg49154

[0188] Pharmacodynamics Up to the 11th visit in patients with ERT experience from Cohort 1 (Figures 27A and 27B): • Alanine aminotransferase (ALT) levels decreased in 5 out of 8 patients, and normalized in 4 out of 4 patients with elevated baseline levels. Aspartate aminotransferase (AST) levels decreased in 6 out of 8 patients, and normalized in 3 / 4 of patients with elevated baseline levels. • Creatine phosphokinase (CPK) levels decreased in 6 out of 8 patients, and normalized in 2 out of 6 patients with elevated baseline levels. • Urinary glucose tetrasaccharide (HEX4) levels decreased in all 8 patients.

[0189] By week 4, all four biomarker levels had decreased in two patients in the treatment-naive cohort (Cohort 3) (Figures 27C and 27D).

[0190] In Figures 27A to 27D, the data is expressed as mean ± standard error.

[0191] safety No serious adverse events (AEs) or infusion-related reactions were reported in any patient after a total of 155 or more infusions. • AEs resulting from the procedure, reported in 11 / 13 (84%) of patients, were generally mild and temporary. Treatment-related adverse events reported in 7 / 13 (53%) of patients were: nausea (n=1), fatigue (n=1), headache (n=1), tremor (n=2), acne (n=1), tachycardia (n=1), and hypotension (n=1).

[0192] conclusion ATB200 alone and in combination with miglustat have been safe and well-tolerated, and no fluid-related reactions have been reported to date. ATB200 alone showed a dose-proportional increase in exposure, which was further enhanced with miglustat, suggesting a chaperone-stabilizing effect on ATB200. After switching from standard treatment to ATB200 / miglustat, patients generally showed improvement in muscle injury biomarkers, with many showing normalization by week 18. The first two previously untreated patients treated with ATB200 / miglustat showed a significant reduction in all biomarkers of muscle damage.

[0193] The embodiments described herein are intended to illustrate the compositions and methods of the present invention and are not intended to limit the scope of the invention. Various modifications and changes consistent with this specification as a whole and readily apparent to those skilled in the art are intended. The appended claims should not be limited by any specific embodiment described in the examples, and should be interpreted in the broadest way consistent with the overall description.

[0194] Patents, patent applications, publications, product descriptions, GenBank accession numbers, and protocols are referenced throughout this application, and these disclosures are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for producing recombinant human lysosomal protein, Culturing host cells in a bioreactor that secretes recombinant human lysosomal proteins; Removing the culture medium from the bioreactor; The culture medium is filtered to provide a filtrate; The filtrate is packed into an anion exchange chromatography (AEX) column to capture the lysosomal protein; and To elute the first protein product from the AEX column, A method that includes this.

2. The method according to claim 1, wherein the recombinant human lysosomal protein is recombinant human α-glucosidase (rhGAA).

3. The method according to claim 2, wherein the rhGAA comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

4. Packing the first protein product onto an immobilized metal affinity chromatography (IMAC) column; and Elute the second protein product from the IMAC column. The method according to any one of claims 1 to 3, further comprising:

5. Packing the second protein product into a third chromatography column; and Elute the third protein product from the third chromatography column. The method according to claim 4, further comprising:

6. The method according to claim 5, wherein the third chromatography column is selected from a cation exchange chromatography (CEX) column and a size exclusion chromatography (SEC) column.

7. The method according to any one of claims 1 to 6, wherein the filtration of the culture medium is selected from alternating tangential flow filtration (ATF) and tangential flow filtration (TFF).

8. The method according to any one of claims 1 to 7, further comprising inactivating a virus in one or more of the first protein product, the second protein product, and the third protein product.

9. The method according to any one of claims 1 to 8, further comprising filtering the second protein product or the third protein product to provide a filtered product, and filling the filtered product into a vial.

10. The method according to any one of claims 1 to 9, further comprising freeze-drying the filtered product.

11. The method according to any one of claims 1 to 10, wherein the host cells include Chinese hamster ovary (CHO) cells.

12. The method according to any one of claims 1 to 11, wherein the host cells include the CHO cell line GA-ATB-200 or ATB-200-001-X5-14 or a subculture thereof.

13. (i) At least 90% of the first protein product, the second protein product, or the third protein product is bound to a cation-independent mannose-6-phosphate receptor (CIMPR), or (ii) The method according to any one of claims 1 to 12, wherein at least 90% of the first protein product, the second protein product, or the third protein product contains an N-glycan having mono-mannose-6-phosphate (mono-M6P) or bis-mannose-6-phosphate (bis-M6P).

14. The method according to any one of claims 1 to 13, wherein the rhGAA comprises seven potential N-glucosylation sites, at least 50% of the rhGAA molecule comprises an N-glycan unit having two mannose-6-phosphate residues at a first site, at least 30% of the rhGAA molecule comprises an N-glycan unit having one mannose-6-phosphate residue at a second site, at least 30% of the rhGAA molecule comprises an N-glycan unit having two mannose-6-phosphate residues at a fourth site, and at least 20% of the rhGAA molecule comprises an N-glycan unit having one mannose-6-phosphate residue at a fourth site.

15. Recombinant protein product prepared by the method described in any one of claims 1 to 14.

16. A pharmaceutical composition comprising the recombinant protein product described in claim 15 and a pharmaceutically acceptable carrier.

17. Use of the pharmaceutical composition according to claim 16 for treating lysosomal storage disorders.

18. The use according to claim 17, wherein the lysosomal storage disorder is Pompe disease and the recombinant protein is rhGAA.

19. The use according to claim 18, wherein a pharmacological chaperone of α-glucosidase is co-administered to the patient within 4 hours of administration of the pharmaceutical composition containing the rhGAA product.

20. The use according to claim 19, wherein the pharmacological chaperone is selected from 1-deoxynojirimycin and N-butyl-deoxynojirimycin.

21. The use according to claim 19 or 20, wherein the pharmacological chaperone is co-formulated with the rhGAA product.

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